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DNA polymerases in developing rat brain Chiu, Jen-fu 1972

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DNA POLYMERASES IN DEVELOPING RAT BRAIN by JEN-FU CHIU B.Pharm., Taipei Medical College, 1964 M.Sc, National Taiwan University, 1967 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in the Department of Biochemistry We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA Apr i l , 1972 I n p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f the r e q u i r e m e n t s f o r an advanced degree a t the U n i v e r s i t y o f B r i t i s h C olumbia, I agree t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p urposes may be g r a n t e d by t h e Head o f my Department o r by h i s r e p r e s e n t a t i v e s . ' I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l n o t be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . Department The U n i v e r s i t y o f B r i t i s h Columbia Vancouver 8, Canada - i -ABSTRACT DNA polymerase activity in soluble extracts from developing rat brain described here shows many simil a r i t i e s to other animal DNA polymerases. Two DNA polymerases, A and B, have been separated and purified from 10-day-old rat brain by ammonium sulfate fractionation and column chromatography on DEAE-cellulose. Some properties of these two enzymes differed considerably. Enzyme A was 2-3 times more active with heat-denatured DNA than with native DNA as template and Enzyme B almost always used native DNA as 2+ template. The optimal concentration of Mn was 0.05 mM for Enzyme A and 0.3 mM for Enzyme B. ATP stimulated only Enzyme A, but EDTA stimulated Enzyme B and showed l i t t l e or no effect on Enzyme A. Enzyme B was strongly stimulated by KC1. In the case of Enzyme A, however, salt gave no activation but rather a marked inhibition. Enzyme A was more sensitive to dithiothreitol and sulfhydryl-blocking agents than Enzyme B. The incorporation was linearly proportional to the Enzyme A concentration, but Enzyme B showed a sigmoidal slope in i t s enzyme concentration curve. Enzyme A sediments at around 9 S on sucrose gradients and Enzyme B at around 3-4 S. The pattern of developmental changes i n two DNA polymerases of rat brain was studied. In the cerebellum, DNA polymerase A i s the more active in very young animals but peaks - i i -at around the 6th day a f t e r b i r t h . DNA polymerase B i s more ac t i v e than Enzyme A i n the c e r e b e l l a of older animals. In the cerebral cortex DNA polymerase A a c t i v i t y i s higher than that of Enzyme B i n the f e t a l stage but the a c t i v i t y of polymerase A i s much lower than that of polymerase B at a l l post-natal ages studied. An extremely high a c t i v i t y of DNA polymerase B was s o l u b i l i z e d from the nuclear membrane-chromatin complex i s o l a t e d from r a t b r a i n . The r e l a t i v e amounts of soluble and p a r t i c u l a t e forms of DNA polymerase i n the c e r e b e l l a of r a t s change with age. Much of the a c t i v i t y i s i n the soluble form i n younger r a t s , but i n the adult r a t , the DNA polymerase e x i s t s almost e x c l u s i v e l y i n a p a r t i c u l a t e form which i s i n a c t i v e unless s o l u b i l i z e d as described. In younger r a t b r a i n , DNA polymerase A only e x i s t s i n the soluble form and i s l o c a l i z e d both i n the nucleoplasm and cytoplasm f r a c t i o n . However, there i s no, or very l i t t l e , DNA polymerase A a c t i v i t y i n adult r a t b r a i n . DNA polymerase B e x i s t s i n both the soluble and par-t i c u l a t e form i n younger r a t b r a i n . The soluble form of DNA p o l y -merase B i s l o c a l i z e d i n the cytoplasm and nucleoplasm. The major part of DNA polymerase B e x i s t s as the p a r t i c u l a t e form i n the n u c l e i at older stages. A p r o t e i n f a c t o r was i s o l a t e d from DNA polymerase B. The f a c t o r stimulated DNA polymerase B a c t i v i t y with double stranded DNA as template and showed no e f f e c t on DNA synthesis with s i n g l e - i i i -stranded DNA as template. It seems that this factor i s specific for DNA polymerase B. The stimulatory effect was not due to the activity of a nuclease or nickase. The polyamines, i.e. spermine, spermidine and putrescine, enhanced the DNA polymerase activity in extracts of brain from 10-day-old rats by more than 50% and stimulated purified DNA polymerase B about 100%. However, polyamines showed l i t t l e or no effect on DNA polymerase A activity. The mechanism of sper-midine stimulation of DNA polymerase B activity is presumably through a f a c i l i t a t i o n or stabilization of the association of enzyme and the protein factor. - iv -ACKNOWLEDGEMENTS I am most grateful to my supervisor, Professor S.C. Sung, for his careful guidance and encouragement throughout the course of this work. I should lik e to express my sincere thanks to Professors S.H. Zbarsky and J.F. Richards for a c r i t i c a l reading of the manuscript. I should lik e to extend general thanks to my laboratory colleagues, particularly Dr. E.G. McGeer and Mr. W.P. Popow for their valuable discussions. I am sincerely grateful to Miss Joanne Allan for typing the manuscript. I thank, most of a l l , my wife Lucia for her encouragement, forbearance and enthusiasm during the research and especially for her help in arranging this manuscript. Looking back into the past I must convey my thanks to my parents for their support, encouragement and stimulating my interest i n the f i e l d of biological science. I wish to thank the Medical Research Council of Canada for financial assistance through grants made to Professor Sung. - V -TABLE OF CONTENTS Page I. INTRODUCTION 1 1.1. Background 1 1.2. General Properties of DNA Polymerase 2 1.2a. DNA primer 3 1.2b. Divalent cation s p e c i f i c i t y 5 1.2c. Effect of monovalent ion 5 1.2d. Optimal pH 6 1.2e. Deoxyribonucleoside triphosphates 6 1.3. Replication of DNA 7 1.3a. Semiconservative mechanism of replication 7 1.3b. Nature of the product 9 1.3c. Starting point and direction of synthesis 10 1.3d. Discontinuous mechanism 11 1.3e. Polynucleotide ligase 13 1.3f. Replication and c e l l membrane 14 1.3g. In vitro DNA replication 18 1.4. The Intracellular Location and Multiple Forms of DNA Polymerase 23 1.5. DNA and Its Synthesis in Developing Brain 27 1.6. The Regulation of DNA Synthesis 29 1.7. Polyamines and DNA Biosynthesis 32 1.8. The Present Investigation 33 - v i -TABLE OF CONTENTS (Continued) Page II. MATERIALS AND METHODS 35 2.1. Experimental Animals 35 2.2. Chemicals 35 2.3. Assay for Enzyme Activities 37 2.3a. DNA polymerase (replicative deoxyribo-nucleotidyl transferase) 37 2.3b. Terminal deoxynucleotidyl transferase .. 39 2.3c. Alkaline DNase 40 2.3d. Nickase 41 2.3e. Phosphodiesterase activity 42 2.3f. ATPase activity 43 2.4. Characterization of The Product 44 2.5. Preparation of Brain Nuclei 45 2.6. Preparation of Enzyme Extracts from Rat Brain . 45 2.7. Preparation of DEAE-Cellulose Column 46 2.8. Preparation of Sephadex G-100 and Sephadex G-200 Columns 46 2.9. Determination of Radioactivity 47 2.10. Purification of DNA Polymerase from 10-day-old Rat Brain 47 2.10a. Ammonium sulfate fractionation 47 2.10b. DEAE-cellulose column chromatography ... 49 - v i i -TABLE OF CONTENTS (Continued) Page 2.11. Fractionation of Enzyme on Sephadex Gel F i l t r a t i o n 49 2.11a. Column chromatography of 10-day-old cerebellar DNA polymerase on Sephadex G-100 .' 49 2.11b. Column chromatography of DNA polymerase B on Sephadex G-200 50 2.12. Neutral Sucrose Density Gradient Centrifugation .. 50 2.13. Subcellular Fractionation of Rat Brain 52 2.14. Other Analytical Methods 53 III. RESULTS AND DISCUSSION 54 3.1. Preliminary Studies on DNA Polymerase in Rat Brain Extract 54 3.1a. Extracts of DNA polymerase from developing rat brain 54 3.1b. Characteristics of DNA polymerase of rat brain 54 3.1c. The properties of DNA polymerase 56 3.Id. The effects of mixing extract from different ages 62 3.1e. Regional changes of DNA polymerase activity of rat brain at different ages 65 3.If. Column chromatography of crude cerebellar DNA polymerase on Sephadex G-100 67 - v i i i -TABLE OF CONTENTS (Continued) Page 3.2. Separation and Characterization of DNA Polymerases A and B from Developing Rat Brain 70 3.2a. Partial purification of two DNA polymerases A and B from rat brain extract 70 (1) Preparation of extract 71 (2) Ammonium sulfate fractionation 71 (3) DEAE-cellulose column chromatography.... 73 3.2b. Properties of par t i a l l y purified enzymes A and B 75 (1) Requirements for the part i a l l y purified DNA polymerases from rat brain 77 (2) Template preference 82 (3) The effect of bivalent cations 84 (4) The effect of monovalent cations 84 (5) PH optimum 87 (6) Time course 87 (7) Enzyme concentration curve 90 3.3. Pattern of Developmental Changes in DNA Polymerases A and B of Rat Brain 93 3.3a. The sedimentation profile of DNA polymerase A and B of rat brain 94 3.3b. Developmental changes i n DNA polymerases A and B of rat cerebellum 97 3.3c. Pattern of developmental changes i n DNA polymerases A and B of rat cortex 97 - i x -TABLE OF CONTENTS (Continued) Page 3.4. P a r t i c u l a t e Form of DNA Polymerase i n Rat B r a i n 102 3.4a. S o l u b i l i z a t i o n of the p a r t i c u l a t e form of DNA polymerase from a d u l t r a t b r a i n n u c l e i ... 103 3.4b. C h a r a c t e r i s t i c s of s o l u b i l i z e d DNA p o l y -merase from a d u l t r a t b r a i n n u c l e i 104 3.4c. P a t t e r n of p a r t i c u l a t e and s o l u b l e forms of DNA polymerases A and B i n r a t b r a i n 109 3.4d. I n t r a c e l l u l a r d i s t r i b u t i o n of DNA polymerases A and B i n r a t b r a i n 113 3.5. P r o t e i n F a c t o r s of DNA Polymerase B i n Developing Rat B r a i n 118 3.5a. Separation of DNA polymerase B i n t o a c t i v a t o r and p a r t i a l l y i n a c t i v e enzyme 120 3.5b. Nature of s t i m u l a t i n g f a c t o r ( s ) 123 3.5c. Nuclease a c t i v i t y of the f a c t o r ( s ) 123 3.5d. Time-course of s t i m u l a t i o n 129 3.5e. The e f f e c t of f a c t o r on DNA polymerases A and B w i t h v a r i o u s DNAs as template 129 3.6. The E f f e c t of Polyamines on DNA Polymerase A c t i v i t y . 133 3.6a. The e f f e c t of polyamines on DNA polymerase of r a t b r a i n e x t r a c t 133 3.6b. The e f f e c t of spermidine on p a r t i a l l y p u r i -f i e d DNA polymerases A and B 136 TABLE OF CONTENTS (Continued) Page 3.6c. The effect of spermidine depends on the template 138 3.6d. The effect of spermidine on enzymes i n -hibiting DNA synthesis 139 3.6e. The effect of spermidine on DNA polymerase B in different concentrations of bivalent cations 141 3.6f. The effect of spermidine on DNA synthesis using nucleohistone as template 144 3.6g. The mechanism of the stimulatory effect of spermidine on DNA polymerase 147 IV. CONCLUSION 153 V. BIBLIOGRAPHY 159 - x i -LIST OF TABLES Page Table I. Requirements for rat brain DNA polymerase 55 Table II. Characterization of the product polymerized by rat brain DNA polymerase 57 Table III. The effect of mixing extracts from different ages of rat 64 Table IV. Regional changes of DNA polymerase activity of rat brain at different ages 66 Table V. Pa r t i a l purification of DNA polymerase from rat brain 72 Table VI. Requirements of DNA polymerases A and B 78 Table VII. Terminal deoxynucleotidyl transferase activity i n enzyme fractions A and B 81 Table VIII. Nature of template and priming a b i l i t y 83 Table IX. Solubilization of DNA polymerase from adult rat brain nuclei 105 Table X. The effects of various factors upon solubilized DNA polymerase act i v i t y 106 Table XI. Nature of template and priming a b i l i t y for solubilized nuclear DNA polymerase 107 Table XII. Intracellular distribution of DNA polymerases in 10-day-old rat brain 115 Table XIII. Intracellular distribution of DNA polymerases i n adult rat brain 116 Table XIV. Partial loss of DNA polymerase B activity during purification 121 Table XV. Nature of activator of DNA polymerase B 124 Table XVI. Nuclease activity of factor 126 Table XVII. Priming activity of DNA with or without pre-incubation with factor 128 - x i i -LIST OF TABLES (Continued) Page Table XVIII. Factor effect on Enzymes A and B with various DNA as template 132 Table XIX. The effect of polyamines on DNA polymerase of rat brain extract 134 Table XX. The effect of polyamines on DNA polymerases A and B 137 Table XXI. Spermidine effects on DNA polymerase fractions II and I I V 142 a b Table XXII. The effect of spermidine on ATPase and phospho-diesterase ac t i v i t i e s ...... 143 2+ Table XXIII. Effect of spermidine and Mg on DNA poly-merase B activity 145 Table XXIV. The effect of spermidine on DNA polymerase ac-t i v i t i e s i n the presence of bivalent cations.. 146 Table XXV. The effect of spermidine and histone on DNA polymerase activity 148 Table XXVI. The effect of spermidine on DNA polymerase A and B using nucleohistone as template 149 Table XXVII. Spermidine effect on DNA polymerases A and B with protein factor isolated from enzyme B ... 152 - x i i i -LIST OF FIGURES Page Figure 1. The effects of magnesium ions on the activity of DNA polymerase from 10-day-old rat brain 58 Figure 2. The effect of pH on the DNA polymerase act i v i t y from rat brain 60 3 Figure 3. The time-course of the incorporation of [ H]dTTP of 10-day-old rat cerebellar extracts 61 Figure 4. The s t a b i l i t y of cerebellar DNA polymerase 63 Figure 5. Column chromatography of crude cerebellar DNA polymerase on Sephadex G-100 69 Figure 6. Column chromatography of Fraction II on DEAE-cellulose 74 Figure 7. Column chromatography of Fraction 11^ on DEAE-cellulose 76 Figure 8. The effect of MgC^ on the DNA polymerase A and B act i v i t i e s 85 Figure 9. The effect of MnC^ on polymerase A and B activities86 Figure 10. The effect of KCI on DNA polymerase A and B act i v i t i e s 88 Figure 11. The effect of pH on DNA polymerase A and B acti v i t i e s 89 3 Figure 12. Time-course of the incorporation of [ H]dTTP of DNA polymerases A and B 91 3 Figure 13. Variation of incorporation of [ H]dTTP into DNA with different amounts of DNA polymerases A and B 92 Figure 14. Sucrose density gradient centrifugation studies on DNA polymerases of rat brain 96 Figure 15. Developmental changes in DNA polymerases A and B of rat brain cerebellum 98 Figure 16. Developmental changes in DNA polymerases A and B of rat cerebral cortex 99 - x i v -LIST OF FIGURES (Continued) Page Fig u r e 17. The sucrose g r a d i e n t c e n t r i f u g a t i o n p r o f i l e of DNA polymerases A and B of f e t a l r a t c e r e b r a l c o r t e x 100 Fi g u r e 18. Enzyme c o n c e n t r a t i o n curve f o r s o l u b i l i z e d n u c l e a r DNA polymerase 108 Fig u r e 19. Sucrose d e n s i t y g r a d i e n t c e n t r i f u g a t i o n pro-f i l e of s o l u b i l i z e d n u c l e a r DNA polymerase ... 110 Fi g u r e 20. Developmental changes i n p a r t i c u l a t e and s o l u b l e forms of DNA polymerases i n r a t cerebellum 112 Fi g u r e 21. Sucrose d e n s i t y g r a d i e n t c e n t r i f u g a t i o n p ro-f i l e o f DNA polymerase from 10-day-old r a t b r a i n mitochondria 117 Fi g u r e 22. Sucrose d e n s i t y g r a d i e n t c e n t r i f u g a t i o n p r -f i l e of DNA polymerases from r a t b r a i n n u c l e i 119 Fi g u r e 23. Column chromatography of DNA polymerase B on Sephadex G-200 122 Figure 24. A l k a l i n e sucrose g r a d i e n t c e n t r i f u g a t i o n p ro-f i l e of n a t i v e a s c i t e s X^C-DNA a f t e r i n c u -b a t i o n w i t h p r o t e i n f a c t o r 127 Fi g u r e 25. K i n e t i c s t u d i e s on s t i m u l a t i o n of DNA p o l y -merase B by F a c t o r 130 Fig u r e 26. The time-course of the a c t i o n of spermidine on DNA polymerase i n 10-day-old r a t b r a i n e x t r a c t 135 Fig u r e 27. The e f f e c t o f spermidine on DNA polymerase a c t i v i t y w i t h d i f f e r e n t DNAs as template ... 140 Fig u r e 28. The e f f e c t of spermidine on enzyme concen-t r a t i o n curve of DNA polymerase B 150 - X V -LIST OF ABBREVIATIONS DNA RNA DNase RNase dNTP dATP dGTP dCTP dTTP or TTP ATP ATPase NAD spd NEM Tris EDTA TCA DEAE-T m cpm nmole pmole O.D. A280 ° r A260 UV deoxyribonucleic acid ribonucleic acid deoxyribonuclease ribonuclease deoxynucleoside triphosphates deoxyadenosine 5'-triphosphate deoxyguanosine 5'-triphosphate deoxycytidine 5'-triphosphate deoxythymidine 5'-triphosphate adenosine 5'-triphosphate adenosine triphosphatase nicotinamide adenine dinucleotide spermidine N-ethylmaleimide t r i s (hydroxymethyl) aminomethane ethylenediaminetetraacetate trichloroacetic acid d i e thy1amino e thy1-midpoint of thermal denaturation counts per minute (radioactivity) _9 nanomole (1 x 10 mole) -12 picomole (1 x 10 mole) optical density absorbance at wave length of 280 nm or 260 nm ultraviolet light - 1 -INTRODUCTION 1.1. Background The observation by Reichard and Estborn (1) that thymidine was taken up into DNA only stimulated research on thymidine metabolism. A major breakthrough in the understanding of DNA biosynthesis occurred in 1957 with the work of Kornberg and his collaborators (2) who reported the preparation of a c e l l -free extract from Escherichia c o l i which brought about the phosphorylation of thymidine to thymidine triphosphate and another extract which brought about the incorporation of the triphosphate into polydeoxyribonucleotides. They also purified the enzyme which catalyzed the latter reaction and named this enzyme DNA polymerase (DNA deoxyribonucleotidyltransferase, EC 2.7.7.7) (3,4). The reaction required the presence of DNA 2+ template, Mg ions, and deoxyribonucleoside 5'-triphosphates (dATP, dGTP, dCTP and dTTP) in addition to the enzyme. The poly-deoxynucleotide produced by the reaction was shown to resemble the primer DNA in base composition (5). Inorganic pyrophosphate was also produced i n proportion to the total amount of deoxy-nucleoside triphosphate used. DNA polymerase catalyzed the for-mation of polydeoxyribonucleotides as represented in the following reaction sequence: - 2 -n, dATP n 2 dGTP n 3 dCTP n, dTTP + DNA Enzyme n1 dAMP xi2 dGMP n 3 dCMP n4 dTMP -DNA + (n± + n 2 + n 3 + n4> PP DNA polymerase has been par t i a l l y or extensively purified and characterized from a number of sources including bacteria (2,4,6-8), bacteriophage-infected bacteria (9-11), calf thymus (12-14), rat thymus (15), regenerating rat l i v e r (16-19), rat li v e r nuclei and mitochondria (20,21), sea urchin (22), mammalian neoplasms (23-28) and certain c e l l lines grown in tissue culture -HeLa cells (29-31), BHK 21 cells (32), KB cells (33,34), RK cells (35) and L cells (36,37). DNA polymerase activity of some of these cultured cells has also been examined after infection with adenovirus (33), vaccinia virus (31,33), pseudorabies virus (35) and herpes simplex virus (32,38). 1.2. General Properties of DNA Polymerase DNA polymerase a c t i v i t i e s which have been par t i a l l y purified from a number of different sources a l l apparently catalyze the overall reaction given above. However, there i s s t i l l no evidence to show that DNA polymerases derived from bacteria are identical with any of those prepared from mammalian tissues. The enzyme purified from E_. c o l i has been shown to di f f e r in immunological cross-reaction from the enzyme prepared after coliphage i n -fection (9). Some of the differences in properties among the - 3 -various DNA polymerase ac t i v i t i e s include the requirements for DNA templates, the physical state of the templates, requirements for primers, the divalent cation s p e c i f i c i t y , and the sensitivity to inhibition by monovalent ions. 1.2a. DNA primer Although the DNA polymerase reaction w i l l not proceed i n the absence of DNA as primer or template (13,19,37,39), the E_. c o l i DNA polymerase catalyzes a de_ novo synthesis of polydeoxynucleotide from deoxynucleoside triphosphates i n the absence of DNA primer (40). The polymers appear only after a long period of incubation during which no acid insoluble polydeoxynucleotide i s detectable. How-ever, no comparable activity has been described for DNA polymerases from mammalian sources. Denatured DNA i s substantially more active in supporting synthesis in most systems than i s native DNA (13,24,37,41). Thermal, chemical or enzymatic degradation of DNA structure i s necessary before incorporation of triphosphate into new polymer molecules can be detected. However, the enzymes purified from regenerating l i v e r (19) and sea urchin (22) appear to prefer native DNA as primer. It would be particularly important i f this phenomenon could be demonstrated in the complete absence of a contaminating nuclease. - 4 -I t has been shown (42) that s m a l l o l i g o d e o x y r i b o n u c l e o t i d e s , as short as 15-20 monomer u n i t s , serve as primers i n c a l f thymus DNA polymerase r e a c t i o n . The " t e r m i n a l a d d i t i o n " and " r e p l i c a t i o n " a c t i v i t i e s have been s u c c e s s f u l l y separated i n c a l f thymus p r e p a r a t i o n s (43), but s i m i l a r a c t i v i t i e s i n p u r i f i e d c o l i polymerase p r e p a r a t i o n s seem to be due to the same enzyme. The r a t e s of the E_. c o l i , c a l f thymus, Landschutz a s c i t e s , and Walker carcinosarcoma DNA p o l y -merase r e p l i c a t i o n r e a c t i o n s are a l l s t i m u l a t e d by l i m i t e d d i g e s t i o n of the double-stranded DNA template. In the l a t t e r two enzyme systems, 3'-phosphoryl t e r m i n a l o l i g o n u c l e o t i d e s exert an i n h i b i t o r y e f f e c t on the s y n t h e t i c r e -a c t i o n , but the i n h i b i t i o n i s e l i m i n a t e d a f t e r removal of the 3'-phosphoryl groups by the a c t i o n of b a c t e r i a l a l k a l i n e phosphatase. P a r a l l e l experiments w i t h 5'-phosphoryl t e r m i n a l o l i g o n u c l e o t i d e s showed that removal of the 5'-phosphoryl group r e s u l t s i n a r e -d u c t i o n of the s t i m u l a t i o n e x e r c i s e d by these compounds (44,45). Th i s has l e d to a suggestion that the presence of a 5'-phosphoryl t e r m i n a l group and 3'-hydroxyl group might t h e r e f o r e promote the in c r e a s e d a c t i v i t y of DNA polymerase, and i t suggests that the polymerase r e a c t i o n i n these cases r e q u i r e s not only a h i g h polymer DNA template but a l s o an o l i g o m e r i c DNA "primer" or " i n i t i a t o r " . - 5 -1.2b. Divalent cation s p e c i f i c i t y The requirement f o r a d i v a l e n t cation i n the r e a c t i o n i s absolute. The h i g h l y p u r i f i e d b a c t e r i a l DNA polymerases 2+ 2+ require e i t h e r Mg or Mn ion f o r a c t i v i t y . The s u b s t i t u t i o n 2+ 2+ of Mn f o r Mg i n the E_. c o l i polymerase r e a c t i o n r e s u l t s i n a loss of s p e c i f i c i t y f o r deoxyribonucleoside triphosphates and the corresponding ribonucleoside triphosphates are incorporated into the product (46). The Walker tumor DNA polymerase i s also 2+ 2+ a c t i v e with e i t h e r Mg or Mn but not r i b o n u c l e o t i d e i n c o r -2+ poration i s detectable i n the presence of the l a t t e r (25). Mn 2+ w i l l not replace Mg f o r the polymerase r e a c t i o n catalyzed by 2+ extracts of regenerating r a t l i v e r (19). I t i s c l e a r that Mg 2+ ions are most e f f i c i e n t of a l l at about 5 mM, that Ca ions are 2+ v i r t u a l l y incapable of promoting any synthesis, and that Mn ions give a r e l a t i v e l y low optimum response at 1 mM and at higher con-centrations are i n h i b i t o r y . 1.2c. E f f e c t of monovalent ion Walwick and Main (15) recognized that monovalent cations exert a profound and s p e c i f i c e f f e c t on the DNA polymerase r e a c t i o n i n preparations from r a t thymus, and the optimal concentrations f o r + + + + + L i , Na , Rb , Cs and NH^ ions are at approximately 12 mM, 45 mM, 55 mM, 60 mM, 65 mM and 105 mM r e s p e c t i v e l y . High concentrations - 6 -of some of the ions s e r i o u s l y i n h i b i t the c a l f thymus DNA p o l y -merase r e a c t i o n (13). At lower c o n c e n t r a t i o n s , K + and N a + ions exert a co n s i d e r a b l e s t i m u l a t o r y e f f e c t that i s p a r t i c u l a r l y marked w i t h K + ions i n the enzyme systems from c a l f thymus and Landschutz a s c i t e s tumor c e l l s (24,47). However, the Walker tumor DNA polymerase was found to be i n h i b i t e d by 20 mM concen-t r a t i o n s of ions i n the f o l l o w i n g i n c r e a s i n g order of i n h i b i t i o n -+ + + + _ _ _ _ _ c a t i o n s : NH^ , K , Na , L i ; anions: Br , N0 3 , C l , HC0 3 , F (48). 1.2d. Optimal pH The optimum pH of DNA polymerase i s about 7.5 i n s e v e r a l p a r t i a l l y p u r i f i e d systems (13,19,37). However, Chang and Bollum (49) reported that there are two DNA polymerases i n mammalian systems, one has a n e u t r a l pH optimum, and the other has an a l k a l i n e pH optimum. The DNA polymerase w i t h an a l k a l i n e pH optimum was f i r s t observed by Howk and Wang (50). S i m i l a r pH optima have been reported by Leung and Zbarsky (51) , and Gold and H e l l e i n e r (37). 1.2e. Deoxyribonucleoside triphosphates A l l four deoxyribonucleoside 5'-triphosphates (dATP, dCTP, dGTP and dTTP) are r e q u i r e d f o r o p t i m a l a c t i v i t y . Omission of one or more of these triphosphates r e s u l t s i n r e d u c t i o n of a c t i v i t y - 7 -(13,19,37). However, in common with most animal DNA polymerases (48) the KB c e l l and sea urchin embryo DNA polymerases do not display a s t r i c t requirement for a l l four deoxyribonucleoside triphosphates. The Ehrlich ascites enzyme is an exception i n that, as in the case of the bacterial and phage polymerases, synthesis drops to very low levels i f one deoxynucleoside t r i -phosphate i s omitted. The KB c e l l and sea urchin enzymes can synthesize at 25-50% of the normal rate with only three triphos-phates (22,33,34). 1.3. Replication of DNA 1.3a. Semiconservative mechanism of replication The absolute requirements of the enzyme for DNA and the four deoxyribonucleoside 5'-triphosphates give a strong indication that the DNA strands are being replicated during the reaction. On the basis of the Watson-Crick model for linear molecules of DNA (52) replication proceeds by a semiconservative mechanism (53) in which only one half of the parental structure i s passed on to i t s daughter molecules. This model of replication contrasts with conservative and dispersive replication (54). Experimental evidence by Meselson 15 14 and Stahl (55), who analyzed the banding pattern of N/ N hybrid E_. c o l i DNA after centrifugation in CsCl density gradients, sup-ported this mechanism of replication. Meselson and Stahl cultured —' c°li f ° r a number of generations i n medium containing heavy - 8 -nitrogen (^N). After incubating these bacteria for different periods of time i n a medium containing lighter isotopes of nitrogen 14 ( N), they then studied the density of the bacterial DNA in cesium chloride gradients. They observed that DNA of high, intermediate and low density appeared in a sequence consistent with a semi-conservative mode of DNA replication. Furthermore, they showed that DNA of intermediate density can be separated by heating at 100°C for 30 min, into a light and heavy fraction, suggesting that this was hybride DNA formed by a light and heavy chain. Results comparable to those of Meselson and Stahl (55) i n E_. c o l i were also obtained i n human c e l l lines (56,57), DNA of intermediate density from a mammalian c e l l line (ERK-1) was also separated into heavy and light strands (58). The experiments of Taylor et ftl (59) were able to provide clear evidence of the semi-conservative segregation DNA. They 3 used thymidine (Tdr) labelled with tritium ( H), an isotope emitting very weak beta radiations, and thus obtained high resolution autoradiographs. Using their improved technique, they flashed labelled root tips of Vicia faba and l e f t the ce l l s to duplicate DNA in the presence of colchicine and non-radioactive media. At the f i r s t cycle of duplication, after labelling, chromosomes showed both chromatids labelled, while at the second f u l l cycle of duplication, after labelling, only one chromatid of each chromosome was labelled. This indicated that each - 9 -chromatid is formed by two functional units that reduplicate* and then segregate at anaphase together with their replica. 1.3b. Nature of the product The replication of a h e l i c a l DNA template by _E. c o l i DNA polymerase produced a macromolecule which showed the molar pro-portion of bases expected of a complementary copy of the tem-plate (3). While i t has not been possible to determine the complete sequence of nucleotides in DNA, as yet, a procedure which i s termed "nearest neighbour frequency analysis" has been devised by Josse et a l (60). This technique permitted an e s t i -mate of the relative frequencies with which pairs of nucleotides l i e next to each other to be made. By this technique, i t was possible to determine that the base order of the synthetic DNA product was specified by the base sequence of the primer. The "nearest neighbour" technique also showed that the two strands of the product had opposite polarities. This result showed that DNA synthesis proceeded i n opposite directions along each of the two strands of template DNA. This would, of course, suggest that synthesis occurs from opposite ends of the double helix or that a separate (and as yet undiscovered) enzyme exists which is specific for synthesis in the opposite direction. Mitra and Romberg (61) have shown that DNA polymerase is only able to replicate a DNA strand, in vitro , in a 5 1 to 3' direction. At - 10 -present no enzyme has been demonstrated which can replicate i n the 3' to 5' direction. 1.3c. Starting point and direction of synthesis It has been known for many years that the chromosome of E_. c o l i i s replicated sequentially from a starting point or origin at a fixed position. This was f i r s t shown by Cairns (62) using an autoradiographic technique. From Cairns study, i t appeared that replication proceeded in a circular manner from one fixed point and in only one direction. Yoshikawa and Sueoka (63) have also presented indirect evidence for a single replication point per chromosome in Bacillus s u b t i l i s . Furthermore firm evidence for unique starting points for replication comes from studies of the timing of duplication of specific v i r a l and bacterial genes (64,65). With phage X, DNA replication always i n i t i a t e s upon the parental DNA duplex very near gene 0 and synthesis proceeds outward from this point (the origin) i n both clockwise and counter-clockwise directions (64,65). An electron micrographic denaturation study on replicating molecules of phage, similar to the X study, again identifies a unique origin (66). Studies with E. c o l i show that DNA replication begins at a fixed point near the arginine H gene (at about 8 O'clock on the genetic map), with some recent experiments suggesting that the growth also proceeds in both directions around the c i r c l e (67-70). This result caused much s u r p r i s e at f i r s t s i n c e v i r t u a l l y every one had assumed t h a t growth would proceed i n only one d i r e c t i o n (62). B i - d i r e c t i o n a l s y n t h e s i s w i t h X i s reminiscent of the a l t e r n a t i n g d i r e c t i o n of r e p l i c a t i o n observed i n the eukaryote chromosome (71). 1.3d. Discontinuous mechanism Since the strands of DNA are a n t i - p a r a l l e l , the newly s y n t h e s i z e d complementary strands would be expected to grow w i t h opposite p o l a r i t y . However, i n v i t r o s t u d i e s w i t h DNA polymerase i n d i c a t e that t h i s enzyme i s capable of c a r r y i n g out DNA r e p l i c a t i o n only by the i n c o r p o r a t i o n of deoxyribonucleo-t i d e s i n a 5' > 3' d i r e c t i o n . A model to circumvent t h i s d i f f i c u l t y has been proposed by Okazaki e_t a_l (72-75). I n t h i s model, DNA polymerase would i n i t i a t e chains a t i n t e r v a l s at the growing p o i n t and r e p l i c a t e DNA i n a 5' > 3' d i r e c t i o n on both strands. This mechanism f o r disco n t i n u o u s s y n t h e s i s of DNA would l e a d to a temporary formation of fragments (7-11S) which would be elongated by DNA polymerase. When juxta p o s e d , these fragments could be j o i n e d by p o l y n u c l e o t i d e l i g a s e . Evidence i n support of t h i s d iscontinuous model of DNA r e p l i -c a t i o n has been presented. A f t e r an extremely short p u l s e w i t h l a b e l l e d thymidine and d e n a t u r a t i o n , p u l s e l a b e l l e d E_. c o l i was found to sediment more s l o w l y than u n i f o r m l y l a b e l l e d DNA. The s m a l l fragments of DNA appeared to be p r e c u r s o r of the p a r e n t a l - 12 -DNA s i n c e they were lengthened and e v e n t u a l l y cosedimented w i t h p a r e n t a l DNA. S i m i l a r r e s u l t s were obtained i n experiments w i t h a number of E. c o l i s t r a i n s (72,75-77). B. s u b t i l i s (72,75,78,79), phage i n f e c t e d c e l l s (72,75-77,80), regenerating r a t l i v e r (81, 82), Chinese hamster c e l l s (83-85), mouse mammary carcinoma c e l l s (86), E h r l i c h and K r e b s - 2 - a s c i t e s tumor c e l l s (87,88), HeLa c e l l s (89-92), a human h e t e r o p l o i d c e l l l i n e c a l l e d EUE (93), and i s o l a t e d HeLa c e l l n u c l e i (94). Recently however there has been some concern about the p o s s i b i l i t y t h a t the s m a l l pieces of DNA may r e s u l t from a f r a g -mentation mechanism (95-97). The suppression of T ^ - l i g a s e mu-t a t i o n by r l l mutation (98-101) or chloramphenicol a l s o appears to support t h i s mechanism (98,101-103). This mechanism i s s t i l l not c o n s o l i d a t e d , s i n c e there i s another e x p l a n a t i o n i n the ex-periments w i t h E_. c o l i DNA-ligase mutation (104). I n a d d i t i o n , n e i t h e r r l l mutation nor chloramphenicol prevents the u s u a l f o r -mation o f nascent s h o r t chains (101,102) and a pu l s e l a b e l i s predominantly a t the 3 1 end of nascent chains, which would not be expected to r e s u l t from a fragmentation mechanism (105). An E_. c o l i l i g a s e mutant accumulates low molecular weight DNA, es-p e c i a l l y at the non-permissive temperatures (106,107), s i m i l a r to the r e s u l t s w i t h T^ l i g a s e - d e f i c i e n t mutant (72,95,98,104,108-110). These r e s u l t s support the discontinuous mechanism of DNA r e p l i c a t i o n . - 13 -1.3e. P o l y n u c l e o t i d e l i g a s e P o l y n u c l e o t i d e l i g a s e was f i r s t detected as an a c t i v i t y necessary f o r the s u c c e s s f u l r e p l i c a t i o n of phage (111). When E_. c o l i c e l l s are i n f e c t e d w i t h t h i s phage a l a r g e f r a c t i o n o f the phage l i n e a r DNA molecules are converted i n t o a c o v a l e n t l y bonded c i r c u l a r form. G e f t e r , Becker and Hurwitz (112) p u r i f i e d the enzyme and found that i t a l s o c a t a l y z e d the conversion of X DNA w i t h s i n g l e - s t r a n d breaks to the c i r c u l a r duplex form, t h a t i s i t can a l s o r e j o i n i n t e r n a l breaks i n the duplex s t r u c t u r e . Both the enzyme s p e c i f i e d by phage during i t s i n f e c t i o n of b a c t e r i a , and the enzyme of E_. c o l i i t s e l f have been s t u d i e d i n some d e t a i l (95,113-115). The enzyme induced by T^ i n b a c t e r i a l i n f e c t i o n r e q u i r e s ATP as a c o f a c t o r (116-119) , w h i l s t the E_. c o l i enzyme r e q u i r e s NAD f o r a c t i v i t y (120,121). With both en-zymes the r e a c t i o n occurs i n two phases. The f i r s t i s the f o r -mation of an enzyme-AMP complex. I n c u b a t i o n of the ligase-AMP complex from e i t h e r source w i t h duplex DNA c a r r y i n g s i n g l e - s t r a n d breaks r e s u l t s i n a s e a l i n g of the breaks and concomitant r e l e a s e of 1 mole of AMP f o r every phosphodiester bond formed. I t seems probable t h a t each enzyme d i s p l a y s the same r e a c t i o n i n t h i s phase. DNA p r e p a r a t i o n s c o n t a i n i n g s i n g l e - s t r a n d breaks w i t h 3'-OH and 5'-P end groups are a c t i v e i n causing breakdown of the complex, but breaks w i t h 3'-P and 5'-OH end groups are not r e p a i r e d . This requirement i s very s p e c i f i c . The use of a phosphatase enzyme - 14 -under conditions where i t removes the i n t e r n a l phosphate from the 5'-P end group abolishes a c t i v i t y of the l i g a s e . The enzyme-AMP complex binds to 5'-P end and AMP reacts with the free phosphate group. Attack by 3'-OH group on t h i s moiety then forms a phos-phodiester bond and seals the gap (95,113,114,119). Polynucleotide l i g a s e s have been i d e n t i f i e d i n E_. c o l i (111,112,121), , T^ and T^ bacteriophage-infected E_. c o l i (116,117,119), and i n mammalian c e l l s (118). The discovery of DNA l i g a s e s was an important f a c t o r i n the i n v i t r o synthesis of the f i r s t b i o l o g i c a l l y a c t i v e DNA (122). 1.3f. R e p l i c a t i o n and c e l l membrane Although b a c t e r i a do not possess a system of the complexity of the m i t o t i c apparatus which ensures the even segregation of chromosomes i n higher organisms, some system must be responsible for the d i s t r i b u t i o n of newly r e p l i c a t e d chromosomes in t o the two daughter c e l l s of a d i v i s i o n . I t seems probable that t h i s system must involve an a s s o c i a t i o n between the chromosome and the c e l l surface - most l i k e l y the membrane. Various models have been proposed upon t h i s b a s i s , and there i s now evidence i n favor of a connection between the genome and the c e l l membrane (123,124), and a d d i t i o n a l forms and properties of complexes between the two have been described i n many sources i n c l u d i n g c e l l s i n f e c t e d by phage cb x 174 (125,126), X (126-128), P 2 2 (129), T 4 (123,130-133), B. s u b t i l i s (134-135), E. c o l i (131,136-139), B. megatherium (131, 140), Pneumococcus (141), HeLa c e l l s (142,143), heteroploid human - 15 -amnion c e l l s (144), c a l f thymus n u c l e i (145), L c e l l mitochondria (146) and trypanosomal k i n e t o p l a s t s (147). Jacob et al (148) proposed the concept of the r e p l i c o n as a b a s i c u n i t of DNA r e p l i c a t i o n to e x p l a i n a number of phenomena r e -l a t e d to DNA s y n t h e s i s i n E_. c o l i . They proposed that r e p l i c a t i o n of the b a c t e r i a l chromosome i s i n i t i a t e d at a s p e c i f i c s i t e termed the r e p l i c a t o r , and that the r e p l i c a t o r i s attached to a s p e c i f i c l o c a t i o n on the b a c t e r i a l c e l l membrane. The attachment of DNA to the c e l l membrane provides a mechanism both f o r the c o n t r o l of i n i t i a t i o n of DNA r e p l i c a t i o n and f o r the s e p a r a t i o n and s e gregation of the daughter chromosomes to opposite poles of the c e l l (136,149). M o r p h o l o g i c a l s t u d i e s (136) on both E_. c o l i and B_. s u b t i l i s have shown that there i s indeed an a s s o c i a t i o n between the b a c t e r i a l 3 n u c l e a r complex and the c e l l membrane. I f [ H]thymidine i s added to c u l t u r e s i n e x p o n e n t i a l growth, i t f i r s t appears i n the DNA of t h i s membrane f r a c t i o n (150,151). The a d d i t i o n of a chase of c o l d thymidine caused the t r i t i u m to assume the same p r o f i l e as the b u l k of the DNA. T his suggests t h a t the membrane f r a c t i o n comprises the newly s y n t h e s i z e d DNA at the r e p l i c a t i o n p o i n t . Sueoka and Quinn (134) have shown the membrane attachment of the r e p l i c a t i o n o r i g i n as w e l l as of the r e p l i c a t i o n p o i n t i n _B. s u b t i l i s using a combined g e n e t i c and b i o c h e m i c a l a n a l y s i s . More evidence has accumulated that the c e l l membrane c o n t r o l s and i n i t i a t e s DNA r e p l i -c a t i o n i n b a c t e r i a (131,137,152-161). The recent i s o l a t i o n of E. - 16 -c o l i mutants which are defective in DNA replication and which have alterations in their membranes seems li k e l y to make possible an investigation of the part which the membrane components play in the replication complex. Jacob and coworkers (162) have recently isolated two types of temperature sensitive replication mutants and have shown that the membranes of wild type and mutant cells have different properties at the high temperature, but are indistinguishable at the low permissive temperature. They have shown that this difference is due to a 60,000 dalton protein com-ponent of the membrane (162-164). Comings (124) has recently attempted to apply this idea to eukaryotic c e l l s , and Comings and Kakefuda (144) have presented some evidence for the i n i t i a t i o n of DNA replication at the nuclear membrane. Some evidence also suggests that in eukaryotes, DNA synthesis i s initiated i n association with nuclear membrane or nuclear membrane-chromatin complex (143,165-171). In contrast to procaryots, several reports (124,172-177) have shown that in eukaryotic cells chromosomes are attached to the nuclear membrane at multiple sites and that this predetermines the arrangement of chromatin fibers as the chromosomes condense and pass into mitosis. Comings e_t al (144) have also shown that the replication is initi a t e d on the nuclear membrane and nucleolus at the commencement of the DNA synthetic phase, but the growing point i s not prone to be on - 17 -the membrane and migrates from the i n i t i a t i o n site on the membrane towards the center of the nucleus. Recently an attempt has been made to detect and isolate a membrane or membrane-chromatin com-plex containing bound DNA polymerase (168,178,179). However, this complex carried out limited replication i n v i t r o . Although several models (134,136,149,180) were proposed for chromosome replication on the c e l l membrane, for several years i t has been crystallized around two conflicting models, i.e. the swivel mechanism proposed by Cairns (181) and the r o l l i n g c i r c l e model devised by Gilbert and Dressier (182) and by Jacob and co-workers (183). The Cairns model envisages that replication starts at a fixed origin from which i t proceeds i n either one or both directions as the parental strands are rotated and separated by some swivel mechanism, and a 6-shaped replicating form i s generated. The ro l l i n g c i r c l e model, by contrast, envisages that one parental strand i s nicked to expose a 3'-hydroxyl group and a 5'-phosphate group which becomes attached to the membrane; nucleotides are then polymerized at the 3'-end, giving r i s e to a DNA chain longer than either parental chain and simultaneously displacing the 5'-end of the nicked parental strand which can thus act as a template for synthesis of a complementary daughter strand. As replication proceeds, the complex assumes a p-shaped structure. To date, the most compelling evidence for the rol l i n g - 18 -c i r c l e model has come almost e x c l u s i v e l y from s t u d i e s of r e p l i -c a t i n g cbx 174 DNA, w h i l e i n v e s t i g a t i o n s of r e p l i c a t i n g E. c o l i chromosomes and the chromosomes of c e r t a i n bacteriophages have i n g e n e r a l support from the Cairns model which has now apparently been r e i n f o r c e d by the DNA tumor v i r o l o g i s t s (184,70). More r e c e n t l y , Champoux and Dulbecco (185) presented evidence from mouse embryo c e l l s to support C a i r n s ' s w i v e l mechanism. 1.3g. I n v i t r o DNA r e p l i c a t i o n Meselson has pointed out (186) that the o n l y f a c t known f o r c e r t a i n about DNA d u p l i c a t i o n i s that i t i s semiconservative and that the two strands of a DNA duplex segregate, one going to each daughter molecule. For s e v e r a l y e a r s , the only candidate f o r the DNA d u p l i c a t i o n has been the DNA polymerase enzyme c h a r a c t e r i z e d by Kornberg and h i s c o l l e a g u e s , but, there have always been o b j e c t i o n s to t h i s i d e a . DNA s y n t h e s i s i n v i t r o , u sing the c l a s s i c p u r i f i e d DNA polymerase system of Kornberg (187), d i f f e r s from r e p l i c a t i o n i n v i v o i n s e v e r a l r e s p e c t s : E. c o l i DNA polymerase I i_n v i t r o o r d i n a r i l y forms only nonsense DNA, which i s branched, non-denaturable, and b i o l o g i c a l l y i n a c t i v e (61,108, 188); no net semiconservative s y n t h e s i s i s achieved i n v i t r o w i t h double stranded DNA as template (189) ; mutants of E_. c o l i have been i s o l a t e d which are d e f e c t i v e i n DNA d u p l i c a t i o n but have nor-mal Kornberg polymerase a c t i v i t y (190-195) ; Kornberg enzyme can only s y n t h e s i z e one of the two strands of DNA duplex con-t i n u o u s l y from one end to the o ther (186) ; the low i n v i t r o - I n -a c t i v i t y of the enzyme compared to the In v i v o r a t e of r e p l i c a t i o n (196); the i n a b i l i t y of the enzyme to i n i t i a t e new strands (197). Further evidence i s found i n the experiments of de L u c i a and Cairns (198) and Gross and Gross (199) demonstrating that the am-ber mutant of _E. c o l i (W 3110 t h y - p o l A, P 3478) which has dras-t i c a l l y reduced l e v e l s of the usu a l polymerase I a c t i v i t y r e p l i -cates normally the non-permissive s i t u a t i o n . A l l t h i s evidence i n -d i c a t e s that the Kornberg enzyme i s not the enzyme which r e p l i -cates DNA i n c o l i c e l l s . Since the Cairns mutant has l e s s than one per cent of the DNA polymerase a c t i v i t y present i n w i l d type c e l l s , but can grow and can r e p l i c a t e i t s DNA normally, v a r i o u s procedures have been used to t r y to i s o l a t e from the Cairns mutant other .enzyme(s) which can r e p l i c a t e DNA, and i t now seems c e r t a i n that r e p l i c a t i o n i n v i v o i s c a t a l y z e d by an enzyme which i s a s s o c i a t e d w i t h the c e l l membrane (155). This complex can perform s e v e r a l f u n c t i o n s c h a r a c t e r i s t i c of DNA r e p l i c a t i o n and produces new DNA semi-c o n s e r v a t i v e l y at a p o l y m e r i z a t i o n r a t e c l o s e to that observed f o r DNA r e p l i c a t i o n i n v i v o (154). DNA polymerase I I , a new, membrane-bound DNA polymerase, was s o l u b i l i z e d and c h a r a c t e r i z e d from c e l l s of the Cairns mutant by s e v e r a l workers (156-159,200,201). This enzyme works best w i t h double stranded DNA as template f o r i t s p o l y m e r i z a t i o n of deoxy-- 20 -nucleoside triphosphate, which takes place in 5' to 3' direction and requires a free 3'-hydroxyl group. The DNA polymerase II activity i s ten times less sensitive to sulfhydryl reagents; i t is completely inhibited by 0.2 M KC1 (the DNA polymerase I i s un-affected) and most essentially, i t i s not inhibited by antisera to Korberg's polymerase (DNA polymerase I ) . More recently, Gefter et_ al (202) showed that the third and lowest DNA polymerase activity detected in E_. c o l i i s temperature sensitive when isolated from the complementary E_. c o l i strains 1026 and 1040 (dna E). This implies that the dna E gene product i s polymerase III. It is also confirmed by Nllsslein e_t a l (203) that wild type dna E gene product consists of a DNA polymerase activity with properties similar to polymerase III and certainly different from polymerases I and II. It w i l l come as no great surprise i f DNA polymerases II and III like polymerase I, prove to be repair enzymes as far as DNA duplication i s concerned. But their discovery establishes the idea that there may be several enzymes in E_. c o l i , a l l of which can polymerize DNA, although most of them are not involved i n DNA duplication. If DNA polymerase I i s not involved in DNA duplication, then i t raises the question of what is i t s real function. The properties of this enzyme, so relentlessly pursued by Kornberg and his col -leagues (189,204-207), in particular i t s a b i l i t y to act as a nuclease > - 21 -and excise mismatched regions of DNA as well as to polymerize DNA, suggest that in common with other enzymes i t might have a repair function editing out mismatched sequences, pyrimidine dimers. Monk et a l (208) have just reported a series of genetic experiments which clearly indicate that in E_. c o l i there are two DNA repair mechanisms, only one of which involves polymerase I. The mutants which De Lucia and Cairns have isolated, although they lack the repair function which involves polymerase I, can survive the damage caused by UV irradiation because the second repair mechanism is functional. This second mechanism, which can be defined genetically as the rec A pathway, and is also required for recombination, seems to be able to handle the repair of most of the pyrimidine dimers induced by irradiation. Gross et a l (209) have found i t i s impossible to use genetic crossing to construct strains of E_. c o l i , which carry mutation that i n -activate both Polymerase I and the rec A function. In other words, E_. co l i cells which lack both repair mechanisms are non-viable. More recently, Hurwitz and his co-workers (210,211) have confirmed the existence of two DNA repair mechanisms i n E_. c o l i by isolation and purification of a DNA polymerase which i s dis-tinguishable from the Kornberg enzyme. Several facts (211) suggest that this new DNA polymerase may not be responsible for the replication of DNA iri vivo but has a repair function in the c e l l . - 22 -The a v a i l a b i l i t y of polymerase-deficient s t r a i n s has s t i m u l a t e d i n t e n s i v e i n v e s t i g a t i o n of the mechanism of chromosome r e p l i c a t i o n and has permitted a new approach to the problem. Biochemical s t u d i e s of chromosome r e p l i c a t i o n have been hampered by the u n a v a i l a b i l i t y of an adequate i n v i t r o system w i t h the b a s i c f e a t u r e s of i n v i v o DNA r e p l i c a t i o n . The c r i t e r i a f o r such a system are: (1) semiconservative r e p l i c a t i o n ; (2) normal b i o l o g i c a l a c t i v i t y of newly s y n t h e s i z e d DNA: (3) normal advance-ment of the o r i g i n a l r e p l i c a t i o n f o r k ; (4) r a t e of DNA r e p l i -c a t i o n e q u i v a l e n t to In v i v o ; and (5) expected phenotypic be-h a v i o r of mutants. There are s e v e r a l new systems which can support DNA s y n t h e s i s i n v i t r o used by Smith et al (154) , Knippers ej: a l (155) , and Okazaki et^ al_ (212). These workers employ the washed membranes of g e n t l y l y s e d spheroplasts prepared from poly A E_. c o l i . The membranes e i t h e r immobilized i n agar (154) or i n f r e e s o l u t i o n 2+ (155,212), when incubated w i t h Mg , four deoxynucleoside t r i -phosphates, a s u l f h y d r y l reagent, and ATP, c a r r y out DNA s y n t h e s i s that resembles chromosome r e p l i c a t i o n . Another system i s that of Moses ej: aJL (213) and Mordoh et a l (214) who used toluene to r e n -der E. c o l i c e l l s permeable to s u b s t r a t e s . A f t e r treatment w i t h toluene, the c e l l s w i l l s y n t h e s i z e DNA i f incubated w i t h ATP, f o u r 2+ deoxynucleoside t r i p h o s p h a t e s , and Mg . Of these systems, t o l u e -n i z e d c e l l s seem to be the most promising (215). Toluenized c e l l s - 23 -of E_. c o l i although no longer v i a b l e , m a i n t a i n many of t h e i r p h y s i o l o g i c a l f u n c t i o n s and, according to Moses and Richardson (213), t h e i r a c t i v i t y i n DNA s y n t h e s i s can be d i f f e r e n t i a t e d i n t o r e p a i r f u n c t i o n s and r e p l i c a t i o n proper. Taking t h i s system f u r t h e r , Sueoka and h i s colleagues (216) reported that the DNA synt h e s i z e d by t o l u e n i z e d B_. s u b t i l i s i s made s e m i c o n s e r v a t i v e l y and, because i t can be used f o r t r a n s f o r m a t i o n , must have b i o -l o g i c a l a c t i v i t y . Even more s i g n i f i c a n t l y , DNA s y n t h e s i s con-tinues a f t e r exposure to toluene at the r e p l i c a t i o n f o r k which was p r e v i o u s l y a c t i v e i n the l i v i n g c e l l s . I f a f t e r t o l u e n i z a t i o n the p r e - e x i s t i n g r e p l i c a t i o n f o r k continues to advance along the chromosome making DNA s e m i c o n s e r v a t i v e l y , i t i s u n l i k e l y indeed that t h i s DNA i s a r e p a i r s y n t h e s i s . More r e c e n t l y , Bonhoeffer and h i s coworkers (217,218) have r e p o r t e d an i n v i t r o system f o r DNA r e p l i c a t i o n which uses a h i g h l y concentrated l y s a t e of DNA polymerase I d e f i c i e n t E_. c o l i b a c t e r i a . This i n v i t r o DNA syn-t h e s i s resembles i n v i v o r e p l i c a t i o n i n many aspects. I t i s semi-c o n s e r v a t i v e . Furthermore the DNA i s sy n t h e s i z e d i n s m a l l pieces which become j o i n e d together upon prolonged i n c u b a t i o n . Treatments which s p e c i f i c a l l y i n h i b i t i n v i v o r e p l i c a t i o n a f f e c t the r a t e of i n v i t r o s y n t h e s i s . 1.4. The I n t r a c e l l u l a r L o c a t i o n and M u l t i p l e Forms of DNA Polymerases In the b a c t e r i a l system, as mentioned i n S e c t i o n 1.3f, the membrane f r a c t i o n o f c e l l s contains a s u b s t a n t i a l p r o p o r t i o n of the - 24 -t o t a l c e l l DNA polymerase and DNA biosynthesis takes place i n a membrane-bound f r a c t i o n . At l e a s t three DNA polymerases have been i s o l a t e d , p u r i f i e d and characterized i n E_. c o l i (156-159, 200-202,219). In t h i s Section only mammalian systems w i l l be emphasized. Bollum and Potter (16) surveyed mammalian tis s u e s for DNA polymerase a c t i v i t y and, somewhat s u r p r i s i n g l y , found that most of the measurable a c t i v i t y was located i n the u l t r a c e n t r i f u g a l supernatant f r a c t i o n of t i s s u e homogenates and only low enzyme a c t i v i t y was found i n the corresponding nuclear f r a c t i o n (220). Keir e_t a l (18) and Behki et_ a l (221) prepared n u c l e i from r e -generating r a t l i v e r and Novikoff hepatoma using the non-aqueous solvent technique (222) and observed s u b s t a n t i a l a c t i v i t y of a DNA polymerase i n the nuclear f r a c t i o n and lower but ap-p r e c i a b l e amounts i n the cytoplasm. However, loss of enzyme a c t i v i t y occurred i f the n u c l e i were prepared i n an aqueous s o l u t i o n , presumably by leaching out into the soluble f r a c t i o n during i s o l a t i o n of the n u c l e i . Canellakis and h i s colleagues (14) have improved the aqueous ext r a c t i o n method by adding C a C ^ to the buffer s o l u t i o n . By applying t h i s method, Main and Cole (223) demonstrated that DNA polymerase a c t i v i t y was evenly d i s -t r i b u t e d between the nuclear and the cytoplasmic f r a c t i o n following high-speed c e n t r i f u g a t i o n of a homogenate of r a t thymus t i s s u e . S i m i l a r r e s u l t s were obtained by B i r n i e e_t al_ (224) - 25 -with mouse embryo c e l l s , but the s p e c i f i c a c t i v i t y was markedly greater i n the nuclear extract. It i s now f i r m l y established that mitochondria contain species of DNA which d i f f e r from that present i n the n u c l e i (225-227). Mitochondrial DNA appears to be synthesized i n a semiconservative manner which i s r e p l i c a t i v e rather than a r e p a i r process (228,229) and i s independent of the synthesis of nuclear DNA (230). DNA polymerase a c t i v i t y has been reported to occur i n mitochondria i s o l a t e d from yeast (231) and from r a t l i v e r (232). K a l f et ail (233) and Meyer et a l (20) recently reported the p u r i f i c a t i o n of mitochondrial DNA polymerase from r a t l i v e r . Their studies suggest that the enzymes d i f f e r from nuclear DNA 2+ polymerase with respect to chromatographic p r o p e r t i e s , Mg requirement, template preference, s o l u b i l i t y and k i n e t i c para-meters (234,235). Recently, i t has been found that eukaryotic c e l l s contained m u l t i p l e forms of DNA polymerase (51,236-239). DNA polymerase i s o l a t e d from mammalian n u c l e i usually prefers native DNA as template (34,240-242). However, mammalian c e l l s contain another class of DNA polymerase, which i s di s t i n g u i s h e d from the above clas s by i t s molecular weight and i t s template requirements (24,27,130,241-243). This enzyme has a higher molecular weight than the DNA polymerases that prefer native DNA and u s u a l l y shows - 26 -a high preference for denatured DNA. Both of these two enzymes require a l l four deoxynucleoside triphosphates to obtain optimal activity, and polymerize deoxynucleoside triphosphates under template direction. However, in mammalian systems, there is another enzyme which incorporates mononucleotide units into DNA without template direction. The enzyme was f i r s t detected by Krakow e_t al_ (14) in calf thymus nuclei and by Bollum from the soluble fraction of calf thymus (244). It displays requirements for heated DNA 2+ primer, Mg and cysteine. The incorporation of mononucleotidyl residues (e.g. dTMP) is not stimulated by the addition of the other three deoxyribonucleoside 5'-triphosphates to the reaction mixtures. This enzyme differs from the two enzymes mentioned above and is called "terminal deoxynucleotidyl transferase" or "terminal addition enzyme" (48). The soluble fractions of calf thymus terminal deoxynucleotidyl transferase have been more ex-tensively purified and studied (43,245). Recently, Leung and Zbarsky (246) have also isolated and purified terminal deoxynucleo-t i d y l transferase from nuclei of rat intestinal mucosa. The properties of their enzyme are similar to those mentioned above for calf thymus enzyme. At present the biological role of terminal DNA transferase has not been determined. Keir (48) has proposed that the mammalian DNA polymerase i s composed of several subunits and terminal deoxynucleotidyl transferase i s one catalytic sub-unit of complex DNA polymerase molecules. However, the question of whether this enzyme exists i n vivo or is derived from DNA polymerase remains unanswered. 1.5. DNA and Its Synthesis in Developing Brain In the central nervous system, as i n other organs, DNA is almost entirely localized i n the nuclei. However, small amounts have been isolated from mitochondria (247). The DNA content i n mammalian adult brains, i n general, is between 6.1 - 7.1 pg/cell (248). In the intact brain, DNA increases during c e l l multi-plication (mitosis) and remains constant when mitosis ceases. The studies by Mandel and co-workers (249,250) i l l u s t r a t e that the temporal changes of the nucleic acids in the developing brain vary with different animals; thus, i n the guinea pig brain DNA remains constant from birth and remains unaltered through adult-hood. In the rat, DNA increases up to the 10th to 15th day post-natally and subsequently remains constant. In the rabbit brain, DNA increases rapidly for 30 days after birth, then i t increases slowly u n t i l the 90th day, at which time DNA reaches adult levels. The dog, cat and chicken brains show a rapid increase in DNA in the f i r s t 30 days of the post-natal period and then remain con-stant. Adams (251) has shown an increase i n total DNA i n rat brains from birth up to 18 days post-natally, after which i t remains constant through adulthood. Sung (252) has also shown a very rapid rate of DNA synthesis (particularly in the rat cere-bellum) which was highest around 6 days after birth and decreased - 28 -rapidly thereafter up to 18 days. Several autoradiographic studies have been carried out in 3 recent years on growing brain exposed to H-thymidine by Altman (253). The uptake of this precursor just before mitosis and i t s indefinite permanence in labelled nuclei have been elegantly ex-ploited i n the identification of proliferating cells and of their migratory and di f f e r e n t i a l pathway. With this technique, Altman found that thymidine incorporation into rat brain DNA, measured after a pulse of 12 h is highest at 6 days post-natally and de-clines rapidly u n t i l the 15th day and more slowly therafter. These data are consistent with Sung's previous observation. DNA polymerase act i v i t y (254) in several regions of developing chicken brain i s similar i n many respects to that described i n other animal systems which show a preference for denatured DNA as opposed to native DNA. The enzyme is dependent upon the presence of a l l four deoxyribonucleoside triphosphates and magnesium ions for activity. The pH optimum is about 8.0. The intensity of the enzymatic re-action appears to be gradually proportional to the rate of DNA increase. DNA polymerase activity in developing rat brain has been studied recently by Brasel er al (255) and Murthy and co-workers (256-258), i t is also similar in many respects to that described i n other vertebrate tissue. Isolation, purification and characterization of two DNA polymerase ac t i v i t i e s i n rat brain was demonstrated by Chiu and Sung (238,239). The results of this study w i l l be presented in this thesis. - 29 -1.6. The Reg u l a t i o n of DNA Synthesis The c o n t r o l of c e l l u l a r p r o l i f e r a t i o n i n t i s s u e s i s c u r r e n t l y being i n v e s t i g a t e d i n terms of b a s i c b i o c h e m i c a l processes l e a d i n g to DNA s y n t h e s i s and the means by which c r i t i c a l steps i n the b i o -s y n t h e t i c sequence may be r e g u l a t e d by precursor a v a i l a b i l i t y , primer s t a t e , l o c a t i o n of e s s e n t i a l components and other p r o t e i n f a c t o r s . The requirement f o r deoxynucleoside triphosphates as the s u b s t r a t e f o r DNA p o l y m e r i z a t i o n makes the enzymatic p r o d u c t i o n of these precursors an important p o i n t of c o n t r o l i n the s y n t h e s i s of DNA (259-261). Evidence that the pro d u c t i o n of TTP i s one of the main r e g u l a t o r y steps i n the s y n t h e s i s of DNA i s accumulating (262). Recently Sung (263) has s t u d i e d thymidine k i n a s e i n developing r a t b r a i n and has found t h a t the a c t i v i t y of thymidine k i n a s e i n the cerebellum of developing r a t b r a i n i s much hi g h e r than t h a t i n the cortex o r other b r a i n r e g i o n s . The peak of a c t i v i t y i n the cerebellum i s around the 6th day a f t e r b i r t h . The a c t i v i t y i n the cerebellum of a d u l t r a t b r a i n was l e s s than one percent of th a t found at 6 days p o s t - n a t a l , whereas i n c e r e b r a l c o r t e x , the hi g h e s t a c t i v i t y was found i n the newborn and decreased g r a d u a l l y u n t i l maturation. A l l these f i n d i n g s are very s i m i l a r to those found i n previous s t u d i e s on DNA s y n t h e s i s i n developing b r a i n (252,264). Although there i s l i t t l e or no DNA synth e s i z e d i n a d u l t b r a i n , the n u c l e a r DNA s y n t h e s i z i n g a c t i v i t y of the a d u l t , h i g h l y - 30 -differentiated tissue i s strikingly modified by exposure to cytoplasm of mitotic tissue. This has been shown in the elegant experiments of Gurdon (265) by nuclear transplantation from brain and other adult organs into enucleated eggs of amphibia. Brain nuclei which are inactive in replicating DNA, start an active synthesis shortly after injection into the egg cytoplasm. The main conclusion to be drawn from these findings is that nuclear a c t i v i t i e s are under the control of cytoplasmic factors. Similar results have also been reported by Harris (266) and Thompson and McCarthy (267). It has been demonstrated that in eukaryotic cells concomitant protein synthesis i s necessary for both the i n i t i a t i o n and con-tinuation of DNA synthesis (268-271). Salas and Green (272) have reported highly promising results in the identification and iso-lation of proteins which may control DNA replication in mouse 3T6 c e l l s . They fractionated c e l l extracts on DNA-cellulose according to the technique developed by Alberts et aJL (273), and then re-solved the DNA-binding proteins on polyacrylamide gel electro-phoreses into eight more or less discrete fractions (PI - P8). Significantly, the amounts of three of these fractions (PI, P2 and P6) depended on whether the i n i t i a l c e l l extracts were pre-pared from resting or dividing c e l l s ; there was more PI and P2 - 31 -i n resting than growing c e l l s , whereas P6 was almost absent from resting cells but was the chief component of eluates from dividing c e l l s . Sales and Green also found that the protein P l , which i s found in resting cells but hardly at a l l i n dividing c e l l s , i s synthesized in excess in cells starved of serum, and stops being synthesized before DNA synthesis begins as c e l l s move from the resting to the dividing state. A l l these results suggest that the protein P l is indeed a molecule which prevents the onset of DNA replication. Conversely, the synthesis of protein P6, which i s almost absent from resting cells but is the chief component of extracts from dividing c e l l s , seems to begin either concomitantly with or a l i t t l e before DNA synthesis i s init i a t e d . This also suggests that P6 may play a role i n the regulation of replication. Similar results were also obtained from E. c o l i 15T~ cells by Markar and Eberle (274). As mentioned above, in mammalian systems, growth inhibitors or stimulators from tissues and tissue culture fluids are reported to be involved (265,272,273,275,276). Recently, Erhan et a l (277) have also isolated a low molecular weight factor, the "wedge", from ascites f l u i d . This factor can stimulate DNA replication -especially with double-stranded DNA as template - and c e l l division. The "wedge" is not a nuclease but might be a "nickase"; however, i t is thought to be essential for the organization and action of - 32 -DNA polymerase (278). There are s t i l l other factors which have been reported and thought to play a r o l e i n r e g u l a t i o n of r e p l i -c ation; such as "unwindase" (279), the product of gene 32 of phage T^ which seems to a s s i s t the denaturation and renaturation of the phage genome. It may be part of a r e p l i c a t i o n complex, i t s function being to help the phage DNA to unwind w h i l s t i t i s r e p l i c a t e d . Other research workers have found that s i m i l a r pro-teins are present i n the meiotic c e l l s of a range of species (280). A p r o t e i n f a c t o r has been p u r i f i e d from E_. c o l i c a l l e d co p r o t e i n which permits r e l a x a t i o n of s u p e r - h e l i c a l turns i n X closed c i r c u l a r DNA (196). This f a c t o r i s heat l a b i l e , i s s e n s i t i v e to protease, and i s not simply a "nickase". The a c t i v i t y of to has so f a r been described only f o r c i r c u l a r X, and i t s i n vivo fun-c t i o n remains unknown. The co f a c t o r could act i n r e p l i c a t i o n to prevent (or r e l i e v e ) twist that would r e s u l t from unwinding a DNA that i s c i r c u l a r , very long, or complex i n s t r u c t u r e . 1.7. Polyamines and DNA Biosynthesis Spermine, spermidine and putrescine are widely d i s t r i b u t e d i n animal t i s s u e s (281-283) and they occur i n high concentrations i n a c t i v e l y p r o l i f e r a t i n g t issues such as regenerating l i v e r (284, 285), growing chick embryo (286) and n e o p l a s t i c c e l l s (287,288). They also stimulate c e l l u l a r p r o l i f e r a t i o n of both animal and plant t i s s u e i n v i t r o (289,290). In s u b c e l l u l a r d i s t r i b u t i o n - 33 -studies, Dion and Herbst (291) have found that the r a t i o of the t o t a l amount of spermidine i n the nucleus to that i n the cytoplasm i s approximately two. The presence of spermine and spermidine has been demonstrated i n mammalian b r a i n (292). In p a r t i c u l a r , sper-mine and spermidine have recently been found i n the cat and chick embryo b r a i n , where they represent, from a q u a n t i t a t i v e point of view, the major amine constituent of t h i s organ (293,294). Shimizu et a l (295) observed that the changes i n the concentration of polyamines i n the developing mouse b r a i n c l o s e l y p a r a l l e l l e d the changes occurring i n the DNA concentration. I t has also been observed that the developmental pattern of n u c l e i c acids i s s i m i l a r to that of polyamine i n the developing embryo (296). Furthermore, the most i n t e r e s t i n g observation i s that the peak of concentration of polyamines i n r a t b r a i n (297) i s s i m i l a r to the peak of a c t i v i t y of DNA synthesis (252). More recent observations (298-300) further confirm t h i s c o r r e l a t i o n between the concen-t r a t i o n s of polyamines and n u c l e i c a c i d . However, the e f f e c t of polyamine on DNA polymerase and the mechanism of s t i m u l a t i o n of polyamines on DNA synthesis i s s t i l l unknown. 1.8. The Present I n v e s t i g a t i o n There i s l i t t l e or no detectable DNA synthesis i n adult rat b r a i n , but considerable DNA synthesis takes place i n the b r a i n of young animals (251,252,264). Although the content of DNA i n the b r a i n during i t s development has been studied (249,251,301), very - 34 -l i t t l e i s known concerning the regional distribution of DNA polymerase activity during brain maturation. The present work was undertaken to study the regional changes in DNA polymerase during maturation of rat brain. Extracts from whole brain as well as from various regions of rat brain at different ages were assayed for the levels of DNA polymerase activity. Purification of this activity was attempted using ammonium sulfate fractionation and column chromatography on Sephadex, DEAE-cellulose. Two major fractions of DNA polymerase activity were obtained. Further characterization studies on these a c t i v i t i e s were performed. Subcellular d i s t r i -bution and developmental changes of these two DNA polymerase acti v i t i e s were also studied and discussed. A protein factor which stimulates one of these a c t i v i t i e s was detected and characterized. The effect of polyamines on DNA polymerase acti v i t i e s was also investigated. - 35 -II. MATERIALS AND METHODS 2.1. Experimental animals Newborn, infant and adult rats of the Wistar strain were obtained from the Vivarium, Department of Zoology, The University of British Columbia, Canada. 2.2. Chemicals A l l common laboratory chemicals were of "reagent grade" and were used without further purification, except DEAE-cellulose which had been further purified according to the method of Peter-son and Sober (302). 3 3 [ H]dTTP and [ H]dCTP were purchased from New England Nuclear Corporation, Boston, U.S.A. Unlabelled dATP, dCTP, dGTP, dTTP and ATP were obtained either from Calbiochem Corporation, Los Angeles, U.S.A. or Sigma Chemical Company, St. Louis, U.S.A. The following chemicals were obtained from Fischer S c i e n t i f i c Company, Vancouver: Ammonium sulfate, trichloroacetic acid, glycerol, sucrose, naphthalene, toluene, dioxane, sodium chloride, potassium chloride, magnesium chloride, mono- and di-sodium phosphates, Tris(hydroxymethyl)aminomethane. - 36 -D i t h i o t h r e i t o l , N-ethylmaleimide, spermidine, spermine, p u t r e s c i n e , serum albumin, cytochrome C, t r y p s i n and t r y p s i n i n h i b i t o r were obtained from Calbiochem. C o r p o r a t i o n , Los Angeles. Potassium s u c c i n a t e and b i s ( p - n i t r o p h e n y l ) p h o s p h a t e were purchased from Sigma Chemical Co., St. L o u i s , U.S.A. DNA was a g i f t from the Laboratory of Dr. E.W. Davie ( U n i v e r s i t y of Washington, S e a t t l e , U.S.A.). C a l f thymus DNA, salmon sperm DNA, _E. c o l i DNA and nucleo-h i s t o n e were obtained from e i t h e r Calbiochem. C o r p o r a t i o n , Los Angeles or from Worthington Biochemical C o r p o r a t i o n , Freehold, N.J., U.S.A. Po l y d(A-T) and Micrococcus l y s o d e i k t i c u s DNA were obtained from M i l e s L a b o r a t o r i e s I nc., Kankakee, I l l i n o i s , U.S.A. P a n c r e a t i c deoxyribonuclease and r i b o n u c l e a s e were purchased from N u t r i t i o n a l Biochemical C o r p o r a t i o n , C l e v e l a n d , U.S.A. g-Mercaptoethanol was obtained from Eastman Kodak Company, Rochester, U.S.A. EDTA was obtained from Baker Chemical Company, P h i l l i p s b u i r g , N. U.S.A. Sodium dodecyl s u l f a t e was bought from the Serva Company, Heidel b e r g , Germany. 2,5-Diphenyloxazole (PPO) and l , 4 - b i s - ( 5 - p h e n y l o x a z o l y l - 2 ) benzene (POPOP) were obtained from F r a s e r M e d i c a l S u p p l i e s L t d . , Vancouver. Actinomycin D was a g i f t from Merck, Sharp and Dohme Canada L t d . , Montreal, P.Q. DEAE-cellulose was purchased from Bio-Rad L a b o r a t o r i e s , Richmond, C a l i f o r n i a . Sephadex G-100 and Sephadex G-200 were obtained from Pharmacia Canada, Montreal. 2.3. Assay f o r Enzyme A c t i v i t i e s 2.3a. DNA polymerase ( r e p l i c a t i v e d e o x y r i b o n u c l e o t i d y l t r a n s f e r a s e ) The assay f o r DNA polymerase a c t i v i t y measures the i n i t i a l r a t e of the i n c o r p o r a t i o n of a given l a b e l l e d n u c l e o t i d y l precursor i n t o an a c i d i n s o l u b l e product. Reagents The r e a c t i o n mixture i n a t o t a l volume of 0.4 ml, contained the f o l l o w i n g : 20 umoles T r i s - H C l b u f f e r , pH 7.4 2 pmoles MgC^ 2 umoles d i t h i o t h r e i t o l 20 nmoles dATP - 38 -20 nmoles dGTP 20 nmoles dCTP 12.8 pmoles [ 3H]dTTP (15.7 c/m mole) 20 ug c a l f thymus DNA ( n a t i v e or heat-denatured) and an appropriate amount of enzyme DNA was d i s s o l v e d i n 0.01 M NaCl at a c o n c e n t r a t i o n of 2 mg/ml and heat-denatured DNA was prepared by keeping the DNA s o l u t i o n at 100°C f o r 10 min and c o o l i n g i t r a p i d l y i n an i c e bath. V a r i a t i o n s i n the components of these assay systems were made from time to time to s u i t the p a r t i c u l a r experiment under study. For example, v a r i o u s amounts of spermidine or KCI were added to the r e a c t i o n mixture f o r studying the e f f e c t of spermidine o r of KCI on the a c t i v i t y of DNA polymerase; phosphate b u f f e r pH 7.4 and [ 3H]dCTP were used i n s t e a d of T r i s - H C l b u f f e r and [ 3H]dTTP on some occasions as w i l l be mentioned l a t e r . Procedure A 30 min i n c u b a t i o n time was used i n the standard assay, during which p e r i o d the i n c o r p o r a t i o n of l a b e l l e d p recursors was at a l i n e a r r a t e . A f t e r the i n c u b a t i o n p e r i o d at 37°C i n a water bath, the mixture was r a p i d l y cooled to 0°C, and the r e -a c t i o n was terminated by the a d d i t i o n of 2.5 ml of 10% t r i c h l o r o -- 39 -a c e t i c a c i d (TCA), and the mixture was mixed w i t h a v o r t e x mixer and was then kept at 0°C f o r 30 min. The TCA-insoluble p r e c i p i -t a t e was spun down and washed 3 times w i t h 5 ml of 5% TCA each time. The p r e c i p i t a t e was then d i s s o l v e d i n 0.2 ml of Hyamine and mixed w i t h s c i n t i l l a t i o n s o l u t i o n and r a d i o a c t i v i t y was measured. 2.3b. Terminal d e o x y n u c l e o t i d y l t r a n s f e r a s e The assay procedure used was s i m i l a r to t h a t r e p o r t e d by Krakow et a l (14). This method measured the c a t a l y t i c a c t i v i t y of an enzyme which p r e f e r e n t i a l l y i n c o r p o r a t e d s i n g l e deoxyribo-n u c l e o s i d e triphosphates i n t o the t e r m i n a l p o s i t i o n of s i n g l e stranded primers. Reagents The r e a c t i o n m i x t u r e , i n a t o t a l volume of 0.4 ml, contained the f o l l o w i n g : 20 ymoles T r i s - H C l b u f f e r , pH 7.4 2 umoles M g C l 2 2 umoles d i t h i o t h r e i t o l 2 ymoles c y s t e i n e 12.8 pmoles [ 3H]dTTP (15.7 c/m mole) 20 yg n a t i v e or heat-denatured c a l f thymus DNA and an a p p r o p r i a t e amount of enzyme - 40 -Procedure The i n c u b a t i o n time and the subsequent procedure were the same as those d e s c r i b e d f o r DNA polymerase. 2.3c. A l k a l i n e DNase A l k a l i n e DNase was assayed by measuring the i n c r e a s e i n o p t i c a l absorbance a t 260 nm i n the a c i d s o l u b l e f r a c t i o n . The procedure f o l l o w e d Sung's method (303) w i t h some m o d i f i c a t i o n . Reagents The standard r e a c t i o n m i x t u r e , i n a t o t a l volume of 2.0 ml, contained the f o l l o w i n g : 25 mM T r i s - H C l b u f f e r , pH 8.9 5 mM MgCl 2 5 mM g-mercaptoethanol 1.0 mg heat-denatured c a l f thymus DNA and an a p p r o p r i a t e amount of enzyme source Procedure The i n c u b a t i o n was c a r r i e d out at 37°C f o r one hour, a f t e r which the tube was cooled i n an i c e bath and 2.0 ml of c o l d 1.4 N p e r c h l o r i c a c i d (PCA) was added. The mixture was mixed by means of a v o r t e x mixer and then kept i n i c e for 10 min. The p r e c i p i t a t e - 41 -was centrifuged down at 10,000 rpm for 10 min. The supernatant fraction was removed with a Pasteur pipette and i t s optical density was measured at 260 nm against a proper blank in a Beckman model DU spectrophotometer. A zero time sample was always carried out simultaneously with each fraction tested and used as blank. One unit of enzyme activity i s defined as the amount of enzyme which w i l l cause an increase of 0.1 O.D. unit at 260 nm. 2.3d. Nickase Nickase is an alkaline endonuclease which produces single strand breaks in DNA. The activity of nickase was assayed by measuring the a b i l i t y of the enzyme to s h i f t the pattern of 14 [ C]DNA from bottom to top on alkaline sucrose density gradient. The procedure followed the method of Bari l at a l (304) with some modification. Reagents The reaction mixture, in total volume of 0.4 ml, contained the following: 20 umoles Tris-HCl buffer, pH 7.4 2 umoles MgC^ 2 umoles dithiothreitol 14 50 ug [ C]ascites tumor DNA and an appropriate amount of enzyme - 42 -14 [ C]Labelled ascites tumor DNA, prepared by the method of Marmur (305), was obtained from Dr. S.C. Sung. DNA was dis-solved in 0.01 M NaCl at a concentration of 2 mg/ml. Procedure After different incubation periods (1 h, 2 h and 4 h), the reaction mixture was layered on a gradient from 5-20% sucrose solution containing 0.3 M NaOH, 0.01 M EDTA and 1.0 M NaCl in a Spinco SW 39 centrifuge tube and was then centrifuged at 36,000 rpm for 16 h at 0°C i n a Spinco Model L ultracentrifuge. After centrifugation, the bottom of the tube was punctured, 15 drop fractions collected, and each fraction was measured for radio-activity. 2.3e. Phosphodiesterase activity Phosphodiesterase activity was measured by the liberation of p-nitrophenol from the synthetic substrate, bis-(p-nitrophenyl) phosphate. The amount of p-nitrophenol could be detected by i t s absorbance at 440 nm (306). Reagents The reaction mixture, a total amount of 1.0 ml, contained the following: 60 ymoles Tris-HCl buffer, pH 8.6 3 umoles MgCl„ - 43 -1 umole bis(p-nitrophenyl)phosphate and an appropriate amount of enzyme Procedure The incubation was carried out at 37°C for 20 min, after that the tube was cooled in an ice bath and 1 ml of 0.2 N NaOH was added and then the solution was allowed to stand at room temperature for 20 min. The absorbancy at 440 nm was deter-mined. The reaction at zero time was used as blank. One unit of enzyme activity = increasing absorbancy at 440 nm of 0.1 O.D. unit. 2.3f. ATPase activity The ATPase activity was measured by the appearance of i n -organic phosphate (P^) from dATP. The total amount of P.^  was detected by the method of Fiske and Subbrow (307). Reagents In a reaction mixture of 0.4 ml containing: 20 ymoles Tris-HCl buffer, pH 7.4 2 ymoles MgCl 2 32 ymoles NaCl 16 ymoles KCI 3 ymoles dATP and an appropriate amount of enzyme solution - 44 -Procedure The r e a c t i o n was s t a r t e d a f t e r 5 min temperature e q u i l i -b r a t i o n at 37°C by the a d d i t i o n of enzyme p r e p a r a t i o n (0.1 ml of b r a i n homogenate or high speed supernatant e x t r a c t ) . A f t e r i n c u b a t i o n f o r 10 min, the r e a c t i o n was stopped by the a d d i t i o n of 2.5 ml of 10% TCA. The t e s t tubes were placed i n i c e f o r 20 min. The p r e c i p i t a t e was c e n t r i f u g e d down, and the super-natant was analyzed f o r i n o r g a n i c phosphate. 2.4. C h a r a c t e r i z a t i o n of the Product The DNA products were s y n t h e s i z e d using the i n v i t r o assay system f o r r a t b r a i n DNA polymerase described e a r l i e r except that each component was present i n 5 times the c o n c e n t r a t i o n of s t a n -dard assay. A f t e r the DNA polymerase r e a c t i o n was stopped by the a d d i t i o n of 10% TCA and washed three times w i t h 5 ml of 5% TCA as d e s c r i b e d i n S e c t i o n 2.3a, the p r e c i p i t a t e was washed twice w i t h 5 ml of 95% a l c o h o l and 5 ml of chloroform-ether. A f t e r removing organic s o l v e n t overnight by vacuum d e s i c c a t o r , the p r e c i p i t a t e was d i s s o l v e d i n 3 ml of T r i s - H C l b u f f e r , pH 7.4 and 0.5 ml a l i q u o t s were incubated f o r one hour at 37°C w i t h DNase or RNase. The samples were then c h i l l e d , p r e c i p i t a t e d w i t h TCA and the r a d i o a c t i v i t y of the p r e c i p i t a t e was counted. 0.1 M N;OH was added to another 0.5 ml a l i q u o t and then b o i l e d f o r 5 min. I t was then c h i l l e d and the sample was p r e c i p i t a t e d w i t h TCA and the r a d i o a c t i v i t y of the p r e c i p i t a t e was counted. - 45 -2.5. Preparation of Brain Nuclei Whole brain nuclei from infant or adult (200-250 gm) Wistar strain rats were prepared by the method of Mandel et^ al_ (308). The animals were k i l l e d by decapitation, the brains excised rapidly and immersed i n an ice cold 2.2 M sucrose solution con-taining 1 mM MgC^ and 10 mM potassium succinate to remove blood contamination. The tissue was transferred to a chilled homogenizing tube, 3 volumes of ice-cold 2.2 M sucrose solution containing 1 mM MgCl 2 and 10 mM potassium succinate were added and homogenized with a Teflon pestle driven by a motor at about 2000 rpm. After homogenization, an additional 2 volumes of 2.2 M sucrose mixture was added and mixed gently. The homogenate was f i l t e r e d through a double layer of cheese cloth followed by centrifugation at 78,000 x g for 60 min at 0°C in a SW 39 rotor in a Spinco model L ultracentrifuge. The nuclei pellets were washed again with 1 M sucrose in 1 mM MgC^ and 10 mM potassium succinate and centri-fuged at 10,000 x g for 30 min. The washed nuclei were used as nuclear sources. 2.6. Preparation of Enzyme Extracts From Rat Brain Animals were decapitated and the cerebellum and cerebral cortex of each brain removed immediately with sharp pointed for-ceps or spatula. The cerebellum and cerebral cortex were rinsed with ice-cold 0.9% NaCl solution to remove blood clotting and then - 46 -homogenized w i t h 10 volumes and 5 volumes, r e s p e c t i v e l y , of i c e -c o l d 0.01 M T r i s - H C l b u f f e r , pH 7.4, c o n t a i n i n g 2 mM B-mercapto-ethanol. The homogenate was c e n t r i f u g e d at 34,800 x g f o r 60 min at 0°C. The supernatant (3-5 mg p r o t e i n per ml) was used as the source of enzyme and designated as F r a c t i o n I (crude enzyme e x t r a c t ) . 2.7. P r e p a r a t i o n of DEAE-Cellulose Column DEAE-cellulose w i t h an exchange c a p a c i t y of 0.66 m i l l i -e q u i v a l e n t per gm was f u r t h e r washed to remove y e l l o w u l t r a v i o l e t absorbing m a t e r i a l as described by Peterson and Sober (302). Approximately 20 gm of DEAE-cellulose were suspended i n about 1 l i t e r of 1.0 N NaOH w i t h constant s t i r r i n g f o r about 10 min. The suspension was f i l t e r e d through a Buchner f u n n e l w i t h s u c t i o n and was then washed thoroughly w i t h water u n t i l n e u t r a l . The washed c e l l u l o s e was re-suspended i n 1 l i t e r of 0.5 N HCl w i t h constant s t i r r i n g f o r about 5 min. The suspension was f i l t e r e d and washed to n e u t r a l as mentioned above. F i n a l l y , the DEAE-c e l l u l o s e was suspended i n 0.01 M phosphate b u f f e r , pH 7.4, and kept i n the c o l d u n t i l r e q u i r e d . When i n use, an a p p r o p r i a t e amount of DEAE-cellulose was a p p l i e d to a column and e q u i l i b r a t e d w i t h 0.01 M phosphate b u f f e r c o n t a i n i n g 2 mM 3-mercaptoethanol and 5% g l y c e r o l . 2.8. P r e p a r a t i o n of Sephadex G-100 and Sephadex G-200 Columns The Sephadex was suspended i n 0.01 M T r i s - H C l b u f f e r , pH 7.4 w i t h 2 mM 0-mercaptoethanol, and s t i r r e d overnight i n a c o l d room - 47 -(4°C). I t was allowed to s e t t l e and the f i n e p a r t i c l e s were removed by s u c t i o n . The Sephadex was kept i n 0.01 M T r i s - H C l b u f f e r s o l u t i o n , pH 7.4, c o n t a i n i n g 2 mM 3-mercaptoethanol i n the c o l d room u n t i l needed. Columns were packed by pouring the swollen g e l s i n t o the column as a s l u r r y and allowed to s e t t l e under g r a v i t y . The column was packed i n a s i n g l e con-tinuous manner to a v o i d t r a p p i n g a i r bubbles. A l l procedures were performed at 4°C as d e s c r i b e d i n the " T e c h n i c a l Data Sheets" which were d i s t r i b u t e d by Pharmacia. The column was e q u i l i b r a t e d by washing w i t h 0.01 M T r i s - H C l b u f f e r , pH 7.4, c o n t a i n i n g 2 mM 3-mercaptoethanol. 2.9. Determination of R a d i o a c t i v i t y R a d i o a c t i v e samples were counted i n a Nuclear-Chicago Mark I l i q u i d s c i n t i l l a t i o n counter w i t h 36-40% e f f i c i e n c y f o r t r i t i u m . Not more than 0.5 ml of the sample was used i n each v i a l , which contained 10 ml of s c i n t i l l a t i o n s o l u t i o n c o n t a i n i n g 15 gm of 2.5 d i p h e n y l o x a z o l (PPO), 150 mg of 1.4-bis-(5-phenyloxazolyl-2) benzene (POPOP) and 240 gm of naphthalene i n a l i t e r each of toluene, dioxane and 95% ethanol. 2.10. P u r i f i c a t i o n of DNA Polymerase From 10-day-old Rat B r a i n 2.10a.Ammonium s u l f a t e f r a c t i o n a t i o n A f i n e powder of ammonium s u l f a t e was added to the enzyme e x t r a c t s to g i v e the d e s i r e d s a t u r a t i o n according to the f o l l o w i n g - 48 -formula (309). W = 0.515 V 1 ( S 2 - S^/ l - S , ^ x 0.272 where W = weight of ammonium s u l f a t e i n gm = volume of s o l u t i o n i n ml at i n i t i a l s a t u r a t i o n S 2 = d e s i r e d s a t u r a t i o n The ammonium s u l f a t e was added s l o w l y to enzyme e x t r a c t , w i t h constant s t i r r i n g at 0-4°C. The f i r s t step of ammonium s u l f a t e f r a c t i o n a t i o n was brought to 25% s a t u r a t i o n . A f t e r s t i r r i n g f o r 30 min, the mixture was c e n t r i f u g e d at 27,000 x g f o r 10 min. The p r e c i p i t a t e was di s c a r d e d and the supernatant was brought to 45% s a t u r a t i o n w i t h ammonium s u l f a t e . The mixture was then c e n t r i f u g e d a f t e r 30 min s t i r r i n g . The p r e c i p i t a t e was c o l l e c t e d by c e n t r i f u g a t i o n and was d i s s o l v e d i n 0.01 M phosphate b u f f e r , pH 7.4, c o n t a i n i n g 2 mM f3-mercaptoethanol and 5% g l y c e r o l . The p r o t e i n s o l u t i o n was d e s a l t e d by d i a l y s i s . The p r o t e i n p r e-c i p i t a t e s which were formed a f t e r d i a l y s i s were removed by c e n t r i -f u g a t i o n and the supernatant was designated F r a c t i o n II„. 45% ammonium s u l f a t e s a t u r a t e d supernatant was then brought to 70% s a t u r a t i o n . The p r e c i p i t a t e was c o l l e c t e d and d i s s o l v e d i n the same b u f f e r as mentioned above. The p r o t e i n s o l u t i o n was de-s a l t e d by d i a l y s i s and designated F r a c t i o n H^. The d i a l y s i s was c a r r i e d out i n the c o l d room (4°C) f o r 12 h against 5 changes o f 1 l i t e r each of 0.01 M phosphate b u f f e r , pH 7.4, c o n t a i n i n g 2 mM 0-mercaptoethanol and 5% g l y c e r o l . - 49 -2.10b. DEAE-cellulose column chromatography A column of DEAE-cellulose (1.9 cm x 25 cm) was prepared and was e q u i l i b r a t e d w i t h 0.01 M phosphate b u f f e r , pH 7.4 con-t a i n i n g 2 mM g-mercaptoethanol and 5% g l y c e r o l . A l l these f r a c t i o n a t i o n procedures were c a r r i e d out i n the c o l d room (4°C) F r a c t i o n I I (82 mg of p r o t e i n ) was a p p l i e d to the column (1.9 cL cm x 25 cm) and allowed to soak i n by g r a v i t y . F i v e ml f r a c t i o n s were c o l l e c t e d and the flow r a t e of t h i s column was between 25 and 30 ml per hour. A f t e r the p r o t e i n s o l u t i o n had completely entered the adsorbant, 150 ml of the same b u f f e r was washed through the column to remove unadsorbed and l o o s e l y adsorbed m a t e r i a l . Absorbed p r o t e i n s were then e l u t e d w i t h KCI i n the same b u f f e r ; the KCI c o n c e n t r a t i o n was i n c r e a s e d stepwise from 0.1 M to 0.2 M, 0.3 M, 0.5 M and 0.7 M r e s p e c t i v e l y . F r a c t i o n I I b (43 mg of p r o t e i n ) was passed through a column (1.5 cm x 21 cm) of DEAE-c e l l u l o s e and processed as above. F r a c t i o n s recovered from the column were measured f o r u l t r a v i o l e t absorbancy at 260 and 280 nm w i t h a Beckman Model DU spectrophotometer. 2.11. F r a c t i o n a t i o n of Enzymes on Sephadex Gel F i l t r a t i o n 2.11a.Column chromatography of 10-day-old c e r e b e l l a r DNA p o l y - merase on Sephadex G-100 A column of Sephadex G-100 (2.0 cm x 23 cm) was prepared and was e q u i l i b r a t e d w i t h 0.005 M T r i s - H C l b u f f e r , pH 7.4, at 4°C. - 50 -1.7 ml of c e r e b r a l e x t r a c t ( c o n t a i n i n g 5.2 mg of p r o t e i n ) from 10-day-old r a t b r a i n was a p p l i e d to the column and allowed to soak i n by g r a v i t y . A l l the f o l l o w i n g steps were c a r r i e d out i n the c o l d room (0-4°C). A f t e r the p r o t e i n s o l u t i o n had completely entered the g e l , the column was g e n t l y washed twice w i t h 1 ml of same b u f f e r , then e l u t e d w i t h the same b u f f e r . 3 ml f r a c t i o n s were c o l l e c t e d at a flow r a t e of about 7-9 ml per hour. Each tube was measured f o r absorbancy at 280 and 260 nm and was assayed f o r DNA polymerase a c t i v i t y . 2 . l i b . Column chromatography of DNA polymerase B on Sephadex G-200 A column of Sephadex G-200 (0.5 cm x 30 cm) was prepared and was e q u i l i b r a t e d w i t h 0.01 M T r i s - H C l b u f f e r , pH 7.4, con-t a i n i n g 2 mM 6-mercaptoethanol and 5% g l y c e r o l i n the c o l d room. DNA polymerase B obtained from DEAE-cellulose column chromatography of F r a c t i o n 11^ was condensed and d i a l y z e d . Concentrated DNA polymerase B (7 mg of p r o t e i n i n 1.5 ml) was a p p l i e d to the Sephadex G-200 column, and then e l u t e d w i t h the same b u f f e r . 3 ml f r a c t i o n s were c o l l e c t e d at a flow r a t e of about 2-3 ml per hour. Each tube was measured f o r absorbancy at,280 and 260 nm and assayed f o r DNA polymerase a c t i v i t y . 2.12. N e u t r a l Sucrose Density Gradient C e n t r i f u g a t i o n A l i n e a r sucrose g r a d i e n t of 5-20% (W/V) c o n c e n t r a t i o n was prepared i n 0.5 x 2 i n c h Beckman SW 39 L c e l l u l o s e n i t r a t e - 51 -tubes f o r the Beckman SW 39 L r o t o r according to the procedure of M a r t i n and Ames (310). The sucrose s o l u t i o n was prepared i n 0.01 M phosphate b u f f e r , pH 7.4 c o n t a i n i n g 2 mM g-mercapto-e t h a n o l , 0.1 M KCI and 6 mM EDTA. 0.2 ml of the enzyme s o l u t i o n was c a r e f u l l y l a y e r e d over the pre-formed g r a d i e n t s . These pr e - l a y e r e d tubes were loaded i n t o an SW 39 L swing bucket r o t o r , and were c e n t r i f u g e d i n a Spinco model L u l t r a c e n t r i f u g e at a r o t o r speed of 36,000 rpm f o r 15 h at 0°C. The time of c e n t r i -f u g a t i o n was taken as the p e r i o d from the s t a r t of a c c e l e r a t i o n of the r o t o r to the s t a r t of d e c e l e r a t i o n . A f t e r c e n t r i f u g a t i o n the bottom of the tube was punctured w i t h a Buchler p i e r c i n g u n i t which was s i m i l a r to the one described by M a r t i n and Ames (310). F i f t e e n drop f r a c t i o n s were c o l l e c t e d i n t e s t tubes, and each f r a c t i o n was assayed f o r DNA polymerase a c t i v i t y . The procedure used f o r a n a l y z i n g p r o t e i n markers was the same as mentioned above, us i n g cytochrome C, bovine serum albumin and glucose oxidase as p r o t e i n markers. The p r o t e i n markers were detected by reading absorbancy at 280 nm. The l i n e a r sucrose g r a d i e n t s were r e p r o d u c i b l e i n d i f f e r e n t tubes when they were prepared under the same c o n d i t i o n s . There-f o r e under s i m i l a r c e n t r i f u g a t i o n a l c o n d i t i o n s , the r a t i o of the di s t a n c e t r a v e l l e d by any two substances from the meniscus would be constant. When a p r o t e i n of known sedimentation c o e f f i c i e n t was used, i t was p o s s i b l e to estimate the "S" value f o r an un-- 52 -known substance. The r a t i o " r " could be determined (310) a f t e r any time of c e n t r i f u g a t i o n as: _ d i s t a n c e t r a v e l l e d from meniscus by unknown di s t a n c e t r a v e l l e d from meniscus by standard The "S" valu e f o r an unknown p r o t e i n w i l l be given by S_. of unknown = S„. of standard x r 20 ,w 20,w 2.13. S u b c e l l u l a r F r a c t i o n a t i o n of Rat B r a i n 10-day-old and a d u l t r a t b r a i n s were c h i l l e d immediately a f t e r removal by immersion i n 0.32 mM sucrose c o n t a i n i n g 3 mM MgCl 2 s o l u t i o n at 0°C. A f t e r removing blood contaminants, the b r a i n s were homogenized w i t h 5 v o l . of 0.32 mM sucrose-3 mM MgC^ s o l u t i o n i n a g l a s s homogenizer under a motor speed of about 1000-1500 rpm. The homogenate was c e n t r i f u g e d i n the S o r v a l l r e f r i g e r a t e d c e n t r i f u g e at 1000 x g f o r 10 min. A f t e r decanting o f f the supernatant f o r f u r t h e r f r a c t i o n a t i o n , the crude n u c l e a r p e l l e t s were washed by re-suspending i n 0.32 M sucrose-3 mM MgC^ s o l u t i o n and c e n t r i f u g i n g at 1000 x g f o r 10 min. The washed p e l l e t was used as the "nu c l e a r f r a c t i o n " . M i t o c h o n d r i a l and microsomal f r a c t i o n s were prepared from the supernatant of the f i r s t c e n t r i f u g a t i o n . M i t o c h o n d r i a were i s o l a t e d by c e n t r i f u g i n g the supernatant at 10,000 x g f o r 10 min. The p e l l e t was re-suspended and r e - c e n t r i f u g e d and the washed p e l l e t used as the "mitochondria f r a c t i o n " . The supernatant obtained i n ' - 53 -the i s o l a t i o n of mitochondria was c e n t r i f u g e d at 100,000 x g f o r 60 min i n a #50 r o t o r of the Spinco u l t r a c e n t r i f u g e . The p e l l e t was used as the "microsome f r a c t i o n " . The high speed supernatant (supernatant obtained i n the i s o l a t i o n of microsome) was used as the "supernatant f r a c t i o n " . 2.14. Other A n a l y t i c a l Methods Ammonium s u l f a t e was determined according to the procedure described by Umbreit, Bunis and S t a n f f e r (311). P r o t e i n was estimated according to the method of Lowry e_t a l (312), using bovine serum albumin as standard. - 54 -I I I . RESULTS AND DISCUSSION 3.1. P r e l i m i n a r y Studies on DNA Polymerase In Rat B r a i n E x t r a c t s I n the i n v e s t i g a t i o n described i n t h i s t h e s i s , DNA polymerase from developing r a t b r a i n was i s o l a t e d and c h a r a c t e r i z e d p r i o r to the d e t a i l e d study of t h i s enzyme. I t was necessary to e s t a b l i s h the assay system and to c h a r a c t e r i z e t h i s enzyme be f o r e attempting to p u r i f y such an enzyme from r a t b r a i n . 3.1a. E x t r a c t of DNA polymerase from developing r a t b r a i n Enzyme e x t r a c t s of 2-, 4-, 6-, 10-, 16-, 17-day-old and ad u l t r a t b r a i n were prepared as des c r i b e d i n Methods. A l l the f o l l o w i n g experiments were c a r r i e d out w i t h f r e s h l y prepared enzyme e x t r a c t s , except f o r the study of the s t a b i l i t y of the enzyme. 3.lb. C h a r a c t e r i s t i c s of DNA polymerase of r a t b r a i n E a r l i e r i n v e s t i g a t o r s (17,38) have shown that the b a s i c r e -quirements f o r mammalian DNA polymerases were e s s e n t i a l l y the same as those p r e v i o u s l y described f o r the b a c t e r i a l system. The a c t i v i t y of DNA polymerase i n s o l u b l e e x t r a c t s from r a t b r a i n presented here a l s o shows many s i m i l a r i t i e s to other animal DNA polymerases (48,254,313). As shown In Table I , DNA polymerase a c t i v i t y of 10-day-old r a t cerebellum r e q u i r e d the a d d i t i o n of 2+ template DNA and Mg . The a c t i v i t y was enhanced by the a d d i t i o n - 55 -Table I . Requirements f o r r a t b r a i n DNA polymerase. [ H] dTTp i n c o r p o r a t i o n (counts/min per assay) Assay system Template: heated DNA Nati v e : DNA Completed system 501 453 - DNA 46 46 - dATP, dGTP, dCTP 100 91 - dATP 135 155 - dGTP 149 199 - dCTP 185 202 - MgCl 2 96 93 - D i t h i o t h r e i t o l 167 220 + T r y p s i n 45 50 + DNase 75 80 + RNase 495 447 - Enzyme 49 23 Enzyme heated 10 min a t 100° 51 45 The composition of the assay mixture i s as described i n the experimental c o n d i t i o n s f o r the standard i n c u b a t i o n mixture. 20 ug of e i t h e r n a t i v e c a l f thymus DNA or heated DNA as template and 0.18 mg of e x t r a c t from the cerebellum of 10-day-old r a t s were used. - 56 -of d i t h i o t h r e i t o l and by the presence of a l l four deoxynucleoside t r i p h o s p h a t e s . With the omission o f u n l a b e l l e d deoxynucleoside 3 triphosphates the i n c o r p o r a t i o n of [ H]dTTP was on l y 20% of that obtained i n the complete system where a l l four deoxynucleoside triphosphates were present, i n d i c a t i n g p r i m a r i l y r e p l i c a t i v e r a t h e r than a t e r m i n a l a d d i t i o n a c t i v i t y . DNA polymerase from r a t b r a i n i s s i m i l a r to other mammalian DNA polymerase systems which have been s t u d i e d (24,37,41) w i t h respect to template pre-ference. The enzyme a c t i v i t y w i t h heat-denatured DNA as template was somewhat higher than that w i t h n a t i v e DNA as template. The 3 a c t i v i t y o f i n c o r p o r a t i o n of [ H]dTTP was destroyed by t r y p s i n and DNase, but not by RNase, i n d i c a t i n g that the i n c o r p o r a t i o n 3 of [ H]dTTP was c a t a l y z e d by enzyme DNA polymerase. A more c r i t i c a l examination of the nature of the r e a c t i o n product was c a r r i e d out by determining i t s s e n s i t i v i t y to DNase, RNase and NaOH treatments as shown i n Table I I . The product could be rendered a c i d - s o l u b l e by p a n c r e a t i c DNase but was unaffe c t e d by p a n c r e a t i c RNase or by 0.1 N NaOH hydrolysis. The product t h e r e f o r e has the p r o p e r t i e s of DNA. 3.1c. The p r o p e r t i e s of DNA polymerase The requirement f o r a d i v a l e n t c a t i o n i n the r e a c t i o n i s absolute as desc r i b e d above. I n F i g u r e 1, the a c t i v i t y of DNA polymerase from r a t b r a i n i s shown at v a r i o u s c o n c e n t r a t i o n s of - 57 -Table II. Characterization of the product polymerized by rat brain DNA polymerase Treatment Acid insoluble radioactivity % Undigested product Untreated 715 100 20 pg DNase 97 13 20 yg RNase 698 97 0.1 N NaOH 734 100 The procedure of this experiment was described in Methods. - 58 -cpm • r i i i J i 1 1 2 3 A 5 6 7 8 M g C l 2 ( m M ) F i g . 1. The e f f e c t of magnesium ions on the a c t i v i t y of DNA polymerase from 10-day-old r a t b r a i n . A A Heated DNA as template • - - - • N a t i v e DNA as template - 59 -2+ 2+ Mg . It is clear that Mg ions at about 5 mM are most efficient of a l l with either native DNA or heated DNA as template. This property is similar to those in other enzyme systems (48). The optimum pH of DNA polymerase was found at 7.5 i n several partially purified systems (13,19,37). However, i n crude extracts of rat brain, the optimal activity of DNA polymerase activity was around pH 8.6 as shown i n Figure 2. The reason that i t showed optimal activity under alkaline conditions could be due to the concomitant DNase or some other contamination in crude extract, or could be due to the tissue spe c i f i c i t y . Recently, Bharucha and Murthy (256) reported that the optimal pH for ac t i v i t i e s of DNA polymerase from new born rat brain was around 7.4 and 8.1. The different optimal pHs obtained are due to different enzyme fractions. The time-course of the incorporation reaction i s shown in Figure 3. An incubation time of 30 min was chosen for the standard assay so as to be well within the period of linearity. 3 The incorporation of [ H]dTTP into DNA by various amounts of crude enzyme extract from 10-day-old rat cerebellum was studied. The reaction was proportional to the enzyme concentration up to 540 pg of enzyme protein. The enzyme concentration of the standard assay was around 180 ug and this was within the range of linearity. F i g . 2. The e f f e c t of pH on DNA polymerase a c t i v i t y from r a t b r a i n . A A Heated' DNA as template • - - - • Native DNA as template - 61 -Time (min.) 3 F i g . 3 . The time-course of the i n c o r p o r a t i o n of [ H]dTTP. 10-day-old c e r e b e l l a r e x t r a c t was used as enzyme source. • • Heated DNA as template o - - - o N a t i v e DNA as template - 62 -The DNA polymerase a c t i v i t y i n the c e r e b e l l a r e x t r a c t was ra t h e r unstable. As shown i n Fig u r e 4, the a c t i v i t y of the crude enzyme e x t r a c t from 10-day-old r a t cerebellum at 0-4°C dropped to about 70% of the o r i g i n a l a c t i v i t y w i t h heated DNA as template, about 50% w i t h n a t i v e DNA as template a f t e r 3 days storage and about 50% w i t h heated DNA as template and about 30% w i t h n a t i v e DNA as template a f t e r 5 days storage. This l o s s of a c t i v i t y was s l i g h t l y lessened by the a d d i t i o n of d i t h i o t h r e i t o l (5 mM) during storage. 3.Id. The e f f e c t o f mixing e x t r a c t from d i f f e r e n t ages When b r a i n e x t r a c t s from r a t s of d i f f e r e n t ages were mixed, an i n h i b i t o r y f a c t o r was n o t i c e d i n e x t r a c t s from o l d e r r a t s . Table I I I shows that when an e x t r a c t of cerebellum from 17-day-o l d r a t s or a d u l t r a t s was added to the e x t r a c t from 4-day-old r a t s , the a c t i v i t y observed (a) was much l e s s than the summation ( b ) . The p o s s i b l e i n h i b i t o r y f a c t o r i n adul t b r a i n was found to be heat-l a b i l e and d i d not l o s e a c t i v i t y a f t e r d i a l y s i s . I n order to t e s t whether t h i s apparent i n h i b i t o r was an enzyme which destroyed one of the r e a c t i o n components, ATPase a c t i v i t y was measured w i t h dATP as s u b s t r a t e (described i n Methods). There was p r a c t i c a l l y no det e c t a b l e ATPase a c t i v i t y i n the c e r e b e l l a r e x t r a c t , though crude c e r e b e l l a r homogenate showed very high ATPase a c t i v i t y . - 63 -o - -— • Heated DNA as template - o N a t i v e DNA as template - 64 -/ Table I I I . The e f f e c t of mixing e x t r a c t s from d i f f e r e n t ages of r a t P r e p a r a t i o n A c t i v i t y Observed (a) (counts/min) Expected (b) R a t i o (a)/(b) I 653 I I 142 I I I 77 I + I I 604 795 0.76 I + I I I 467 730 0.64 Preparations I , I I , and I I I were e x t r a c t e d from 4-day-old, 17-day-old and a d u l t r a t s , r e s p e c t i v e l y . About 0.2 mg of p r o t e i n i n each p r e p a r a t i o n was used i n t h i s experiment. - 65 -Gray et_ a l (261) and Otsuka et a l (314) r e p o r t e d the i n h i -b i t i o n of c e l l u l a r DNA s y n t h e s i s by the r a t l i v e r c e l l e x t r a c t . Sasada and Terayama (315) presented evidence i n d i c a t i n g that there are at l e a s t two i n h i b i t o r y f a c t o r s i n the a d u l t r a t l i v e r e x t r a c t . One component i s arginase and the o t h e r i s thymidine h y d r o l a s e (316). Malignant hepatomas l a c k both a r g i n a s e and thymidine h y d r o l a s e enzymes i n crude e x t r a c t . Sung (303) has shown that deoxyribo-nuclease increased r a p i d l y i n r a t b r a i n during maturation. The increased a c t i v i t y of these degradative enzymes may c o n t r i b u t e to the i n h i b i t i o n of DNA s y n t h e s i s which i s observed. 3.1e. Regional changes of DNA polymerase a c t i v i t y of r a t b r a i n at  d i f f e r e n t ages The a c t i v i t i e s of DNA polymerase i n r a t cerebellum and c o r t e x at 2-, 6-, 10- and 16-days of age were s t u d i e d and the r e s u l t s are shown i n Table IV. I n accordance w i t h Sung's previous study (252) 14 on the i n c o r p o r a t i o n of [ C]thymidine i n t o developing r a t b r a i n DNA, the a c t i v i t y of DNA polymerase i n the cerebellum i s 20-50 times higher than i n the c o r t e x depending on age. The a c t i v i t y i n the cerebellum a t t a i n s the maximum l e v e l at around 6 days of age and then decreases r a p i d l y d u r ing maturation to the a d u l t l e v e l where very l i t t l e a c t i v i t y i s d e t e c t a b l e . The c e r e b e l l a r a c t i v i t y at 16-days of age was only about 28% of that at 6 days. The c o r -t i c a l a c t i v i t y was the highest immediately a f t e r b i r t h and decreased Table IV. Regional changes of DNA polymerase activity of rat brain at different ages Age Brain Activity (units /mg protein) Ratio (days) region Template: heated DNA (a) Native DNA (b) (a)/(b) 2 cerebellum cortex 2.57 + 0.09 0.094 + 0.002 1.54 0.069 + 0.16 + 0.006 1.67 1.36 6 cerebellum cortex 3.52 + 0.12 0.064 + 0.003 2.43 0.058 + 0.09 + 0.01 1.45 1.10 10 cerebellum cortex 2.30 + 0.13 0.048 + 0.004 2.14 0.055 + 0.13 + 0.005 1.07 0.87 i o\ O N 16 cerebellum cortex 0.79 + 0.07 0.043 + 0.003 1.14 0.065 + 0.04 + 0.003 0.69 0.66 1 One unit of DNA polymerase activity was defined as the amount of enzyme 3 required to convert 1 pmole of [ H]dTTp into the acid-insoluble product in 30 min under the assay conditions described in Methods. - 67 -g r a d u a l l y during maturation. The DNA polymerase of newborn r a t s showed a preference f o r heat-denatured DNA as template, but i n o l d e r r a t s there was a p r o g r e s s i v e s h i f t towards n a t i v e DNA which was the p r e f e r r e d template a t 16-days of age. The r a t i o of DNA polymerase a c t i v i t y using heated and n a t i v e DNA as template i s 1.67 i n the 2-day-old r a t cerebellum, and then g r a d u a l l y dropped to 0.69 i n 16-day-old r a t cerebellum. The p o s s i b i l i t y of s h i f t i n g the preference f o r heat-denatured DNA as template to n a t i v e DNA could be due to heterog e n e i t y of DNA polymerase i n r a t b r a i n . Younger r a t b r a i n may c o n t a i n a higher amount of enzyme which p r e f e r s heat-denatured DNA as tem-p l a t e than the enzyme which p r e f e r s n a t i v e DNA as template, and the former enzyme disappeared f a s t e r than the l a t t e r d u r i ng maturation o f r a t b r a i n . So at o l d e r ages, i t contained a higher amount of enzyme which p r e f e r r e d n a t i v e DNA as template. This p o s s i b i l i t y could be confirmed by s e p a r a t i o n of two enzyme a c t i v i -t i e s from column chromatography of Sephadex G-100. 3.If. Column chromatography of crude c e r e b e l l a r DNA polymerase on  Sephadex G-100 When the crude c e r e b e l l a r e x t r a c t from 10-day-old r a t (5.2 mg of p r o t e i n ) was passed through a column (2.0 x 23 cm) of Sephadex G-100, two main peaks of absorbancy at 280 nm (A„ Q ) - 68 -were observed as shown i n F i g u r e 5. The r a t i o of absorbancy at 280 nm to 260 nm (A„0_. /A„.rt ) of t h i s peak was about 0.9 280nm 260nm r i n F r a c t i o n 5 ( F i g . 5) and 1.1 i n F r a c t i o n 9. The second peak showed much higher absorbance at 260 nm than at 280 nm, e.g. the A 0 0 . /A.,rt r a t i o i n F r a c t i o n 17 was 0.5, i n d i c a t i n g the 280nm 260nm presence of n u c l e i c a c i d or n u c l e o t i d e . From the data of determination of p r o t e i n by the method of Lowry et a l (312), i t was seen that o n ly the f i r s t A 0 0_ peak contained p r o t e i n . ZoUnm There was no p r o t e i n contained i n the second A„ o r > peak. zoUnm As shown i n F i g u r e 5, two peaks of DNA polymerase a c t i v i t i e s were observed. The f i r s t enzyme peak c o i n c i d e s w i t h the f i r s t A Q r t peak, having a strong DNA polymerase a c t i v i t y w i t h a preference f o r heat-denatured DNA as template. The second enzyme peak ( F r a c t i o n 9) found between the two major ^280Tfm P e ak s» showed much l e s s a c t i v i t y but e x h i b i t e d a preference f o r n a t i v e DNA as template i n s t e a d of heated DNA. From the data presented here and described i n Table IV, there seems to be two k i n d s of DNA polymerase i n c e r e b e l l a r ex-t r a c t ; one p r e f e r s heated DNA as template and the other p r e f e r s n a t i v e DNA as template. This p o s s i b i l i t y w i l l be proved by i s o l a t i o n , p u r i f i c a t i o n and c h a r a c t e r i z a t i o n of two DNA p o l y -merases from 10-day-old r a t b r a i n , and t h i s w i l l be described i n the f o l l o w i n g pages. F i g . 5. Column chromatography of crude c e r e b e l l a r DNA polymerase on Sephadex G-100. 5.2 mg of 10-day-old r a t c e r e b e l l a r e x t r a c t was a p p l i e d to a column of Sephadex G-100 (2.0 x 23 cm, e q u i l i b r a t e d w i t h 0.005 M T r i s - H C l b u f f e r , pH 7.4) and e l u t e d w i t h the same b u f f e r . F r a c t i o n s of 3 ml each were c o l l e c t e d at a flow r a t e of about 9 ml/h. Each tube was measured f o r absorbancy at 280 and 260 nm and then assayed f o r DNA polymerase a c t i v i t y A280nm DNA polymerase a c t i v i t y w i t h heat-denatured DNA as template - - - - DNA polymerase a c t i v i t y w i t h n a t i v e DNA as template. - 69 -- 70 -3.2. Separation and C h a r a c t e r i z a t i o n of DNA Polymerases A and B  from Developing Rat B r a i n In the previous S e c t i o n , some of the p r e l i m i n a r y s t u d i e s on DNA polymerase from r a t b r a i n e x t r a c t were presented. Evidence f o r the p o s s i b i l i t y of two DNA polymerases being contained i n r a t b r a i n were a l s o d e s cribed. A more d e t a i l e d account of the s e p a r a t i o n , p u r i f i c a t i o n and c h a r a c t e r i z a t i o n of the two DNA polymerases, A and B, from developing r a t b r a i n w i l l be d e s c r i b e d i n t h i s S e c t i o n . 3.2a. P a r t i a l p u r i f i c a t i o n o f two DNA polymerases, A and B, from  r a t b r a i n e x t r a c t Enzymes are found i n nature i n complex m i x t u r e s , u s u a l l y i n c e l l s which perhaps c o n t a i n hundreds or more d i f f e r e n t enzymes; i n most cases, some of the other enzymes present w i l l i n t e r f e r e , e i t h e r by a t t a c k i n g the s u b s t r a t e or by degrading the product. The main pa r t of the p u r i f i c a t i o n w i l l c o n s i s t of a s e r i e s of f r a c t i o n a t i o n s by which the DNA polymerase i s separated from the non-protein m a t e r i a l s (e.g. n u c l e i c a c i d n u c l e o t i d e s ) and other enzymes, such as DNase, phosphodiesterases and t r i p h o s p h a t e s . Some v a r i a t i o n of the procedure to be des c r i b e d has proven e f f e c t i v e i n the p u r i f i c a t i o n of mammalian DNA polymerase a c t i v i t y from c a l f thymus gland (13), reg e n e r a t i n g r a t l i v e r (19), r a t l i v e r n u c l e i and mitochondria (20), r a t i n t e s t i n a l mucosa (51), - 71 -and mammalian neoplasms (16,24-26,28). I t has been found that p u r i f i c a t i o n of DNA polymerase from mammalian t i s s u e i s u s u a l l y more d i f f i c u l t than from b a c t e r i a l systems (13). (1) P r e p a r a t i o n of e x t r a c t E x t r a c t s from 10-day-old baby r a t b r a i n s were prepared by f o l l o w i n g the procedure mentioned i n Methods, except that whole b r a i n was used i n t h i s experiment. F i v e volumes of e x t r a c t i n g medium were added and the mixture was homogenized. The high speed supernatant ( F r a c t i o n I , around 5.6 mg protein/ml) was used as the enzyme source f o r f u r t h e r p u r i f i c a t i o n . A l l the f o l l o w i n g steps were c a r r i e d out at 0-4°C. The p u r i f i c a t i o n procedure i s summarized i n Table V. (2) Ammonium s u l f a t e f r a c t i o n a t i o n Ammonium s u l f a t e f r a c t i o n a t i o n has been used w i d e l y to f r a c t i o n a t e DNA polymerase from d i f f e r e n t t i s s u e e x t r a c t s (13, 16,19,20,24-26,28). I n the present work, F r a c t i o n I from the previous step was f r a c t i o n a t e d to two major f r a c t i o n s , i . e . 25%-45% s a t u r a t i o n f o r F r a c t i o n I I and 45%-70% s a t u r a t i o n f o r a F r a c t i o n I I b > The p r e c i p i t a t e s of 25%-45% s a t u r a t i o n and 45%-70% s a t u r a t i o n were d i s s o l v e d i n 0.01 M phosphate b u f f e r , pH 7.4, co n t a i n i n g 2 mM B-mercaptoethanol and 5% g l y c e r o l and d i a l y z e d a g a i n s t the same b u f f e r to remove ammonium s u l f a t e . The a c t i v i t i e s of DNA polymerase were assayed i n both f r a c t i o n s . - 72 -Table V. P a r t i a l p u r i f i c a t i o n of DNA polymerase from r a t b r a i n T o t a l A c t i v i t y with S p e c i f i c A c t i v i t y F r a c t i o n protein Native DNA Heated DNA ( p m ° l e s [ ^ T M P { m g / \ / / . v r J. / • \ p r o t e i n per min) (mg) (counts/mm) (counts/min) I. Crude extract 572 672 774 0.52 II. (NH 4) 2S0 4 F r a c t i o n II F r a c t i o n II, 286 51 334 1102 645 120 1.54 5.27 III. DEAE-Cellulose Fr a c t i o n A 67 F r a c t i o n B 12 575 1863 1374 157 2.00 18.00 Each assay contained 0.1 - 0.4 mg of enzyme protein.. The s p e c i f i c a c t i v i t y of DNA polymerase was assayed i n the re a c t i o n mixture as mentioned i n Methods except that i t contained 20 nmoles c a r r i e r dTTP, 25.6 pmoles [ 3H]dTTP and 0.4 A u n i t of poly d(A-T) was used as template. - 73 -I t can be seen from Table V that the DNA polymerase a c t i v i t y of F r a c t i o n I I showed twice as much a c t i v i t y w i t h heat-denatured a J DNA as template. However, F r a c t i o n I I , was about 10 times more b a c t i v e w i t h n a t i v e DNA as template than w i t h heat-denatured DNA as template. Furthermore, F r a c t i o n I I was more a c t i v e than F r a c t i o n 11^ w i t h heat-denatured DNA as template, but was much l e s s a c t i v e i f n a t i v e DNA was used as template. The r e s u l t s of these experiments i n d i c a t e the e x i s t e n c e of at l e a s t two DNA polymerases which d i f f e r from each other w i t h respect to template s p e c i f i c i t y . The DNA polymerase which i s present i n the 25%-45% s a t u r a t e d ammonium s u l f a t e shows i t s pre-ference f o r heat-denatured DNA. The other enzyme, which i s present i n 45%-70% s a t u r a t e d ammonium s u l f a t e , i s more a c t i v e w i t h n a t i v e DNA as template than w i t h heat-denatured DNA as template. The t o t a l DNA polymerase a c t i v i t y recovered from both f r a c t i o n s was about 2.5 times g r e a t e r than that found i n the o r i g i n a l e x t r a c t i n d i c a t i n g some i n h i b i t o r ( s ) was removed from t h i s procedure. (3) DEAE-cellulose column chromatography Both F r a c t i o n I I and I I , were f u r t h e r p u r i f i e d by DEAE-a b c e l l u l o s e column chromatography. F i g u r e 6 shows the t y p i c a l e l u t i o n p a t t e r n obtained by chromatography of F r a c t i o n I I on a DEAE-cellulose column. I t w i l l be seen that the major peak of DNA polymerase a c t i v i t y comes o f f the column at the 0.2 M KC1 e l u t i o n s t e p , and one minor DNA polymerase peak comes o f f at the 0.3 M KC1 e l u t i o n step. From i t s p r o p e r t i e s , i t i s suggested that t h i s minor peak could be a Fraction number F i g . 6. Column chromatography of F r a c t i o n I I on DEAE-cellulose. F r a c t i o n I I (82 mg of p r o t e i n ) was a p p l i e d to a column (1.9 x 25 cm) of DEAE-c e l l u l o s e . Arrows i n d i c a t e the change of b u f f e r f r a c t i o n s . A ? f i n A A DNA polymerase a c t i v i t y - 75 -r e t e n t i o n peak of major DNA polymerase which was e l u t e d at 0.2 M KCI. The f r a c t i o n s ( F r a c t i o n s 38-42) of the major enzyme peak were c o l l e c t e d and d i a l y z e d against 0.01 M T r i s - H C l b u f f e r , pH 7.4, c o n t a i n i n g 2 mM g-mercaptoethanol and 5% g l y c e r o l . A f t e r d i a l y s i s , the enzyme f r a c t i o n was designated DNA polymerase A ( F r a c t i o n A ) . As shown i n Table V, enzyme A showed 3 times higher a c t i v i t y w i t h heated DNA than w i t h n a t i v e DNA as template. The t y p i c a l e l u t i o n p a t t e r n of F r a c t i o n 11^ on DEAE-cellulose column chromatography i s shown i n F i g u r e 7. Only one major enzyme a c t i v i t y peak was obtained i n the 0.1 M KCI e l u t i o n step. The peak of major enzyme f r a c t i o n s ( F r a c t i o n s 18-22) were c o l l e c t e d , d i a l y z e d and designated as DNA polymerase B. DNA polymerase B showed a higher a c t i v i t y w i t h n a t i v e DNA as template than w i t h heat-denatured DNA. The s p e c i f i c a c t i v i t y of DNA polymerase B i s 36 times that of the o r i g i n a l e x t r a c t . 3.2b. P r o p e r t i e s of p a r t i a l l y p u r i f i e d enzymes A and B Two enzyme f r a c t i o n s A and B were separated by means of ammonium s u l f a t e f r a c t i o n a t i o n and DEAE-cellulose chromatography. Th e i r preferences f o r DNA template were d i f f e r e n t , as i s shown i n Table V. In order to determine whether these two enzyme f r a c t i o n s are d i s t i n c t enzymes, f u r t h e r c h a r a c t e r i z a t i o n s were c a r r i e d out. 0.1M 0.2M 0.3M 0.5M 0.7M washing KCI KCI KCI KCI K G Fraction number Fig. 7. Column chromatography of Fraction 11^ on DEAE-cellulose. Fraction II. (43 mg of protein) was applied to a column (1.5 x 21 cm) of DEAE-cellulose. Arrows indicate the change of buffer fractions. A280nm A A DNA polymerase activity - 77 -(1) Requirements f o r the p a r t i a l l y p u r i f i e d DNA  polymerases from r a t b r a i n The requirements f o r maximum a c t i v i t y In v i t r o of the p a r t i a l l y p u r i f i e d DNA polymerases, Enzyme A and Enzyme B, of r a t b r a i n are shown i n Table V I . The enzymes r e q u i r e d a comple-2+ ment of the four deoxyribonucleoside triphosphates Mg and DNA as template. A l l these requirements are s i m i l a r to the crude e x t r a c t from r a t b r a i n which was de s c r i b e d p r e v i o u s l y . S u l f -h y d r y l compounds such as d i t h i o t h r e i t o l enhanced both enzyme a c t i v i t i e s ; Enzyme A being s t i m u l a t e d more than Enzyme B. S u l f h y d r y l b l o c k i n g agents such as N-ethylmaleimide i n h i b i t e d Enzyme A by 90% and Enzyme B by 57%. I t has been shown (157, 158) that _E. c o l i DNA polymerase I I was s t r o n g l y i n h i b i t e d by a s u l f h y d r y l - b l o c k i n g agent, which does not a f f e c t polymerase I (204). However, Enzyme A d i f f e r s from E_. c o l i DNA polymerase I I because of i t s strong preference towards heat-denatured DNA as template and r a t h e r Enzyme B i s s i m i l a r to polymerase I , s i n c e Enzyme B r e q u i r e s e x c l u s i v e l y n a t i v e DNA as template. I n c l u s i o n of a s m a l l amount of EDTA i n the DNA polymerase r e a c t i o n mixture i n v a r i a b l y s t i m u l a t e s the a c t i v i t y of the enzyme and the degree of s t i m u l a t i o n i s u s u a l l y f i v e - to t e n - f o l d ; the optimum range of EDTA i n the r e a c t i o n mixture i s 0.3 to 0.5 mM (26). However, EDTA s t r o n g l y s t i m u l a t e d DNA polymerase B from r a t b r a i n , but showed l i t t l e or no e f f e c t on DNA polymerase A (Table V I ) , and - 78 -Table VI. Requirements of DNA polymerases A and B Polymerase A Polymerase B (counts/min) (counts/min) Completed system 1587 1822 - DNA 57 69 - MgCl 2 48 67 - dithiothreitol 545 980 - dithiothreitol + NEM 151 791 - dNTP 165 479 + 0.3 mM ATP 2619 1841 + 3 mM EDTA 1746 2789 + 10 pg Actinomycin D 161 174 + 5 yg Trypsin 65 61 + 5 pg DNase 89 134 + 5 yg RNase 1569 1796 Enzyme A (0.11 mg of protein) was assayed with heat denatured DNA as template. Enzyme B (0.09 mg of protein) was assayed with native DNA as template. - 79 -the optimum range of EDTA f o r Enzyme B i s around 3 mM. The mechanism of a c t i v a t i o n of EDTA on DNA polymerase a c t i v i t y i s p o s s i b l y due to the removal of i n h i b i t o r y c a t i o n s from enzyme f r a c t i o n s and assay reagents by c h e l a t i o n (317). T r y p s i n and DNase, but not RNase, completely i n h i b i t e d 3 the i n c o r p o r a t i o n of [ H]TTP i n t o the a c i d - i n s o l u b l e f r a c t i o n , i n d i c a t i n g DNA s y n t h e s i s c a t a l y z e d by DNA-dependent DNA p o l y -merase; RNA played no d i r e c t r o l e i n the DNA polymerase r e a c t i o n i n v i t r o . ATP (0.3 mM) s t i m u l a t e d Enzyme A by 65% but showed no e f f e c t on Enzyme B. Recently, Moses and Richardson (213) have shown r e p l i c a t i v e DNA s y n t h e s i s i_n v i t r o i n E_. c o l i c e l l s by treatment w i t h toluene. They observed t h a t , w i t h t h i s system, ATP s t r o n g l y s t i m u l a t e d r e p l i c a t i v e s y n t h e s i s , but not r e p a i r s y n t h e s i s and N-ethylmaleimide completely i n h i b i t e d r e p l i c a t i v e s y n t h e s i s without a f f e c t i n g r e p a i r s y n t h e s i s . From t h i s p o i n t of view, i t seems that Enzyme A plays a r o l e i n r e p l i c a t i v e syn-t h e s i s and Enzyme B i s only f o r r e p a i r s y n t h e s i s . Both Enzyme A and Enzyme B r e q u i r e a l l four deoxyribo-n u c l e o s i d e triphosphates f o r maximum a c t i v i t y , i n d i c a t i n g that a predominant pa r t of the a c t i v i t y i s due to the r e p l i c a t i v e form of DNA polymerase. Actinomycin D i n h i b i t e d Enzyme A and Enzyme B by over 90% i n a r e a c t i o n mixture c o n t a i n i n g 10 yg of Actinomycin D. - 80 -Actinomycin D i s understood to i n h i b i t DNA s y n t h e s i s by b i n d i n g w i t h the guanine residues i n the primer DNA so that r e p l i c a t i o n by the b a s e - p a i r i n g mechanism i s s e r i o u s l y hampered (318) ; on the c o n t r a r y , the n o n - r e p l i c a t i v e t e r m i n a l d e o x y n u c l e o t i d y l t r a n s f e r a s e should not be a f f e c t e d by Actinomycin D (48) In order to examine the t e r m i n a l d e o x y n u c l e o t i d y l t r a n s -f e r a s e a c t i v i t y i n both F r a c t i o n s A and B, the t e r m i n a l deoxy-n u c l e o t i d y l t r a n s f e r a s e a c t i v i t y was assayed w i t h F r a c t i o n s A and B by f o l l o w i n g the method of Krakow e_t a_l (14). The r e s u l t s of t h i s assay are shown i n Table V I I . I t shows that i n the ab-sence of d i t h i o t h r e i t o l and three of the deoxynucleoside 3 triphosphates (only [ H]dTTP was added to the r e a c t i o n m i x t u r e ) , 3 a l i m i t e d i n c o r p o r a t i o n of [ H]TMP i n t o DNA occurred. The a d d i t i o n of a l l four deoxynucleoside triphosphates s t i m u l a t e d DNA s y n t h e s i s r a t h e r than i n h i b i t e d i t , and upon the a d d i t i o n of c y s t e i n e , no s t i m u l a t i o n of i n c o r p o r a t i o n occurred f o r any of the enzyme f r a c t i o n s t e s t e d . The s u l f h y d r y l - b l o c k i n g agent (NEM) ab o l i s h e d the a c t i v i t y of both Enzyme A and Enzyme B. This i n a c t i v a t i o n was not reversed by c y s t e i n e . Since the omission of three of the deoxynucleoside triphosphates and the a d d i t i o n of c y s t e i n e could s t r o n g l y s t i m u l a t e the t e r m i n a l deoxy-n u c l e o t i d y l t r a n s f e r a s e (14), i t seems that both F r a c t i o n s A and B contained very s m a l l amounts of t e r m i n a l d e o x y n u c l e o t i d y l t r a n s f e r a s e . - 81 -Table V I I . Terminal d e o x y n u c l e o t i d y l t r a n s f e r a s e a c t i v i t y i n enzyme f r a c t i o n s A and B . % of a c t i v i t y Assay system F r a c t i o n A F r a c t i o n B Complete - d i t h i o t h r e i t o l - d i t h i o t h r e i t o l & dNTP - d i t h i o t h r e i t o l & dNTP + c y s t e i n e - d i t h i o t h r e i t o l & dNTP + NEM - d i t h i o t h r e i t o l & dNTP + NEM + c y s t e i n e 2 mM of c y s t e i n e and 1.3 mM of N-ethylmaleimide were used i n t h i s experiment. 100 35 17 11 100 54 26 26 18 16 - 82 -(2) Template preference Most of the r e p o r t e d mammalian DNA polymerase p r e p a r a t i o n s g e n e r a l l y show a preference f o r heat-denatured or s i n g l e - s t r a n d e d DNA as the template (13,24,37,38). However, the DNA polymerase obtained from r a t l i v e r n u c l e i f r a c t i o n (20,50), the reg e n e r a t i n g l i v e r s o l u b l e f r a c t i o n (19,240), Walker tumor (319), and i s o l a t e d n u c l e i (34,237,241) u s u a l l y p r e f e r n a t i v e DNA as template. The crude p r e p a r a t i o n of polymerase from r a t b r a i n showed a s l i g h t l y g r e a t e r preference f o r heat-denatured DNA over n a t i v e DNA as tem-p l a t e . A f t e r p u r i f i c a t i o n , two enzyme f r a c t i o n s were obtained w i t h d i f f e r e n t DNA template preferences (Table V ) . Re c e n t l y , Wallace e_t al (236) a l s o reported two DNA polymerases present i n mammalian t i s s u e ; one p r e f e r i n g heat-denatured DNA as template and the other p r e f e r r i n g n a t i v e DNA as template. I n order to exclude the p o s s i b i l i t y that template preference i s due only to s p e c i f i c species of DNA, the preference of template f o r Enzyme A and Enzyme B was s t u d i e d w i t h DNA from v a r i o u s sources as shown i n Table V I I I . T his Table shows that a l l heat-denatured DNA enhance the a c t i v i t y of Enzyme A approximately 2-3 f o l d (except E_. c o l i DNA) over that observed w i t h corresponding n a t i v e DNA, and Enzyme B showed 6-30 times higher a c t i v i t y w i t h n a t i v e DNA as tem-p l a t e than w i t h heat-denatured DNA. More c o r r e c t l y , Enzyme B almost always r e q u i r e d n a t i v e DNA as template and very l i t t l e i n c o r p o r a t i o n occurred w i t h heat-denatured DNA as template. The extent of i n c o r -p o r a t i o n v a r i e d to some degree depending on the source of DNA. Table VIII. Nature of template and priming a b i l i t y „ . „ „ Activity of Base ratio i Enzyme A Enzyme B „ _ , r . Template , L / ., \ / 1 / ., \ Enzyme B /. t. ,_ - of template ^ (counts/min) (counts/min) Activity of / A + T/fL r\ w . (A+T/G+C) Enzyme A E. c o l i DNA Native Heated 603 752 3420 113 4.6 1.0 Calf thymus DNA Native Heated 575 1374 1863 157 1.4 1.25 Salmon sperm DNA Native Heated 543 1155 1312 200 1.1 1.45 T^ phage DNA Native Heated 219 983 •903 146 0.9 1.9 * The activity of Enzyme B with native DNA and that of Enzyme A with heated DNA - 84 -I t i s c l e a r that the r a t i o of the a c t i v i t y of Enzyme B to that of Enzyme A i s higher when DNA w i t h a high content of (G + C) i s used as template than when DNA w i t h a high content of (A + T) i s used as template. These r e s u l t s suggest that the mechanism of DNA s y n t h e s i s c a t a l y z e d by Enzyme A may be d i f f e r e n t from that c a t a l y z e d by Enzyme B. (3) The e f f e c t of b i v a l e n t c a t i o n s 2+ Mantsavinos (19) has observed t h a t Mn could not r e p l a c e 2+ the requirement f o r Mg i n the r e a c t i o n s t u d i e d w i t h p a r t i a l l y p u r i f i e d enzyme from regenerating r a t l i v e r . However, i n b r a i n 2+ 2+ e x t r a c t , Mn can p a r t i a l l y r e p l a c e the requirement f o r Mg i o n s . This response i s s i m i l a r to the Landschutz a s c i t e s tumor c e l l s (48). 2+ 2+ The e f f e c t of b i v a l e n t c a t i o n s , Mg and Mn , on p a r t i a l l y p u r i f i e d Enzyme A and Enzyme B was s t u d i e d . The r e s u l t s are shown i n Figures 8 and 9. In F i g u r e 8, the opt i m a l a c t i v i t y i s obtained 2+ w i t h Mg i n the range of 3-5 mM w i t h Enzyme A or Enzyme B, s i m i l a r 2+ to s e v e r a l mammalian systems (48). However, i n response to Mn , Enzyme A was s t i m u l a t e d more than Enzyme B. The opt i m a l c o n c e n t r a t i o n 2+ of Mn i s around 0.05 mM f o r Enzyme A and 0.3 mM f o r Enzyme B ( F i g . 9 ) . (4) The e f f e c t of monovalent c a t i o n s S i m i l a r to the Landschutz a s c i t e s tumor (26) and the r a t l i v e r m i t o c h o n d r i a l DNA polymerase (20) , DNA polymerase B from r a t b r a i n was s t r o n g l y s t i m u l a t e d by KC1; about 100% s t i m u l a t i o n i n the range - 85 -1800 / 1500 1200 £ \ -t—> c O O 900 600 - / 300 5 7 9 MgCI 2 (mM ) 11 13 If F i g . 8. The e f f e c t of MgCl^ on DNA polymerase A and B a c t i v i t i e s . The assay systems are as mentioned i n Methods, except w i t h v a r i e d amounts of MgClg as i n d i c a t e d . DNA polymerase A DNA polymerase B - 86 -1200-0.05 0.1 0.15 0.2 0.3 M n C I 2 ( m M ) F i g . 9. The e f f e c t of MnC]^ on DNA polymerase A and B a c t i v i t i e s . The assay systems were the same as i n Methods except w i t h v a r i a b l e amounts of MnC^ as i n d i c a t e d . A DNA polymerase A • - - - • DNA polymerase B - 87 -0.03 - 0.05 M as shown i n F i g u r e 10. In the case of DNA polymerase A, however, s a l t gave no a c t i v a t i o n but r a t h e r a marked i n h i b i t i o n . The i n h i b i t i o n of DNA polymerase A by KC1 i s s i m i l a r to r a t l i v e r chromosomal DNA polymerase (50). Bharucha and Murthy r e p o r t e d that monovalent ca t i o n s i n h i b i t e d DNA polymerase prepared from nuclear s o l u b l e f r a c t i o n s of new born r a t b r a i n (256) . Th i s phenomenon could be due to the crude enzyme prep a r a t i o n s used by these workers. P u r i f i c a t i o n changes the response of DNA polymerase to KC1. A l t e r n a t i v e l y , the major DNA polymerase i n the n u c l e a r s o l u b l e f r a c t i o n may be DNA polymerase A. (This p o s s i b i l i t y was proved to be t r u e and w i l l be discussed i n F i g u r e 22). (5) pH optimum As shown i n F i g u r e 11, o p t i m a l a c t i v i t y of Enzyme A was observed at pH 7.2 i n T r i s - H C l b u f f e r and at pH 7.2 - 7.5 i n phosphate b u f f e r . On the other hand, the pH optimum f o r Enzyme B was pH 7.5 - 7.8 i n T r i s - H C l b u f f e r and pH 7.5 i n phosphate b u f f e r . S i m i l a r pH optima have been re p o r t e d by Leung and Zbarsky (51), Bollum (13)and Gold and H e l l e i n e r (37). G e n e r a l i z i n g from a number of r e s u l t s , Chang and Bollum ( 4 9 ) suggested that there are two DNA polymerases i n mammalian s p e c i e s ; one has a n e u t r a l pH optimum, and the other has an a l k a l i n e pH optimum. (6) Time course 3 The i n c o r p o r a t i o n of [ H]dTTP i n t o DNA over 2 hours was t e s t e d w i t h A and B enzyme f r a c t i o n s ( F i g . 12). With Enzyme A, - 88 -I I I I I 10 30 50 70 90 KCI (mM) F i g . 10. The e f f e c t of KCI on DNA polymerase A and B a c t i v i t i e s . KCI was added to the standard assay mixture (see Methods). • • DNA polymerase B • - - - • DNA polymerase A - 89 -1500 1200 N 900 F i g . 11. The e f f e c t of pH on DNA polymerase A and B a c t i v i t i e s , The assay systems were the same as i n Methods except f o r d i f f e r e n t b u f f e r s of v a r i e d pH. A A DNA polymerase A i n Tri s - m a l e a t e b u f f e r A A DNA polymerase A i n phosphate b u f f e r • - - - • DNA polymerase B i n Tri s - m a l e a t e b u f f e r o - - - o DNA polymerase B i n phosphate b u f f e r - 90 -i n c o r p o r a t i o n was l i n e a r and reached a maximum at 40 min a f t e r which the l e v e l was maintained. With Enzyme B, a l i n e a r i n c o r -p o r a t i o n of the s u b s t r a t e i n t o DNA f o r a p e r i o d of 2 h was ob-ta i n e d . The l e v e l l i n g o f f of the a c t i v i t y of Enzyme A at 40 min could p o s s i b l y be due to c o n t a i n i n g an i n h i b i t o r i n the enzyme p r e p a r a t i o n , such as p r o t e o l y t i c enzyme or DNase. However, i t i s not because of the template used s i n c e Enzyme A reached a maximum at 40 min w i t h e i t h e r heat-denatured or n a t i v e DNA as template . and Enzyme B a l s o showed a l i n e a r i n c o r p o r a t i o n up to 2 h w i t h heat-denatured DNA as template, except that i t s a c t i v i t y was much lower. (7) Enzyme c o n c e n t r a t i o n curve 3 The i n c o r p o r a t i o n of [ H]dTTP i n t o DNA by Enzyme A was pr o -p o r t i o n a l to the enzyme c o n c e n t r a t i o n ( F i g . 13). However, f o r Enzyme B, the i n c o r p o r a t i o n was p r o p o r t i o n a l to the enzyme concen-t r a t i o n only over 25 pg of p r o t e i n range. The o v e r a l l s i g m o i d a l slope i n the Enzyme B c o n c e n t r a t i o n curve may i n d i c a t e co-o p e r a t i v e b i n d i n g of enzyme a c t i v i t y . I t could be that there are two or more subunits i n Enzyme B, and when Enzyme B was assayed under very d i l u t e c o n d i t i o n s , the subunits were d i s s o c i a t e d and no enzyme a c t i v i t y could be detected. The i s o l a t i o n and c h a r a c t e r i z a t i o n of p r o t e i n f a c t o r ( s ) which a c t i v a t e d Enzyme B w i l l be de s c r i b e d i n - 91 -A A _ _Q o 20 40 60 80 100 ' 120 M i n u t e s F i g . 12. Time-course of the i n c o r p o r a t i o n of [ H]dTTP A A A DNA polymerase A w i t h heat-denatured DNA as template A DNA polymerase A w i t h n a t i v e DNA as template • - - - • DNA polymerase B w i t h n a t i v e DNA as template o - - - o DNA polymerase B w i t h heat-denatured DNA as template - 92 -Protein (ug) 3 F i g . 13. V a r i a t i o n of i n c o r p o r a t i o n of [ H]dTTP i n t o DNA w i t h d i f f e r e n t amounts of DNA polymerases A and B. • • Enzyme B A - - - A Enzyme A S e c t i o n 3.5. Spermidine a c t i v a t e d Enzyme B and changed the enzyme co n c e n t r a t i o n curve to a s t r a i g h t l i n e (see S e c t i o n 3.6). 3.3. P a t t e r n of Developmental Changes i n DNA Polymerases A and B  of Rat B r a i n The r e g i o n a l changes of DNA polymerase a c t i v i t i e s of r a t b r a i n at d i f f e r e n t ages are shown i n Table IV. Table IV shows tha t the a c t i v i t y of DNA polymerase i n the cerebellum a t t a i n s i t s maximum l e v e l at around 6 days of age and then decreases r a p i d l y during maturation to adulthood where very l i t t l e a c t i v i t y could be detected. The c o r t i c a l a c t i v i t y was h i g h e s t immediately a f t e r b i r t h and went down g r a d u a l l y during maturation. The DNA p o l y -merase of newborn r a t s showed a preference f o r heat-denatured DNA as template, but i n o l d e r r a t s there was a p r o g r e s s i v e s h i f t t o -wards n a t i v e DNA which was the p r e f e r r e d template at 16 days of age. Two DNA polymerases, A and B, have been separated and p a r t i a l l y p u r i f i e d from r a t b r a i n , t h e i r p r o p e r t i e s have a l s o been c h a r a c t e r i z e d as d e s c r i b e d i n S e c t i o n 3.2. Enzyme A has a strong preference f o r heat-denatured DNA as template and Enzyme B p r e f e r s n a t i v e DNA as template. Younger r a t s presumably c o n t a i n a higher amount of Enzyme A than Enzyme B and the reverse can be found i n o l d e r r a t b r a i n . This p o s s i b i l i t y was s t u d i e d and w i l l be discussed i n t h i s S e c t i o n . - 94 -3.3a. The sedimentation p r o f i l e f o r DNA polymerases A and B  of r a t b r a i n In order to study the changes i n p a t t e r n of DNA polymerases A and B during maturation, v a r i o u s approaches were i n v e s t i g a t e d . Sucrose d e n s i t y g r a d i e n t c e n t r i f u g a t i o n was found to be the most s a t i s f a c t o r y f o r t h i s study because t h i s method combined accuracy and s i m p l i c i t y f o r the d e t e c t i o n of changes of p a t t e r n of these two enzymes i n r a t b r a i n at d i f f e r e n t ages. DNA polymerases A and B separated and p u r i f i e d from 10-day-o l d r a t b r a i n were l a y e r e d on 5.0 ml of a 5-20% (W/V) l i n e a r sucrose g r a d i e n t . A f t e r 15 h c e n t r i f u g a t i o n at 36,000 rpm at 0°C i n a SW 39 r o t o r of the Spinco model L u l t r a c e n t r i f u g e , the bottom of the tube was punctured and 15 drop f r a c t i o n s were c o l l e c t e d as described by M a r t i n and Ames (310). The f r a c t i o n s were assayed f o r DNA polymerase a c t i v i t y . The r e s u l t s are shown i n F i g u r e 14. Fi g u r e 14a and F i g u r e 14b show c l e a r l y that each sample o f p u r i f i e d DNA polymerase A or B has only one a c t i v i t y peak. T h e i r S val u e s are around 9 S f o r Enzyme A and 3-4 S f o r Enzyme B. Approximate S values f o r the molecular weights were c a l c u l a t e d according to the procedure of M a r t i n and Ames (310), using cytochrome C, serum albumin and glucose oxidase as standards. As shown i n F i g u r e 14c, a combination of DNA polymerases A and B can be c l e a r l y separated i n t o two enzyme a c t i v i t i e s appearing i n the ap p r o p r i a t e f r a c t i o n s . - 95 -F i g u r e 14d shows that a crude e x t r a c t of whole b r a i n from 10-day-o l d r a t s a l s o contains two DNA polymerase a c t i v i t i e s i n s i m i l a r f r a c t i o n s to those of the p u r i f i e d Enzymes A and B. I t was t h e r e f o r e decided to i n v e s t i g a t e d i r e c t l y the p a t t e r n of changes of these two enzyme a c t i v i t i e s w i t h development using crude e x t r a c t s , thereby m i n i m i z i n g l o s s of a c t i v i t y during p u r i -f i c a t i o n procedures. When sucrose gradient c e n t r i f u g a t i o n under the same con-d i t i o n s but o m i t t i n g KCI or EDTA or both from the sucrose g r a d i e n t s was t r i e d , the p u r i f i e d polymerases A and B s e p a r a t e l y showed pro-f i l e s s i m i l a r to those obtained w i t h KCI and EDTA i n the medium. However, e i t h e r the combination of p u r i f i e d polymerases A and B or a crude e x t r a c t of 10-day-old r a t b r a i n ( F i g . 14e) showed o n l y one a s s o c i a t e d peak around 6-7 S. In order to exclude the p o s s i b i l i t y that these two a c t i v i t i e s are due to the same enzyme a s s o c i a t e d w i t h d i f f e r e n t i m p u r i t i e s , sucrose d e n s i t y g r a d i e n t c e n t r i f u g a t i o n was performed i n the presence of 0.05% and 0.1% (W/V) sodium dodecyl s u l f a t e (SDS). Although both a c t i v i t i e s i n the b r a i n e x t r a c t were s t r o n g l y i n h i b i -ted i n the presence of SDS, the p a t t e r n s i n the g r a d i e n t s were i d e n t i c a l to those obtained i n the absence of SDS. Recently, Chang and Bollum (49) have shown the presence of a low molecular weight DNA polymerase i n r a b b i t bone marrow, w i t h i t s S value around 3.3 S w i t h an a l k a l i n e pH optimum. This enzyme seems to be s i m i l a r to DNA polymerase B of r a t b r a i n . F i g . 14. Sucrose d e n s i t y gradient c e n t r i f u g a t i o n s t u d i e s on DNA polymerase of r a t b r a i n . 0.4 ml of p u r i f i e d enzyme pr e p a r a t i o n (approximately 0.5 - 1 mg of p r o t e i n ) or b r a i n e x t r a c t (approximately 3 mg of p r o t e i n ) was l a y e r e d over 5.0 ml of a 5-20% (W/V) l i n e a r sucrose g r a d i e n t . The sucrose s o l u t i o n was prepared i n 0.01 M phosphate b u f f e r (pH 7.4) c o n t a i n i n g 2 mM 3-mercaptoethanol, 0.1 M KC1 and 6 mM EDTA. A f t e r 15 h c e n t r i f u g a t i o n at 36,000 rpm at 0°C i n a SW r o t o r of the Spinco model L u l t r a -c e n t r i f u g e . A f t e r c e n t r i f u g a t i o n the bottom of the tube was punctured and 15 drop f r a c t i o n s were c o l l e c t e d (about 0.2 1 ml). The f r a c t i o n s were assayed f o r DNA polymerase a c t i v i t y (see Methods). Approximate S values were c a l c u l a t e d according to the procedure of M a r t i n and Ames ( 3 ) , using cytochrome c, bovine serum albumin and glucose oxidase as standards; (a) p u r i f i e d DNA polymerase A; (b) p u r i f i e d DNA polymerase B; (c) Enzyme A + Enzyme B; (d) 10-day-old whole b r a i n e x t r a c t ; (e) 10-day-old whole b r a i n e x t r a c t (without 0.1 M KC1 and 6 mM EDTA i n sucrose medium). A * » / \ \ I I vO ON B 5 10 15 20 25 5 10 15 2 0 25 5 10 15 2 0 25 5 10 15 20 25 5 10 15 20 25 J Fract ion n u m b e r - 97 -3.3b. Developmental changes i n DNA polymerases A and B of r a t  cerebellum E x t r a c t s of cerebellum from r a t s aged 2 days, 6 days, 10 days, 16 days and a d u l t were analyzed by sucrose gradient c e n t r i f u g a t i o n as shown i n F i g u r e 15. From t h i s f i g u r e , i t can be seen that Enzyme A i s very a c t i v e i n the c e r e b e l l a of younger r a t s , peaks at around the 6th day a f t e r b i r t h , then decreases r a p i d l y to very low a c t i v i t y i n a d u l t r a t c e r e b e l l a . DNA polymerase B i s l e s s a c t i v e than Enzyme A i n the younger r a t s . However, i t decreases l e s s r a p i d l y than Enzyme A w i t h age and shows a higher a c t i v i t y than Enzyme A i n a d u l t r a t cerebellum. 3.3c. P a t t e r n of developmental changes i n DNA polymerases A and  B of r a t c o r t e x F i g u r e 16 shows the p a t t e r n of DNA polymerases A and B of r a t c e r e b r a l cortex during development from newborn to a d u l t . This p a t t e r n i s d i f f e r e n t from that of the cerebellum ( F i g . 15) at a l l p o s t n a t a l stages s t u d i e d ( F i g . 16a to F i g . 16e) DNA p o l y -merase A i s l e s s a c t i v e than DNA polymerase B. The l a t t e r a c t i v i t y i s q u i t e high i n young r a t s and s l o w l y decreases during maturation. Adult r a t cortex s t i l l c ontains s i g n i f i c a n t amounts of DNA p o l y -merase B but almost no Enzyme A ( F i g . 16e). In f e t a l c o r t e x , however, DNA polymerase A shows extremely high a c t i v i t y compared to that of DNA polymerase B ( F i g . 17). A l l the r e s u l t s described n i O X c E \ -t—I c =3 o o 12 -8-4 -a b c d e i • .A - ft • 1 \ /I J \ / \ IxL—i i S—T 1 • * 1 • « • • \ / \ 1 1 h——^~ A A. I i i i "~~T1 A . l - r - r — r i ^ r t - u oo B 5 10 15 20 25 5 10 15 20 25 5 10 15 20 25 5 10 15 20 25 5 10 15 20 25 J Fraction number F i g . 15. Developmental changes i n DNA polymerases A and B of r a t b r a i n cerebellum. The p r o f i l e of DNA polymerases A and B was analyzed by sucrose gradient c e n t r i f u g a t i o n . Approximately 1.5 - 2 mg of p r o t e i n from c e r e b e l l a r e x t r a c t was used i n each experiment. A l l procedures were as mentioned i n Methods. (a) 2-day-old; (b) 6-day-old; (c) 10-day-old; (d) 17-day-old; (e) a d u l t . cn I O X c £ 4—1 c 3 o o 84 7 6 5 • 4 -3 •• 2-1-V B i 1 H - ^ = * \ I / v \ \ A A \ • A • V / \ A v O V O 5 10 15 20 25 5 10 15 20 25 5 10 15 20 25 5 10 15 20 25 5 10 15 20 25 T Fract ion number Fig. 16. Developmental changes in DNA polymerases A and B of the rat cerebral cortex. The profile of DNA polymerases A and B was analyzed by sucrose gradient centrifugation. Approximately 3 mg protein of cerebral extract was used in each experiment. A l l procedures were as mentioned in Methods, (a) 2-day-old; (b) 6-day-old; (c) 10-day-old; (d) 17-day-old; (e) adult. - 100 -F r a c t i o n n u m b e r F i g . 17. The sucrose g r a d i e n t c e n t r i f u g a t i o n p r o f i l e o f DNA polymerase A and B of f e t a l r a t c e r e b r a l c o r t e x (approximately 3 mg of p r o t e i n ) . G e s t a t i o n p e r i o d of the f e t a l r a t was about 2 weeks. A l l procedures were as mentioned i n Fi g u r e 13. - 101 -above are c o n s i s t e n t w i t h those recorded i n Table IV. That i s , there are two DNA polymerases, A which p r e f e r s heated DNA and B which p r e f e r s n a t i v e DNA. Only i n neonatal r a t b r a i n i s the a c t i v i t y of DNA polymerase A higher than that of DNA polymerase B. This p a t t e r n g r a d u a l l y changes during maturation. The a c t i v i t y r a t i o of Enzyme A/Enzyme B decreases w i t h i n c r e a s i n g age. I n the a d u l t b r a i n , Enzyme B i s higher than Enzyme A. The s h i f t i n the p a t t e r n i s due to the f a c t that DNA polymerase A disappears f a s t e r than DNA polymerase B. The cerebellum of the r a t grows r a p i d l y f o r 16 days a f t e r b i r t h and shows r a p i d c e l l p r o l i f e r a t i o n during t h i s p e r i o d and DNA c o n c e n t r a t i o n a l s o i n c r e a s e s up to 2 weeks (252,264). How-ever, i n the c e r e b r a l cortex of the r a t , c e l l d i v i s i o n i s almost complete at b i r t h and DNA c o n c e n t r a t i o n decreases during maturation and there i s very l i t t l e i f any a c t i v i t y f o r DNA s y n t h e s i s (320). From these data and the p a t t e r n of developmental changes i n DNA polymerases A and B which have been described above, i t seems probable that DNA polymerase A only shows h i g h a c t i v i t y i n a pro-l i f e r a t i n g stage and shows l i t t l e or no a c t i v i t y when m i t o s i s ceases. Ove et a l (242) have reported that only the DNA polymerase which p r e f e r s heated DNA as template increases markedly i n hepatoma. This a l s o suggests that i n d u c t i o n of p r o l i f e r a t i v e a c t i v i t y has i t s major e f f e c t on DNA polymerase A. R e c e n t l y , Chang and Bollum (49) - 102 -a l s o suggested that 6-8 S DNA polymerase from mammalian t i s s u e are r e s p o n s i b l e f o r p r o l i f e r a t i o n . The b i o l o g i c a l f u n c t i o n of DNA polymerase B i s s t i l l unknown, s i n c e s i g n i f i c a n t a c t i v i t y of DNA polymerase B i s found i n o l d e r r a t b r a i n . I t seems that t h i s enzyme may be concerned w i t h r e p a i r or some other f u n c t i o n such as gene a m p l i f i c a t i o n or d i f f e r e n t i a t i o n . 3.4. P a r t i c u l a t e Form of DNA Polymerase i n Rat B r a i n The r e s u l t s i n Table IV and those d e s c r i b e d i n S e c t i o n 3.3 show that there i s very l i t t l e DNA polymerase present i n a d u l t r a t b r a i n . Disappearance of DNA polymerases could be due to the d e s t r u c t i o n or i n a c t i v a t i o n of these enzymes. I t i s p o s s i b l e that i n a c t i v a t i o n of enzymes may be due to the contamination w i t h a high content of i n h i b i t o r s , e.g. DNase (303), thymidine hydrolase and phosphatase e t c . , i n a d u l t r a t b r a i n or due to l o s s of a c t i v a t i o n ( p r o t e i n f a c t o r s ) , e.g. unwindase e t c . A l t e r n a t i v e l y , DNA polymerases may simply bind to chromatin o r membrane to form the p a r t i c u l a t e enzyme, the a c t i v i t y of which may not be detected. Howk and Wang (50) have reported the presence of DNA polymerase a s s o c i a t e d w i t h the a c i d p r o t e i n f r a c t i o n of chromatin. Sev e r a l i n v e s t i g a t o r s have detected DNA polymerase a c t i v i t y i n membrane and membrane-chromatin complex from animal c e l l s (167-170). However, t h i s complex c a r r i e d out l i m i t e d r e p l i c a t i o n i n v i t r o . - 103 -In t h i s S e c t i o n , the s o l u b i l i z a t i o n and c h a r a c t e r i z a t i o n of the p a r t i c u l a t e form of DNA polymerase from a d u l t r a t b r a i n n u c l e i , i n t r a c e l l u l a r d i s t r i b u t i o n of DNA polymerases A and B i n developing r a t b r a i n and the p a t t e r n of the p a r t i c u l a t e forms of DNA p o l y -merases A and B i n r a t cerebellum w i l l be described. 3.4a. S o l u b i l i z a t i o n of the p a r t i c u l a t e form of DNA polymerase  from a d u l t r a t b r a i n n u c l e i Whole b r a i n n u c l e i from a d u l t r a t s of the Wistar s t r a i n (200-250 g) were prepared by the method of Mandel et a l (308). The pure n u c l e i were washed again w i t h 1 M sucrose i n 1 mM MgC^ and 10 mM potassium s u c c i n a t e and c e n t r i f u g e d at 10,000 x g f o r 30 min. The washed pure n u c l e i were homogenized i n 0.01 M T r i s -HCl b u f f e r , pH 7.4, c o n t a i n i n g 2 mM g-mercaptoethanol, and the homogenate was c e n t r i f u g e d . The supernatant was designated "nuclear e x t r a c t I " . The p e l l e t s ( i n s o l u b l e chromatin-membrane complex) were s e q u e n t i a l l y e x t r a c t e d w i t h 0.2 M, 0.5 M and 1 M phosphate b u f f e r , pH 7.4, c o n t a i n i n g 2 mM 3-mercaptoethanol. A f t e r c e n t r i f u g a t i o n , the supematants were d i a l y z e d a g a i n s t 0.01 M T r i s - H C l b u f f e r , pH 7.4, c o n t a i n i n g 2 mM g-mercaptoethanol, and designated " n u c l e a r e x t r a c t I I " , "nuclear e x t r a c t I I I " and " n u c l e a r e x t r a c t IV", r e s p e c t i v e l y . A f t e r s e q u e n t i a l e x t r a c t i o n w i t h phosphate b u f f e r , the p e l l e t s were designated " r e s i d u e " . - 104 -Table IX shows that the nuclear extract II fraction contained extremely high DNA polymerase activity. Its specific activity was 3 about 5 nmole of [ H]dCTP incorporated per mg protein per 30 min incubation, which is about 10 times higher than purified DNA polymerase B (Table V) and about 60 times higher than that of cytoplasmic DNA polymerase activity (see Table XIII). 3.4b. Characteristics of solubilized DNA polymerase from adult rat brain nuclei As shown in Table X, solubilized DNA polymerase required a 2+ complement of four deoxynucleoside triphosphates, Mg and DNA as template. N-Ethylmaleimide inhibited the activity of the enzyme by 55%. ATP showed no effect on the solubilized nuclear enzyme. However, both EDTA and spermidine stimulated the solu-bil i z e d enzyme by over 50%. As shown in Table XI, the solubilized nuclear enzyme, like DNA polymerase B purified from 10-day-old rat brain (Table VIII, required native DNA as template. The extent of incorporation varied to some degree depending on the source of DNA. 3 The incorporation of [ H]dCTP into DNA by solubilized nuclear enzyme was proportional to the enzyme concentration between 10 and 80 yg of protein (Fig. 18). The solubilized DNA polymerase also showed sigmoidal slope in the enzyme concentration curve as exhibited by Enzyme B. - 105 -Table IX. S o l u b i l i z a t i o n of DNA polymerase from a d u l t r a t b r a i n n u c l e i . „ . c- • /o of t o t a l „ . S p e c i f i c a c t i v i t y , F r a c t i o n s , / . . nuclear mumole/mg p r o t e i n . . r a c t i v i t y Nuclear e x t r a c t I (0.01 M T r i s ) 0.15 3.9 Nuclear e x t r a c t I I (0.2 M phosphate) 4.82 93.2 Nuclear e x t r a c t I I I (0.5 M phosphate) 0.14 0.9 Nuclear e x t r a c t IV (1.0 M phosphate) 0.02 0.1 Residue 0.06 0.9 [ H]dCTP was used as l a b e l l e d p r e c u r s o r . - 106 -Table X. The e f f e c t s of v a r i o u s f a c t o r s upon s o l u b i l i z e d DNA polymerase a c t i v i t y System enzyme a c t i v i t y (counts/min) % of a c t i v i t y Completed 6800 100 - DNA 184 3 2 + - Mg 157 3 - d i t h i o t h r e i t o l 4343 64 - d i t h i o t h r e i t o l + NEM 3749 55 - dNTP 2934 43 + 0.3 mM ATP 6857 101 + 0.5 mM Spd 12837 187 + 3 mM EDTA 10366 151 [ H]dCTP was used as l a b e l l e d precursor - 107 -Table X I . Nature of template and priming a b i l i t y f o r s o l u b i l i z e d nuclear DNA polymerase „ ^ c Template sources Nature of _ Template £^ c Q l i D N A phage DNA C a l f thymus DNA cpm cpm cpm Native-DNA 3068 1070 5041 Heated-DNA 123 106 227 H]dCTP was used as l a b e l l e d p recursor - 108 -Protein cone, (ug ) F i g . 18. Enzyme co n c e n t r a t i o n curve f o r s o l u b i l i z e d n u c l e a r DNA polymerase. The a c t i v i t y of DNA polymerase was assayed i n the r e a c t i o n mixture as described i n Methods except dTTP was used as l a b e l l e d precursor and n a t i v e c a l f thymus DNA was used as template. Enzyme (2-175 pg of p r o t e i n ) was added to each i n c u b a t i o n mixture as i n d i -cated. - 109 -Sucrose density gradient centrifugation was also studied on solubilized nuclear DNA polymerase using purified DNA poly-merases A and B as standards. Analytical results of sucrose density gradient centrifugation for a l l enzyme preparations are shown in Figure 19. DNA polymerase A sedimented around 9 S. DNA polymerase B and solubilized nuclear DNA polymerase sedi-mented coincidently around 3-4 S. A l l these data described above suggest that the enzyme which was solubilized from nuclear chromatin-membrane complex is similar to DNA polymerase B. They also suggest that DNA polymerase B may be localized in nuclear chromatin-membrane complex i n a particulate form in the adult animal, but in younger animals i t may exist in both soluble and particulate forms. 3.4c. Pattern of particulate and soluble forms of DNA polymerases  A and B in rat brain In order to test this po s s i b i l i t y , the amount of soluble and particulate forms of DNA polymerases of rat cerebellum during maturation was studied (Fig. 20). In this experiment, cerebella of rat brains of different ages were homogenized with 5 volumes of 0.01 M Tris-HCl buffer, pH 7.4, containing 2 mM g-mercaptoethanol or with 5 volumes of 0.2 M phosphate buffer, pH 7.4, containing 2 mM g-mercaptoethanol, followed by centri-- 110 -9-6 3 8S \ A i i i i 7 9 o c *E CO c o O 6 3 4 3 S 2 1 S • / \ \ I l 9-6 3-- — .._ — * 1 ' ' 15 20 5 10 Fraction no. 25 Fig. 19. Sucrose density gradient centrifugation profile of solubilized nuclear DNA polymerase. Each sample of DNA polymerase A (a), B (b) and solubilized nuclear enzyme (c) was analyzed by sucrose density gradient centrifugation as described in Methods. - I l l -fugation at 34,800 x g for 1 h at 0°C. The supernatant fractions were dialyzed against 0.01 M Tris-HCl buffer, pH 7.4 - 2 mM 0-mercaptoethanol, and then loaded onto 5-20% sucrose linear gradients. Here, the Tris-buffer extract contains only the soluble form of DNA polymerase, while the phosphate buffer extract contains both soluble and particulate DNA polymerases. The difference between the act i v i t i e s in the two extracts therefore represents the par-ticulate enzyme activity. Figure 20 shows clearly that DNA polymerase A is present only in the soluble form at a l l ages and i s very active in younger rats but shows no activity in the adult. However, DNA polymerase B occurs as 45% soluble form and 55% particulate form in 10-day-old rats, 22% soluble form and 78% particulate form i n 17-day-old rats and only 7% soluble form and 93% particulate form in adult rat cerebellum. These results show clearly that adult rat brain con-tains a high amount of DNA polymerase present in a particulate form which has not been detected previously. Independently, Murthy and Bharucha (257) have just reported that they also found a particulate form of DNA polymerase present in new born baby rat brain, and the ratio of the soluble form of DNA polymerase to the particulate form was 0.73. These data agreed with our results. More recently, Long and Garren (321) also ob-served particulate DNA polymerase in beef adrenal nuclei. However, both these two groups described the finding of DNA polymerase - 112 -F i g . 20. Developmental changes i n p a r t i c u l a t e and s o l u b l e forms of DNA polymerases i n r a t cerebellum. The p r o f i l e of DNA polymerases was analyzed by sucrose g r a d i e n t c e n t r i -f u g a t i o n . Approximately 1.5 - 2 mg of p r o t e i n of cere-b e l l a r e x t r a c t was used i n each experiment. (a) 2-day-o l d ; (b) 10-day-old; (c) 17-day-old; (d) a d u l t . • • 0.01 M T r i s - H C l b u f f e r e x t r a c t • 0.2 M phosphate b u f f e r e x t r a c t - 113 -activity in broken nuclear pellets or suspension and they also have not yet characterized this particulate DNA polymerase. 3.4d. Intracellular distribution of DNA polymerase A and B  in rat brain The data given in Figure 20 clearly show that total DNA polymerase B (soluble form plus particulate form) concentration at different ages was almost similar. However, the difference is only in the proportion of the soluble form and particulate form. It is possible that in younger rat brain, the soluble form of DNA polymerase B present in either nucleoplasm or cyto-plasm and then shifts to nuclei and binds to chromatin. There-fore, i t would be very interesting to study the intracellular distribution of DNA polymerases in rat brain at different ages. 10-day-old and adult rat brain were used in this experiment. Subcellular fractionation of whole brain was performed as des-cribed i n Methods. Washed nucelar, mitochondrial and microsomal fractions were extracted with 0.01 M Tris-HCl buffer, pH 7.4, containing 2 mM 3-mercaptoethanol, followed by centrifugation at 10,000 x g for 30 min. The pellets were then extracted with 0.2 M phosphate buffer, pH 7.4, containing 2 mM B-mercaptoethanol. Both Tris-buffer extracts and phosphate buffer extracts were dialyzed before assaying the DNA polymerase a c t i v i t i e s . - 114 -As shown i n Table X I I , 10-day-old r a t b r a i n contains 95% of the t o t a l enzyme a c t i v i t y i n the cytoplasmic f r a c t i o n and 41% i n the nuclear f r a c t i o n . However, t h i s d i s t r i b u t i o n i n a d u l t r a t b r a i n (Table X I I I ) was j u s t reversed. I t contains only 21% i n the cytoplasmic and 79% i n the nuclear f r a c t i o n . I t i s a l s o i n t e r e s t i n g to see that the major DNA polymerase a c t i v i t y i n a d u l t r a t b r a i n i s l o c a l i z e d i n the n u c l e a r f r a c t i o n as p a r t i c u l a t e form. However, i n 10-day-old r a t b r a i n , the major DNA polymerase ( i n c l u d i n g A + B) i s present as s o l u b l e form and i s l o c a l i z e d i n the cytoplasmic f r a c t i o n . The DNA polymerase a c t i v i t y i n 10-day-old r a t b r a i n m i t o -chondria i s very h i g h , but there i s l i t t l e a c t i v i t y i n a d u l t mito-chondria. The a c t i v i t i e s of DNA polymerase i n 10-day-old r a t b r a i n mitochondria are about 9% of the t o t a l a c t i v i t y . DNA p o l y -merase (s) i s o l a t e d from 10-day-old r a t b r a i n mitochondria were a l s o s t u d i e d on sucrose d e n s i t y g r a d i e n t c e n t r i f u g a t i o n as compared to p u r i f i e d DNA polymerases A and B. The r e s u l t s are shown i n Figure 21. S u r p r i s i n g l y , the major DNA polymerase i n mitochondria was d i f f e r e n t from p u r i f i e d DNA polymerase A on sucrose g r a d i e n t c e n t r i f u g a t i o n p r o f i l e . I t s h i f t e d about 4 f r a c t i o n tubes towards the top. Recently, Meyer and Simpson (20,234,235) reporte d t h a t mitochondria DNA polymerase i s d i f f e r e n t from n u c l e a r DNA polymerase by means of p r e c i p i t a t i o n at d i f f e r e n t concentrations of ammonium s u l f a t e , e l u t i o n from DEAE-cellulose column at d i f f e r e n t s a l t con-- 115 -Table XII. Intracellular distribution of DNA polymerases in 10-day-old rat brain Fractions Specific activity (mumole/mg) Total * activity (mpmole) % of total activity Cytoplasmic fraction 1) Supernatant 0.24 2) Microsome (a) Tris extract 0.19 (b) Phosphate extract 1.14 3) Mitochondria (a) Tris extract 0.14 (b) Phosphate extract 0.53 35.05 23.12 1.94 4.33 2.27 3.39 59 II. Nuclear fraction 24.88 41 (a) Tris extract 0.90 3.16 (b) Phosphate extract 5.30 21.72 * 21.8 gm of whole brains of 24 rats aged 10 days were processed by subcellular fractionation as described i n Methods. - 116 -Table XIII. Intracellular distribution of DNA polymerase in adult rat brain. Fractions Specific a c t i v i t y (rapmole/mg) Total * a c t i v i t y (mymole) % of total activity Cytoplasmic fraction 1) Supernatant 0.08 2) Microsome (a) Tris extract 0.11 (b) Phosphate extract 0.24 3) Mitochondria (a) Tris extract 0.04 (b) Phosphate extract 0.06 3.01 2.33 0.2 0.35 0.08 0.05 21 II. Nuclear fraction 11.21 79 (a) Tris extract 0.15 0.45 (b) Phosphate extract 4.82 10.76 6.8 gm of whole brains of 4 adult rats were processed by subcellular fractionation as described in Methods. - 117 -9--6 -3 - / \ 7 9-o 6-£ CO ° c o O 2-- c A / \ / \ 10 15 20 Fraction number 25 F i g . 21. Sucrose d e n s i t y g r a d i e n t c e n t r i f u g a t i o n p r o f i l e of DNA polymerases from 10-day-old r a t b r a i n mitochondria, (a) p u r i f i e d DNA polymerase A; (b) p u r i f i e d DNA polymerase B; (c) m i t o c h o n d r i a l DNA polymerases. - 118 -concentrations and op t i m a l f u n c t i o n at d i f f e r e n t s a l t c o n c e n t r a t i o n s . However, preference of template f o r mitochondria DNA polymerase i s c o n t r a d i c t o r y . Meyer and Simpson re p o r t e d that mitochondria DNA polymerase p r e f e r s heated DNA as template (235), but K a l f and Chih reported that i t p r e f e r s n a t i v e DNA as template (233). The T r i s - b u f f e r e x t r a c t and phosphate b u f f e r e x t r a c t of nu c l e a r DNA polymerases were a l s o s t u d i e d on sucrose-density gradient c e n t r i f u g a t i o n . Here, T r i s - b u f f e r e x t r a c t contains a nucleoplasmic s o l u b l e form of DNA polymerase, and phosphate b u f f e r e x t r a c t contains only the n u c l e a r p a r t i c u l a t e form of DNA p o l y -merase (s i n c e n u c l e i were e x t r a c t e d w i t h 0.01 M T r i s - b u f f e r f i r s t to remove the s o l u b l e nucleoplasmic enzyme and then e x t r a c t e d w i t h phosphate b u f f e r ) . I t i s very i n t e r e s t i n g to see that the major p a r t of the s o l u b l e form of DNA polymerase i n n u c l e i contains mostly DNA polymerase A w i t h low amounts of DNA polymerase B, w h i l e the p a r t i c u l a t e form of DNA polymerase i n n u c l e i i s s i m i l a r to DNA polymerase B as shown i n Fi g u r e 22. 3.5. P r o t e i n Factors of DNA Polymerase B i n Developing Rat B r a i n I n order to study the enzymatic r e g u l a t i o n mechanism and f u n c t i o n of DNA polymerase B, f u r t h e r p u r i f i c a t i o n s of DNA p o l y -merase B e i t h e r by column chromatography of Sephadex G-200, phospho-c e l l u l o s e or hydroxyapetite were performed. However, an unexplained F i g . 22. Sucrose d e n s i t y g r a d i e n t c e n t r i f u g a t i o n of DNA polymerase from r a t b r a i n . (a) T r i s - b u f f e r e x t r a c t of 10-day-old r a t b r a i n n u c l e i ; (b) phosphate-buffer e x t r a c t of 10-day-old r a t b r a i n n u c l e i ; (c) T r i s - b u f f e r e x t r a c t of a d u l t r a t b r a i n n u c l e i ; (d) phosphate-buffer e x t r a c t of a d u l t r a t b r a i n n u c l e i . 10 8 6 4 2 10 8 6 4 ? 2 o x 110 co 8 » § 6 o O 4 2 10 8 6 4 2 a - 119 -Fraction number - 120 -l o s s of enzymic a c t i v i t y has o f t e n been encountered during f u r t h e r p u r i f i c a t i o n . Subsequently, i t was found that the l o s s of a c t i v i t y was manifested only at a l a t e r stage of the p u r i f i c a t i o n procedure. Table XIV shows a t y p i c a l p a t t e r n of s p e c i f i c a c t i v i t y of r a t b r a i n DNA polymerase B at d i f f e r e n t stages of p u r i f i c a t i o n . The s p e c i f i c a c t i v i t y was increased from 0.52 i n F r a c t i o n I to 3 18 pmoles [ H]dTMP/mg protein/min i n F r a c t i o n I I I . F u rther p u r i f i c a t i o n r e s u l t e d i n a p r e c i p i t o u s l o s s of a c t i v i t y ( F r a c t i o n I V ) . Pronounced s t i m u l a t i o n was observed on the a d d i t i o n of f r a c t i o n e l u t e d from Sephadex G-200. I t was concluded that some f a c t o r ( s ) of DNA polymerase were removed from the enzyme during the course of p u r i f i c a t i o n . 3.5a. Separation of DNA polymerase B i n t o a c t i v a t o r and p a r t i a l l y  i n a c t i v e enzyme Fig u r e 23 shows the e l u t i o n p a t t e r n of Enzyme B from a Sephadex G-200 column and DNA polymerase B a c t i v i t y i n i t s stimu-l a t e d and non-stimulated s t a t e . The a c t i v i t y of DNA polymerase B was p a r t i a l l y i n a c t i v a t e d a f t e r passing through the column. However, the combination of tubes 10 and 12 r e s t o r e d the a c t i v i t y of DNA polymerase. Presumably tube 12 contained an a c t i v a t i n g f a c t o r ( s ) and i t s p r o p e r t i e s were c h a r a c t e r i z e d . - 121 -Table XIV. P a r t i a l l o s s of DNA polymerase B a c t i v i t y d u r i n g p u r i f i c a t i o n . S p e c i f i c a c t i v i t y F r a c t i o n s . , R - J , J M W „ / ^ • / • s (pmoles L HJ dTMP/mg protein/nun) I . Crude e x t r a c t 0.52 I I . (NH.) SO F r a c t i o n a t i o n 5.27 (0.45-0.7 s a t u r a t i o n ) I I I . DEAE-cellulose chromatography 18.0 IV. Sephadex G-200 f i l t r a t i o n " 0.21 - 122 -Fraction number F i g . 23 Column chromatography of DNA polymerase B on Sephadex G-200 Absorbancy at 280 nm P a r t i a l i n a c t i v a t e d DNA polymerase a c t i v i t y - - - A c t i v a t e d DNA polymerase a c t i v i t y - 123 -3.5b. Nature of s t i m u l a t i n g f a c t o r ( s ) The a c t i v i t y of DNA polymerase B was assayed w i t h or without a c t i v a t o r o r t r e a t e d a c t i v a t o r as shown i n Table XV. The a c t i -v a t o r was exposed to the a c t i o n of t r y p s i n , before assaying f o r s t i m u l a t o r y a c t i v i t y (Table XV). The untreated a c t i v a t o r i n -creased DNA polymerase B a c t i v i t y 3 - f o l d . A f t e r treatment w i t h t r y p s i n f o l l o w e d by a t r y p s i n i n h i b i t o r , however, the s t i m u l a t o r y a c t i v i t y was l o s t . C o n t r o l s c o n t a i n i n g (1) a c t i v a t o r p r e t r e a t e d w i t h t r y p s i n f o l l o w e d by t r y p s i n i n h i b i t o r and (2) untreated a c t i v a t o r plus t r y p s i n plus t r y p s i n i n h i b i t o r , were each i n c u -bated w i t h DNA polymerase to measure t h e i r e f f e c t s on the assay system. This experiment proved t h a t t h i s a c t i v a t o r i s a p r o t e i n and not a n u c l e i c a c i d . The a c t i v a t o r i s h e a t - l a b i l e . Thompson and McCarthy (267) reporte d that the a d d i t i o n of a s c i t e s c e l l cytoplasm to mouse l i v e r n u c l e i caused a 15-20 f o l d i n c r e a s e i n the r a t e of DNA s y n t h e s i s , and the a d d i t i o n of L - c e l l cytoplasm to the hen e r y t h r o c y t e n u c l e i caused an i n c r e a s e of approximately 1 0 - f o l d i n the t o t a l i n c o r p o r a t i o n . They suggested that a s t i m u l a t o r of DNA s y n t h e s i s was present i n the cytoplasmic f r a c t i o n . However, t h e i r s t i m u l a t o r s are not s i m i l a r to the a c t i v a t o r described here, s i n c e t h e i r f a c t o r s are heat-stable. 3.5c. Nuclease a c t i v i t y of the f a c t o r ( s ) The p o s s i b i l i t y of the f a c t o r s being nucleases was s t u d i e d . - 124 -Table XV. Nature of a c t i v a t o r of DNA polymerase B Treatment + A c t i v a t o r ( t r e a t e d w i t h t r y p s i n then t r y p s i n i n h i b i t o r added) + A c t i v a t o r ( t r e a t e d w i t h t r y p s i n and t r y p s i n i n h i b i t o r ) ,o A c t i v i t y cpm None 154 + A c t i v a t o r 662 218 604 + A c t i v a t o r + t r y p s i n + t r y p s i n i n h i b i t o r 631 + A c t i v a t o r (heated at 100 C f o r 10 min) 165 The a c t i v i t y of DNA polymerase B obtained from the Sephadex G-200 column was assayed w i t h an a c t i v a t o r or t r e a t e d a c t i v a t o r . T r y p s i n 25 pg and t r y p s i n i n h i b i t o r 5 0 u g were used i n t h i s experiment. - 125 -60 pg p r o t e i n f a c t o r was incubated w i t h a s c i t e s [ C]DNA at 37°C f o r 1 h. The r a d i o a c t i v i t y of TCA p r e c i p i t a t e and supernatant was measured. The r e s u l t s are shown i n Table XVI. Table XVI shows c l e a r l y that the f a c t o r i s not a nuclease. However, i t s t i l l cannot be r u l e d out that t h i s f a c t o r i s a s p e c i f i c endo-nuclease or n i c k a s e . In order to detect the a c t i v i t y of a s p e c i f i c endonuclease or nickase which makes a n i c k on DNA f o r DNA p o l y -merase, a l k a l i n e sucrose d e n s i t y g r a d i e n t c e n t r i f u g a t i o n was 14 c a r r i e d out. A s c i t e s [ C]DNA was incubated w i t h the f a c t o r at 37°C f o r 1 h, 2 h and 4 h r e s p e c t i v e l y . A f t e r i n c u b a t i o n , the r e a c t i o n mixture was l a y e r e d over 5-20% a l k a l i n e sucrose g r a d i e n t and c e n t r i f u g e d a t 36,000 rpm f o r 16 h at 0°C i n a Spinco model L u l t r a c e n t r i f u g e . A n a l y t i c a l r e s u l t s of c e n t r i f u g a t i o n were shown 14 i n F i g u r e 24. A l l a s c i t e s [ C]DNA p r e t r e a t e d w i t h f a c t o r at v a r i o u s times of i n c u b a t i o n showed a s h i f t of only a tube f r a c t i o n from c o n t r o l towards the top. This suggests that the f a c t o r i s not a n i c k a s e . The one tube s h i f t from the c o n t r o l experiment may be due to contamination by a very s m a l l amount of endonuclease. The f a c t o r was pre-incubated w i t h DNA f o r 0, 7 and 15 min r e s p e c t i v e l y before assaying the DNA polymerase a c t i v i t y by adding Enzyme B and then i n c u b a t i n g at 37°C f o r another 30 min. The r e s u l t s are shown i n Table XVII. Table XVII shows c l e a r l y that there i s no d i f f e r e n c e i n a c t i v i t y w i t h v a r i o u s p r e - i n c u b a t i o n s . These a c t i v i t i e s were independent of the p r e - i n c u b a t i o n time, - 126 -Table XVI. Nuclease a c t i v i t y of F a c t o r . R a d i o a c t i v i t y (cpm) Supernatant P r e c i p i t a t e 34 56 9050 8967 8978 8035 9234 9163 474 436 440 1295 Sample Experiments Factor (60 yg) 5 yg DNase 2 yg DNase a b a b 1 yg DNase A f t e r Factor or DNase was incubated w i t h 20 ymole T r i s - H C l b u f f e r , 14 0.15 mg a s c i t e s C-DNA, 2 ymole MgC^ and 2 ymole d i t h i o t h r e i t o l a t 37°C f o r 1 h r , 10% TCA was added and the mixture was c e n t r i f u g e d . The r a d i o a c t i v i t y of supernatant and p r e c i p i t a t e was measured. - 1 2 7 -F i g . 2 4 . A l k a l i n e sucrose gr a d i e n t c e n t r i f u g a t i o n p r o f i l e of n a t i v e - ^ C - a s c i t e s DNA a f t e r i n c u b a t i o n f o r 0 , 1 , 2 and 4 h r s . i n the presence of the p r o t e i n f a c t o r . A l l procedures as described i n Methods. • • Zero time i n c u b a t i o n f o r c o n t r o l A - - - A 1 h i n c u b a t i o n A - - - A 2 h i n c u b a t i o n o - o 4 h i n c u b a t i o n - 128 -Table XVTI. Priming ac t i v i t y of DNA with or without preincubation with Factor. Preincubation time (min) Activity (cpm) 0 1219 7 1189 15 1242 Factor preincubated w i t h DNA i n r e a c t i o n mixture, except without adding enzyme B, f o r 0,7 and 15 minutes r e s p e c t i v e l y . A f t e r p r e i n c u b a t i o n , enzyme B was added to the r e a c t i o n mixture and incubated a t 37°C f o r another 30 min. - 129 -i n d i c a t i n g that a c t i v a t i o n was not a c a t a l y t i c process. This suggests a l s o that the f a c t o r i s not a nickase s i n c e i f the f a c t o r were a nickase i t would be expected that d i f f e r e n t pre-i n c u b a t i o n times would r e s u l t i n v a r i o u s degrees of breakage of the template which would have increased v a r i o u s degrees of template a c t i v i t y i n the DNA polymerase assay system. 3.5d. Time course of s t i m u l a t i o n 3 I t has been shown th a t l i n e a r i n c o r p o r a t i o n of the ( H]dTTP i n t o DNA c a t a l y z e d by DNA polymerase B f o r a p e r i o d of 2 h was obtained as desc r i b e d i n F i g u r e 12. However, the i n c o r p o r a t i o n c a t a l y z e d by f u r t h e r p u r i f i e d Enzyme B (Sephadex G-200 f r a c t i o n ) l e v e l l e d o f f at 10 min i n c u b a t i o n . The a d d i t i o n of 25 pg of p r o t e i n f a c t o r i n t o the r e a c t i o n mixture c o n t a i n i n g 50 ug p r o t e i n of Sephadex-fraction of Enzyme B caused a marked s t i m u l a t i o n and l i n e a r i n c o r p o r a t i o n as shown i n Fi g u r e 25. No d e t e c t a b l e l a g was observed a f t e r the a d d i t i o n of the f a c t o r . F i g u r e 25 a l s o shows that the f a c t o r s t i m u l a t e s i n c o r p o r a t i o n at i n i t i a l time. This a l s o suggests that the mechanism of s t i m u l a t i o n i s not a c a t a l y t i c process. 3.5e. The e f f e c t of f a c t o r on DNA polymerases A and B w i t h v a r i o u s  DNAs as template The f a c t o r was added to the r e a c t i o n mixture of Enzyme A w i t h v a r i o u s kinds of DNA as template. The r e s u l t s as shown i n - 130 -12-10-8-x c 6 £ c o 4-> c g 4 O / / / / / / / / / / / / / / / 7 1' 20 30 T i m e (min.) 40 F i g . 25. K i n e t i c s t u d i e s on s t i m u l a t i o n of DNA polymerase B by f a c t o r . 25 yg of f a c t o r was added to the r e a c t i o n mixture at 10 and 20 min r e s p e c t i v e l y a f t e r i n c u b a t i o n commenced. Arrows i n d i c a t e the time of a d d i t i o n of f a c t o r . o without f a c t o r k A w i t h f a c t o r - 131 -Table XVIII c l e a r l y show that no matter what the source of DNA used as template, the f a c t o r cannot s t i m u l a t e DNA polymerase A a c t i v i t y . However, DNA polymerase B was s t i m u l a t e d by the f a c t o r w i t h a l l the DNA templates which were t e s t e d . These r e s u l t s i n d i c a t e that the f a c t o r i s a s t i m u l a t o r s p e c i f i c f o r DNA polymerase B, but not f o r DNA polymerase A. Table V I I I shows that DNA polymerase B i s more a c t i v e w i t h DNA of high (G + C) content as template. In accordance w i t h t h i s r e s u l t , the percentage o f s t i m u l a t i o n of DNA polymerase B a c t i v i t y by f a c t o r i s higher w i t h DNA of high G + C content as template. The p r o p e r t i e s of p r o t e i n f a c t o r described here, are very s i m i l a r to "the wedge" described by Erhan et a l (277). The wedge i s a s m a l l molecular weight f a c t o r from a s c i t i c f l u i d and stimu-l a t e s DNA r e p l i c a t i o n . This f a c t o r only s t i m u l a t e s DNA p o l y -merase a c t i v i t y w i t h double stranded DNA as template; i t shows no e f f e c t on DNA s y n t h e s i s w i t h s i n g l e stranded DNA as template. Presumably the wedge i s an unwindase; however, the p r o t e i n i s o l a t e d from r a t b r a i n described i n t h i s S e c t i o n seems not to be an unwindase because i t cannot s t i m u l a t e DNA polymerase A a c t i v i t y w i t h n a t i v e DNA as template. Since t h i s p r o t e i n a l s o cannot s t i m u l a t e DNA polymerase B a c t i v i t y w i t h s i n g l e stranded DNA as template, i t i s suggested that t h i s p r o t e i n f a c t o r i s s p e c i f i c f o r DNA polymerase B but i t s a c t i o n mechanism i s s t i l l unknown. - 132 -Table XVTII. Factor e f f e c t on Enzymes A and B w i t h v a r i o u s DNA as template. DNA Template Enzyme C a l f thymus Salmon sperm M.Lysodeikticus (1) (2) (1) (2) (1) W cpm cpm cpm cpm cpm cpm A 263 227 375 415 191 125 B 556 980 411 1014 286 1237 The a c t i v i t i e s of enzymes A and B were assayed as mentioned i n Methods, u s i n g v a r i o u s DNAs as template, i n the absence (1) or presence (2) of the a d d i t i o n of Factor i n the r e a c t i o n mixture. - 133 -3.6. The E f f e c t of Polyamines on DNA Polymerase A c t i v i t y Although there are many r e p o r t s d e a l i n g w i t h the c o r r e l a t i o n between the concentrations of polyamines and n u c l e i c a c i d s y n t h e s i s (281-283), l i t t l e i s known about the e f f e c t of polyamines on DNA polymerase as there i s i n s u f f i c i e n t evidence to suggest a mechanism by which polyamines act on DNA polymerase. The experimental r e s u l t s d iscussed i n t h i s S e c t i o n concern the e f f e c t of polyamines on DNA polymerase a c t i v i t y and the mechanisms of t h e i r a c t i o n . 3.6a. The e f f e c t of polyamines on DNA polymerase of r a t b r a i n  e x t r a c t In t h i s experiment, 10-day-old whole b r a i n e x t r a c t was used as the enzyme source. Spermine, spermidine and p u t r e s c i n e , when added to the complete assay system, enhance DNA polymerase a c t i v i t y by more than 50% (Table XIX). The c o n c e n t r a t i o n of polyamines r e q u i r e d to produce t h i s enhancement i s i n the range of 0.5 to 1.0 mM spermidine, 0.05 to 0.1 mM spermine, and 2 to 3 mM p u t r e -s c i n e . A higher c o n c e n t r a t i o n of polyamines s t r o n g l y i n h i b i t e d DNA polymerase a c t i v i t y . The a c t i v a t i o n and i n h i b i t i o n of DNA polymerase a c t i v i t y by spermidine was seen from the i n i t i a l time of the i n c u b a t i o n as shown i n F i g u r e 26. Of the polyamines tes t e d the order of s t i m u l a t i o n of DNA polymerase B a c t i v i t y was spermine > spermidine > p u t r e s c i n e . That i s , spermine which contains U n i t r o g e n atoms s t i m u l a t e s DNA Table XIX. The e f f e c t of polyamines on DNA polymerase of r a t b r a i n e x t r a c t . Spermidine „. Spermine „ Putrescine added(mM) ° p m /o added (mM) C p m added (mM) C p m 0 3750 100 0 4305 100 0 3622 100 0.5 6375 170 0.05 6113 142 1.0 4238 117 1.0 6710 179 0.075 6199 144 2.5 5216 144 3.0 3375 90 0.10 6686 153 3.8 4926 136 5.0 2512 67 0.25 4110 93 5.0 4129 114 - 135 -T i m e ( m i n ) F i g . 26. The time-course of the a c t i o n of spermidine on DNA p o l y -merase i n 10-day-old r a t b r a i n e x t r a c t . • • C o n t r o l (without a d d i t i o n of spermidine) A — . — A 1 mM of spermidine added to r e a c t i o n mixture A - - - A 5 mM of spermidine added to r e a c t i o n mixture - 136 -polymerase B a c t i v i t y more e f f e c t i v e l y than spermidine which con-t a i n s 3 n i t r o g e n atoms which i n turn i s more e f f e c t i v e than pu-t r e s c i n e which contains only 2 n i t r o g e n atoms. This o b s e r v a t i o n suggests that polyamines which c o n t a i n more n i t r o g e n atoms are more e f f e c t i v e on DNA polymerase a c t i v i t y . The order of stimu-l a t i o n i s a l s o p a r a l l e l l e d by the order of molecular s i z e of the polyamines. 3.6b. The e f f e c t of spermidine on p a r t i a l l y p u r i f i e d DNA p o l y - merases A and B Brewer and Rusch (322) have reported s t i m u l a t i o n by spermine on DNA polymerase i n i s o l a t e d n u c l e i of Physarum polycephalum s u p p l i e d w i t h exogenous source of DNA and deoxynucleoside t r i -phosphates. This f i n d i n g i s s i m i l a r to the r e s u l t obtained i n the present study using c e l l - f r e e e x t r a c t as d e s c r i b e d above (previous S e c t i o n s ) . However, O'Brien, O l e n i c k and Hahn r e p o r t e d an i n h i b i t i o n by spermine of the a c t i v i t y of p a r t i a l l y p u r i f i e d DNA polymerase i n the presence of n a t i v e DNA primer. Here, the e f f e c t o f spermidine on p a r t i a l l y p u r i f i e d DNA polymerases A and B separated from 10-day-old r a t b r a i n w i l l be r e p o r t e d . . Optimal co n c e n t r a t i o n s of polyamines, 1 mM of spermidine, 0.1 mM of spermine and 3 mM of p u t r e s c i n e were added i n each assay system of DNA polymerases A and B. As shown i n Table XX, polyamines h i g h l y s t i m u l a t e d the a c t i v i t y of Enzyme B, but had - 1 3 7 -Table XX. The e f f e c t of polyamines on DNA polymerases A and B (mM) (counts/min) % (counts/mm) % — 1 4 0 5 1 0 0 1 2 2 4 1 0 0 Spermidine 1 1 3 2 6 9 4 2 2 6 3 1 8 5 Spermine 0 . 1 1 5 5 0 1 1 0 2 2 0 3 1 8 0 P u t r e s c i n e 3 1 4 3 1 1 0 2 1 7 1 3 1 4 0 - 138 -no e f f e c t on Enzyme A. This r e s u l t i s d i f f e r e n t from the r e s u l t s reported by O'Brien et_ al_ (323). However, s i n c e they used E. c o l i DNA polymerase as the enzyme source, t h i s response may be species s p e c i f i c . Another p o s s i b i l i t y i s that they f r a c t i o n a t e d E_. c o l i DNA polymerase w i t h ammonium s u l f a t e between 30-60% s a t u r a t e d , and t h i s f r a c t i o n probably shows some p r o p e r t i e s s i m i l a r to Enzyme A. O'Brien e_t al_ a l s o showed that spermine at a low c o n c e n t r a t i o n (0.1 mM) s l i g h t l y s t i m u l a t e d E_. c o l i DNA polymerase and higher concentrations of spermine s t r o n g l y i n h i b i t e d the enzyme which i s s i m i l a r to the behavior of p a r t i a l l y p u r i f i e d DNA polymerase A. 3.6c. The e f f e c t of spermidine depends on the template By studying the e f f e c t of spermine on the Tm of DNA, Mandel (324) found that the extent of the i n c r e a s e i n Tm was found to be a f u n c t i o n of the adenine-thymine content of the DNA sample. L i q u o r i e_t a_l s t u d i e d the complexes between DNA and polyamines and b u i l t a molecular model d e r i v e d on the b a s i s of the r e s u l t s obtained from the X-ray a n a l y s i s of the c r y s t a l s t r u c t u r e of spermine h y d r o c h l o r i d e . They a l s o concluded t h a t i n aqueous s o l u t i o n polyamines are very l i k e l y to be concerntrated w i t h i n regions r i c h i n A-T p a i r s (327). - 139 -The v a r i o u s templates of DNA, ranging i n base composition from 50-100% o f A + T content were used as template i n the r e -a c t i o n of DNA polymerase w i t h v a r i o u s amounts of spermidine. The r e s u l t s , as shown i n Fi g u r e 27, suggest that spermidine s t i m u l a t i o n of DNA polymerase a c t i v i t y depends on the DNA tem-p l a t e used. Using T^ phage DNA as template, the a c t i v i t y of DNA polymerase was s t r o n g l y i n h i b i t e d by spermidine. This e f f e c t could be due to the f a c t that T^ phage DNA contains g l u c o s y l a t e d hydroxymethyldeoxycytidine or 5-hydroxymethyldeoxycytidine ra t h e r than c y t o s i n e (325,326) and t h i s group shows a s p e c i f i c s t e r e o - s t r u c t u r e d i f f e r e n t from E_. c o l i and c a l f thymus DNA. Po l y d(A-T) which contains 100% A + T, showed l e s s a c t i v a t i o n by spermidine than E. c o l i (50% A + T) and c a l f thymus (58% A + T) DNA, because spermidine binds to poly d(A-T) more s t r o n g l y than these two DNAs (324,327). 3.6d. The e f f e c t of spermidine on enzymes i n h i b i t i n g DNA s y n t h e s i s The i n t e r a c t i o n between DNA and polyamines have been s t u d i e d by many i n v e s t i g a t o r s . This i n t e r a c t i o n between DNA and polymerase has the a b i l i t y o f p r o t e c t i n g DNA (281-283). In order to determine whether the s t i m u l a t i o n of DNA s y n t h e s i s by spermidine i s due to i n h i b i t i o n of the enzymes which are i n h i b i t o r y f o r DNA s y n t h e s i s , the e f f e c t of spermidine on DNase, ATPase and phosphodiesterase was s t u d i e d . Ammonium s u l f a t e f r a c t i o n s I I and I I , of 10-day-old - 140 -f ' 1 1 ' 1 0.5 1.0 1.5 2.0 2-5 S p e r m i d i n e ( m M ) F i g . 27. The e f f e c t of spermidine on DNA polymerase a c t i v i t y w i t h d i f f e r e n t DNAs as template. 10-day-old r a t b r a i n e x t r a c t was used as enzyme source. • • C a l f thymus DNA; A A E. c o l i DNA • — - • Pol y d(A-T); i k T, phage DNA - 141 -b r a i n e x t r a c t were used as enzyme sources. The e f f e c t of spermidine on the a c t i v i t y of these two enzyme f r a c t i o n s , II ^  and 11^, was s t u d i e d . As shown i n Table XXI, spermidine could not s t i m u l a t e the DNA polymerase a c t i v i t y of F r a c t i o n I I , but s t r o n g l y stimu-3 . l a t e d the a c t i v i t y i n F r a c t i o n I I , . However, F r a c t i o n I I contained b a higher a c t i v i t i e s of DNase and phosphodiesterase than F r a c t i o n 11^, i n d i c a t i n g that the s t i m u l a t i n g a c t i v i t y of spermidine i s due not only to the i n h i b i t i o n of DNase and phosphodiesterase. A c t u a l l y , 0.5 mM spermidine does not i n h i b i t ATPase and phosphodiesterase a c t i v i t i e s as shown i n Table X X I I . 3.6e. The e f f e c t of spermidine on DNA polymerase B i n d i f f e r e n t  c o ncentrations of b i v a l e n t c a t i o n s R e c e n t l y , Takeda (328) r e p o r t e d that polyamines can r e p l a c e 2+ Mg ions i n p r o t e i n s y n t h e s i s and suggested (329) that polyamines a f f e c t aminocyl-tRNA synthetase by a c t i v a t i n g the enzyme. The 2+ p o s s i b i l i t y of replacement of Mg ions by spermidine i n the DNA polymerase r e a c t i o n systems was s t u d i e d . As shown i n Table XXII 2+ 2+ spermidine cannot r e p l a c e Mg i o n s . In the absence of Mg i o n s , spermidine cannot s t i m u l a t e the r e a c t i o n , even w i t h the a d d i t i o n of 2 mM spermidine. The higher c o n c e n t r a t i o n of p o l y -amines s t r o n g l y i n h i b i t e d DNA polymerase a c t i v i t y . The peak of s t i m u l a t i o n of spermidine i s s t i l l around 0.5 to 1 mM at any con-2+ 2+ c e n t r a t i o n of Mg i o n s , and the o p t i m a l c o n c e n t r a t i o n of Mg i s Table XXI. Spermidine effects on DNA polymerase Fractions Ila and l i b . Enzyme preparation DNA polymerase activities Native DNA as Template Heated DNA as Template without with 0.5 mM without with 0.5 mM spermidine spermidine spermidine spermidine DNase activity unit, '/assay Phospho -diesterase activity unit "/fog cpm cpm cpm cpm Fraction Ila 519 502 1405 1326 1.2 0.9 Fraction l i b 2186 3222 154 246 0.4 0.6 - 143 -Table XXII. The e f f e c t of spermidine on ATPase and phosphodiesterase a c t i v i t y P r e p a r a t i o n Enzyme a c t i v i t y (unit/mg) without spermidine w i t h 1 mM spermidine ATPase 1.0 1.04 Phosphodiesterase 1.4 1.45 10-day-old b r a i n e x t r a c t s were used as enzyme p r e p a r a t i o n 1 u n i t of ATPase = 1 pg of P^ l i b e r a t e d 1 u n i t of phosphodiesterase = reading at 440 nm of 0.1 O.D. u n i t . - 144 -always 5 mM at any concentration of spermidine. This also i n d i -2+ cates that spermidine cannot par t i a l l y replace Mg ions. That i s , at the optimal spermidine concentration of 1 mM, the net increase in DNA polymerase B activity was constant at a l l con-2+ centrations of Mg within the range 1.0 to 7.0 mM. This i n d i -cates that the mechanism of stimulation by polyamines is not the 2+ same as that of Mg , since the presence of a polyamine even at 2+ optimal concentrations of Mg results in an additive stimulation of DNA polymerase activity. Table XXIV shows that spermidine stimulates DNA polymerase B 2+ by about 100% in the presence of 5 mM Mg . However, spermidine 2+ stimulated DNA polymerase B by about 800% i n the presence of Mn but 2+ not Mg ions. Spermidine shows no effect on DNA polymerase A i n the presence of either Mg ions or.Mn ions. 3.6f. The e f f e c t of spermidine on DNA sy n t h e s i s using nucleo- h i s t o n e as template Rec e n t l y , Schwimmer (330) has st u d i e d the e f f e c t of p u t r e s c i n e and cadaverine on p a r t i a l l y p u r i f i e d _E_. c o l i DNA polymerase a c t i v i t y w i t h c l e a n DNA, nu c l e o h i s t o n e , and chromatin as template. He con-cluded t h a t the s t i m u l a t i n g e f f e c t of spermidine i s due to r e -moving h i s t o n e from DNA. In order to study t h i s p o s s i b i l i t y , the e f f e c t s of spermidine on DNA polymerase a c t i v i t y i n the presence of h i s t o n e and on the nucleohistone-primed polymerase a c t i v i t y - 145 -Table X X I I I . E f f e c t of spermidine and Mg on DNA polymerase B a c t i v i t y Spermidine (mM) Mg ions c o n c e n t r a t i o n (mM) 0 1.0 3.0 5.0 7.0 cpm cpm cpm cpm cpm 0 67 352 579 876 677 0.5 85 1228 1467 1555 1086 1 115 1361 1535 1747 1244 2 85 543 1302 1597 1511 4 50 98 249 194 239 DNA polymerase B was obtained from DEAE-cellulose chromatography of F r a c t i o n I I , - 146 -Table XXIV. The e f f e c t of spermidine on DNA polymerase a c t i v i t y i n the presence of bi v a l e n t cations Native DNA as template Heated DNA as template without with 0.5 mM without with 0.5 mM Bivalent DNA „ , „ , Spd. Spd. Spd. Spd. Cations polymerase 1249 1075 82 147 Mn"' A 408 489 498 518 B 435 3332 189 371 Mg 2 + A 615 749 B 1065 2037 - 147 -were studied. As shown in Table XXV, spermidine stimulated DNA polymerase B by 72% in the absence of histone. However, spermidine showed inhibition of DNA polymerase B i n the presence of histone rather than stimulation. In Table XXVI, i t is also shown that spermidine stimulates DNA polymerase B much more with clean DNA as template than with nucleohistone. The results of these two experiments indicate that the stimulatory effect of spermidine i s not only due to removal of histone from DNA. Schwimmer used a fresh preparation of chromatin and nucleohistone from rat l i v e r , rat thymus and pea as template. The stimulatory effect of poly-amine which he observed could be due to contamination of nucleo-histones with particulate DNA polymerase and this enzyme was stimulated. 3.6g. The mechanism of the stimulatory effect of spermidine on  DNA polymerase It has been shown that the enzyme concentration curve of DNA polymerase was a sigmoidal slope (Fig. 13). The effect of spermidine on the enzyme concentration curve of DNA polymerase B was studied, and the results are shown i n Figure 28. Sper-midine stimulated DNA polymerase B in i n i t i a l time and showed linear proportionality to the enzyme concentration. Stimu-lation of DNA polymerase B activity by spermidine was sig n i -ficantly greater at low concentrations of enzyme protein than at higher concentrations (Fig. 28). The sigmoidal slope of DNA - 148 -Table XXV. The e f f e c t of spermidine and h i s t o n e on DNA polymerase a c t i v i t y Spermidine h i s t o n e added (yg)  a d d e d 0 10 20 30 40 — 1593 1380 1017 262 126 0.5 mM 2732 1692 851 179 119 - 149 -Table XXVI. The e f f e c t of spermidine on DNA polymerases A and B using nucleohistone as template Enzyme A Enzyme B Template Without w i t h 0.5 mM Without w i t h 0.5 mM Spd. Spd. Spd. Spd. H e a ^ c a l f t h y m u S 1040 1002 44 71 DNA Native c a l f thymus m ^ m 4 ^ DNA C a l f thymus ^ m 4 1 5 5 5 2 2 3 2 nucleohistone - 150 -10 20 Protein ( u g ) F i g . 28. The e f f e c t of spermidine on the enzyme c o n c e n t r a t i o n curve of DNA polymerase B. A A without spermidine _ • w i t h 1 mM spermidine - 151 -polymerase B concentration curve could be due to separation of p r o t e i n f a c t o r ( s ) (see Section 3.5) from the enzyme i n a d i l u t e d condition and t h i s p r o t e i n f a c t o r i s p o s s i b l y e s s e n t i a l f o r DNA polymerase a c t i v i t y . Spermidine may f u n c t i o n as a c a t i o n bridge s t a b i l i z i n g the i n t e r a c t i o n between DNA polymerase B and i t s f a c t o r . The r o l e of spermidine i n t h i s system i s presumably s i m i l a r 2+ 2+ to the Mg e f f e c t on ribosome (331). When the Mg concentration i s r a i s e d , 50 S and 30 S ribosomal subunits become associated to form 70 S ribosomes. The p o s s i b i l i t y of t h i s mechanism was also supported by the r e s u l t s recorded i n Table XXIV. P a r t i a l l y i n a c t i v a t e d DNA polymerase B (Sephadex f r a c t i o n ) was extremely stimulated by spermidine i n the presence of f a c t o r . E i t h e r spermidine or p r o t e i n f a c t o r alone showed much less s t i m u l a t i o n than i n the presence of both. The f a c t o r exhibited no DNA poly-merase a c t i v i t y i n the presence or absence of spermidine. Neither p r o t e i n f a c t o r nor spermidine could stimulate DNA poly-merase A. Table XXVII. Spermidine e f f e c t on DNA polymerases A and B with p r o t e i n fac tor i s o l a t e d from enzyme B Preparat ion Without spermidine With 0.5 mM spermidine heated DNA n a t i v e DNA heated DNA n a t i v e DNA cpm cpm cpm cpm Enzyme A 320 201 309 194 Enzyme B (Sephadex f r a c t i o n ) 50 127 80 238 Factor 58 26 ' 35 22 Enzyme A + f a c t o r 330 200 335 191 Enzyme B + f a c t o r 69 300 74 1090 Enzyme A was obtained from D E A E - c e l l u l o s e chromatography of F r a c t i o n II 3. Enzyme B was obtained from Sephadex G-200 f i l t r a t i o n of DNA polymerase B P r o t e i n fac tor was obtained as shown i n Sect ion 3.5. - 153 -IV. CONCLUSION DNA polymerase (deoxynucleoside t r i p h o s p h a t e : DNA deoxy-n u c l e o t i d y l t r a n s f e r a s e E.C. 2.7.7.7) a c t i v i t y i n s o l u b l e e x t r a c t s from developing r a t b r a i n described here shows many s i m i l a r i t i e s to other animal DNA polymerases (48,108,196,313). DNA polymerase 2+ from i n f a n t r a t b r a i n r e q u i r e d the presence of template DNA Mg and a l l four deoxynucleoside t r i p h o s p h a t e s . The a c t i v i t y was enhanced by the a d d i t i o n of d i t h i o t h r e i t o l . The o p t i m a l con-2+ c e n t r a t i o n of Mg was around 5 mM. The optimum pH of DNA p o l y -merase of crude r a t b r a i n e x t r a c t was around pH 8.6. The i n c o r -p o r a t i o n continued l i n e a r l y w i t h b r a i n e x t r a c t f o r 60 min. DNA polymerase i n r a t b r a i n e x t r a c t i s unstable during storage. L i k e most of the animal DNA polymerases (48,108,196,313), the enzyme i n the crude e x t r a c t from developing r a t b r a i n p r e f e r s heat-denatured DNA as template, except when o l d e r (Table I V ) , where the a c t i v i t y seems to be higher w i t h n a t i v e DNA as template. Furthermore, as shown i n F i g . 5, there seem to be two kinds of DNA polymerase i n c e r e b e l l a r e x t r a c t ; one p r e f e r s the heat-denatured DNA as template and the other p r e f e r s n a t i v e DNA as template. I n accordance w i t h Sung's previous study on the i n c o r p o r a t i o n 14 of [ C]thymidine i n t o developing r a t b r a i n DNA (252) , the a c t i v i t y of DNA polymerase i n the cerebellum i s 20-50 times higher than that - 154 -i n the c o r t e x , depending on the age. The a c t i v i t y i n the cere-bellum a t t a i n s i t s maximum l e v e l at around 6 days of age and then decreases r a p i d l y during maturation to a d u l t where l i t t l e or no a c t i v i t y could be detected. The a c t i v i t y i n the co r t e x was h i g h e s t immediately a f t e r b i r t h and decreased g r a d u a l l y . S i m i l a r to the r e s u l t s of B e l l a i r , using regenerating r a t l i v e r (237), and Leung and Zbarsky using r a t i n t e s t i n a l mucose (51), two DNA polymerase a c t i v i t i e s , A and B, have been found i n 10-day-old r a t b r a i n . The a c t i v i t i e s of both f r a c t i o n s depended on the presence of a l l four deoxyribonucleoside t r i -2+ phosphates, Mg and DNA as template. N e i t h e r enzyme A nor enzyme B are t e r m i n a l n u c l e o t i d y l t r a n s f e r a s e ( s ) . The o p t i m a l 2+ co n c e n t r a t i o n of Mg was 3-5 mM. Optimal pH was around 7.2-7.5 f o r enzyme A and 7.5-7.8 f o r enzyme B. However, some p r o p e r t i e s of these two enzymes d i f f e r e d c o n s i d e r a b l y . Enzyme A showed a 2-3 times preference f o r heat-denatured DNA and enzyme B almost always used n a t i v e DNA as template. The o p t i m a l concen-2+ t r a t i o n of Mn was 0.05 mM f o r enzyme A and 0.3 mM f o r enzyme B. ATP s t i m u l a t e d only enzyme A, but EDTA s t i m u l a t e d enzyme B and showed l i t t l e or no e f f e c t on enzyme A. Enzyme B was s t r o n g l y s t i m u l a t e d by K C l . In the case of enzyme A, however, s a l t gave no a c t i v a t i o n but r a t h e r a marked i n h i b i t i o n . Enzyme A was more s e n s i t i v e to d i t h i o t h r e i t o l and s u l f h y d r y l - b l o c k i n g agents than - 155 -enzyme B. The r e a c t i o n l e v e l l e d o f f a f t e r 40 min i n c u b a t i o n w i t h enzyme A, but i n c o r p o r a t i o n was l i n e a r f o r at l e a s t 2 h w i t h enzyme B. The i n c o r p o r a t i o n was l i n e a r l y p r o p o r t i o n a l to the enzyme A c o n c e n t r a t i o n , but enzyme B showed a s i g m o i d a l slope i n i t s enzyme co n c e n t r a t i o n curve. However, the most d i s t i n c t p r o p e r t i e s of these two enzymes are t h e i r molecular weight and s u b c e l l u l a r l o c a t i o n . Enzyme A sediments at around 9 S on sucrose g r a d i e n t s and enzyme B at around 3-4 S ( F i g . 14). N e u t r a l sucrose d e n s i t y g r a d i e n t c e n t r i f u g a t i o n was used as a t o o l i n the study of the p a t t e r n of developmental changes i n two DNA polymerases of r a t b r a i n . I n the cerebellum, DNA polymerase A i s the more a c t i v e i n very young animals but peaks at around the 6th day a f t e r b i r t h . DNA polymerase B i s more a c t i v e than enzyme A i n the c e r e b e l l a of o l d e r animals. In the c e r e b r a l c o r t e x DNA p o l y -merase A a c t i v i t y i s higher than that of enzyme B i n the f e t a l stage but the a c t i v i t y of polymerase A i s much lower than that of polymerase B at a l l post n a t a l ages s t u d i e d . Since DNA polymerase A showed high a c t i v i t y d u r i ng r a p i d c e l l p r o l i f e r a t i o n i n r a t b r a i n , i t seems probable t h a t DNA polymerase A plays a r o l e i n the i n d u c t i o n of p r o l i f e r a t i v e a c t i v i t y . The b i o l o g i c a l f u n c t i o n of DNA polymerase B i s s t i l l not known. Since s i g n i f i -cant a c t i v i t y of DNA polymerase B i s found i n o l d e r r a t b r a i n , i t seems th a t t h i s enzyme may be concerned w i t h r e p a i r or some other f u n c t i o n such as gene a m p l i f i c a t i o n or d i f f e r e n t i a t i o n . - 156 -An extremely high a c t i v i t y of DNA polymerase was s o l u b i l i z e d from the n u c l e i c membrane-chromatin complex i s o l a t e d from a d u l t r a t b r a i n . The p r o p e r t i e s of the s o l u b i l i z e d n u c l e a r p a r t i c u l a t e DNA polymerase were s i m i l a r to those of DNA polymerase B from developing r a t b r a i n , such as template preference, s a l t e f f e c t , pH optimum, EDTA and spermidine s t i m u l a t i o n , and sucrose d e n s i t y g r a d i e n t sedimentation e t c . The r e l a t i v e amounts of s o l u b l e and p a r t i c u l a t e forms of DNA polymerase i n the c e r e b e l l a of r a t s change w i t h age. Much of the a c t i v i t y i s i n the s o l u b l e form i n younger r a t s but, i n the a d u l t r a t , the DNA polymerase e x i s t s almost ex-c l u s i v e l y i n a p a r t i c u l a t e form which i s i n a c t i v e unless s o l u -b i l i z e d as d e s c r i b e d . I n younger r a t b r a i n , DNA polymerase A e x i s t s only i n s o l u b l e form and i s l o c a t e d both i n the nucleoplasm and cytoplasm f r a c t i o n . However, there i s very l i t t l e or no DNA polymerase A a c t i v i t y i n a d u l t r a t b r a i n . DNA polymerase B e x i s t s i n both a s o l u b l e and p a r t i c u l a t e form i n younger r a t b r a i n . The s o l u b l e form of DNA polymerase B i s l o c a t e d i n both the cytoplasm and the nucleoplasm. The major p a r t of DNA polymerase B e x i s t s as p a r t i c u l a t e form i n the n u c l e i at o l d e r stages. M i t o c h o n d r i a l DNA polymerase i s d i s t i n g u i s h e d from DNA polymerases A and B by i t s behavior on sucrose d e n s i t y g r a d i e n t s . There i s a high a c t i v i t y i n 10-day-old r a t b r a i n mitochondria but very low or no a c t i v i t y i n a d u l t r a t b r a i n mitochondria. - 157 -Fur t h e r p u r i f i c a t i o n of DNA polymerase B of r a t b r a i n r e s u l t e d i n a p a r t i a l l o s s of a c t i v i t y , due to d i s s o c i a t i o n of an a c t i v a t o r (or f a c t o r ) from the enzyme. The f r a c t i o n obtained i n the l a s t stage of p u r i f i c a t i o n was d e f i c i e n t i n the f a c t o r , and DNA p o l y -merase was r e l a t i v e l y i n a c t i v e unless supplemented w i t h an exo-genous f a c t o r . The f a c t o r was s e n s i t i v e to p r o t e o l y t i c enzymes and h e a t - l a b i l e . This i n d i c a t e s that the f a c t o r i s a p r o t e i n . The s t i m u l a t o r y e f f e c t was not due to the a c t i v i t y of a nuclease or n i c k a s e . The f a c t o r only s t i m u l a t e d DNA polymerase B a c t i v i t y w i t h double stranded DNA as template and showed no e f f e c t on DNA s y n t h e s i s w i t h s i n g l e stranded DNA as template. I t seems that t h i s f a c t o r i s s p e c i f i c f o r DNA polymerase B. I t s mechanism of a c t i o n i s s t i l l unknown. The e f f e c t s of polyamines, i . e . spermine, spermidine and 3 p u t r e s c i n e , on [ H]dTTP i n c o r p o r a t i o n i n t o DNA have been s t u d i e d . The polyamines enhanced the DNA polymerase a c t i v i t y i n e x t r a c t s of b r a i n from 10-day-old r a t s by more than 50% and s t i m u l a t e d p u r i f i e d DNA polymerase B around 100%. However, polyamines showed l i t t l e or no e f f e c t on DNA polymerase A. DNA polymerase B showed d i f f e r e n t responses to spermidine depending on the sources of DNA. DNA polymerase B was s t r o n g l y i n h i b i t e d by spermidine using T^ phage DNA as template. Various degrees of s t i m u l a t i o n were observed w i t h the other DNA templates which were t e s t e d , suggesting that the - 158 -nature of DNA i s important f o r the e f f e c t of spermidine. S t i m u l a t i o n of DNA polymerase a c t i v i t y by spermidine i s not simply due to pro-t e c t i o n from enzymes i n h i b i t o r y to DNA s y n t h e s i s , such as DNase, ATpase and phosphodiesterase. I t i s a l s o not due to replacement 2+ of Mg ions by spermidine i n DNA s y n t h e s i s . The p o s s i b i l i t y of spermidine s t i m u l a t i o n a c t i n g by d i s p l a c i n g h i s t o n e s from DNA was a l s o excluded. Presumably, the mechanism of spermidine s t i m u l a t i o n of DNA polymerase B a c t i v i t y by f a c i l i t a t i n g or s t a b i l i z i n g the a s s o c i a t i o n of enzyme and'factor. - 159 -V. BIBLIOGRAPHY 1. Reichard, P. and Estbom, B. J . B i o l . Chem. 188, 839 (1951). 2. Kornberg, A., Lehman, I.R., Bessman, M.J. and Simms, E.S. Biochim. Biophys. Acta 21> 1 9 7 (1956). 3. Lehman, I.R., Bessman, M.J., Simms, E.S. and Korberg, A. J . B i o l . Chem. 233, 163 (1958). 4. Richardson, C C , S c h i l d k a r a u t , C.L. and Aposhian, H.V. J . B i o l . Chem. 239, 222 (1964). 5. Lehman, I.R., Zimmerman, S.B., A d l e r , J . , Bessman, M.J. and Simms, E.S. Proc. N a t l . Acad. S c i . U.S. 44, 1191 (1958). 6. Okazaki, T. and Kornberg, A. J . B i o l . Chem. 239, 259 (1964). 7. Zimmerman, B.K. J . B i o l . Chem. 241, 2035 (1966). 8. Litman, R.M. J . B i o l . Chem. 242, 6 2 2 2 (1968). 9. Aposhian, H.V. and Kornberg, A. J . B i o l . Chem. 237, 519 (1962), 10. Goulian, M., Lucas, Z.J. and Kornberg, A. J . B i o l . Chem. 243, 627 (1968). 11. Orr, C.W.M., H e r r i o t t , S.T. and Bessman, M.J. J . B i o l . Chem. 240, 4652 (1965). 12. F r i e d k i n , M., T i l s o n , D. and Roberts, D. J . B i o l . Chem. 220, 627 (1956). 13. Bollum, F.J. J . B i o l . Chem. 235, 2399 (1960). 14. Krakow, J.S., Coutsogeorgopoulosi, C. and C a n e l l a k i s , E.S. Biochim. Biophys. A c t a 5_5, 639 (1962) 15. Walwick, E.R. and Main, R.K. Biochim. Biophys. Acta 61, 876 (1962). - 160 -16. Bollum, F.J. and P o t t e r , V.R. J . B i o l . Chem. 233, 478 (1958). 17. Mantsavinos, R. and C a n e l l a k i s , E.S. J . B i o l . Chem. 234, 635 (1959). 18. K e i r , H.M., S m e l l i e , R.M. and S i e b e r t , G. Nature 196, 752 (1962). 19. Mantsavinos, R. J . B i o l . Chem. 239, 3431 (1964). 20. Meyer, R.R. and Simpson, M.V. Proc. N a t l . Acad. S c i . U.S. 61, 130 (1968). 21. Lynch, W.E., Brown, R.F., Umeda, T., Langreth, S.G. and Lieberman, I . J . B i o l . Chem. 245, 3911 (1970). 22. Loeb, L.A. J . B i o l . Chem. 244, 1672 (1969). 23. Davidson, J.N., S m e l l i e , R.M.S., K e i r , H.M. and McArdle, A.N. Nature 182, 589 (1958). 24. K e i r , H.M., B i n n i e , B. and S m e l l i e , R.M.S. Biochem. J . 8_2, 493 (1962). 25. F u r l o n g , N.B. Biochim. Biophys. Acta 108, 489 (1965). 26. Shepherd, J.B. and K e i r , H.M. Biochem. J . 99, 443 (1966). 27. Roychoudhury, R. and Bloc h , D.P. J . B i o l . Chem. 244, 3359 (1969). 28. S m e l l i e , R.M.S., Gray, E.D., K e i r , H.M., Richards, J . , B e l l , D. and Davidson, J.N. Biochim. Biophys. Acta 3_7, 243 (1960). 29. H a r f o r d , C G . and Romberg, A. Fed. Proc. 17, 515 (1958). 30. Bach, M.K. Proc. N a t l . Acad. S c i . U.S. 48_, 1031 (1962). 31. Magee, W.E. V i r o l o g y 17, 604 (1962). - 161 -32. K e i r , H.M. and Gold, E. Biochim. Biophys. Acta 72_, 263 (1963). 33. Green, M., P i n a , M. and Chagoya, V. J . B i o l . Chem. 239, 1188 (1964). 34. Greene, R. and Korn, D. J . B i o l . Chem. 245, 254 (1970). 35. Nohara, H. and Kaplan, A.S. Fed. Proc. 22, 615 (1963). 36. L i t t l e f i e l d , J.W., McGovern, A.P. and Morgeson, K.B. Proc. N a t l . Acad. S c i . U.S. 49, 102 (1963). 37. Gold, M. and H e l l e i n e r , CW. Biochim. Biophys. Acta 80, 193 (1964). 38. R u s s e l l , W.C, Gold, E., K e i r , H.M. , Omura, H. , Watson, D.H. and Wildy, P. V i r o l o g y 22, 103 (1964). 39. Mantsavinos, R. and C a n e l l a k i s , E.S. Cancer Res. J-9_, 1239 (1959). 40. Radding, CM. and Romberg, A. J . B i o l . Chem., 237, 2877 (1962). 41. BoHum, F.J. J . B i o l . Chem. 234, 2733 (1959). 42. Bollum, F.J. J . B i o l . Chem. 237, 1945 (1962). 43. Yoneda, M. and Bollum, F.J. J . B i o l . Chem. 240, 3385 (1965). 44. S m e l l i e , R.M.S. E x p t l . C e l l Res. Suppl. _9, 245 (1963). 45. F u r l o n g , N.B. Biochim. Biophys. Acta 114, 491 (1966). 46. Berg, P., Fancer, H. and Chamberlin, M. i n Vo g e l , H.J., Bryson, V. and Lampen, J.O., " I n f o r m a t i o n a l Macromolecules", p. 467, Academic P r e s s , New York (1963). 47. K e i r , H.M., Shepherd, J.B. and Hay, J . Biochem. J . 89, 9P (1963). - 162 -48. Keir, H.M. in Davidson, J.N. and Cohn, W.E. "Progress in Nucleic Acid Research and Molecular Biology", Vol. 4, p. 81, Academic Press, New York (1965). 49. Chang, L.M.S. and Bollum, F.J. J. Biol. Chem. 246, 5835 (1971). 50. Howk, R. and Wang, T.Y. Arch. Biochem. Biophys. 133, 238 (1969) . 51. Leung, F.Y.T. and Zbarsky, S.H. Can. J. Biochem. 48, 529 (1970) . 52. Watson, J.D. and Crick, F.H.C. Nature 171, 737 (1953). 53. Watson, J.D. and Crick, F.H.C. Nature 171, 964 (1953). 54. Delbruck, M. and Stent, G.S. in McElroy, W.D. and Glass, B. "The Chemical Basis of Heredity", p. 699, Johns Hopkins Press, Baltimore (1957). 55. Meselson, M. and Stahl, F. Proc. Natl. Acad. Sci. U.S. 44, 671 (1958). 56. Djordjevic, B. and Szybalski, W. J. Exptl. Med. 112, 509 (1960). 57. Simon, E.H. J. Mol. Biol. _3. 101 (1961). 58. Chun, E.H.L. and L i t t l e f i e l d , J.W. J. Mol. Biol. 3, 668 (1961). 59. Taylor, J.H., Woods, P.S. and Hughes, W.L. Proc. Natl. Acad. Sci. U.S. 43, 122 (1957). 60. Josse, J., Kaiser, A.D. and Kornberg, A. J. Biol. Chem. 236, 864 (1961). - 163 -61. M i t r a , S. and Kornberg, A. J . Gen. P h y s i o l . 49, 59 (1966). 62. C a i r n s , J . Cold Spring Harbor Symp. Quant. B i o l . 28, 43 (1963). 63. Yoshikawa, H. and Sueoka, N. Proc. N a t l . Acad. S c i . U.S. 49, 559 (1963). 64. Schnos, M. and Inman, R.B. J . Mol. B i o l . _51, 61 (1970). 65. Makover, S. Proc. N a t l . Acad. S c i . U.S. 59, 1345 (1968). 66. Schnos, M. and Inman, R.B. J . Mol. B i o l . 55_, 31 (1971). 67. Caro, L.G. and Berg, CM. Cold Spring Harbor Symp. Quant. B i o l . 33, 559 (1968). 68. Wolf, B., Pato, M.L. , Ward, C B . and G l a s e r , D.A. Cold Spring Harbor Symp. Quant. B i o l . 33, 575 (1968). 69. Cerda-Olmedo, C. and Hanawalt, P.C Cold Spring Harbor Symp. Quant. B i o l . 33_, 599 (1968). 70. Master, M. and Broda, P. Nature New Bio l o g y 232, 137 (1971). 71. Huberman, J.A. and Riggs, A.D. J . Mol. B i o l . 32, 327 (1968). 72. Okazaki, R., Okazaki, T., Sakabe, K., Sugimoto, K., Kainuma, R., Sugino, A. and I w a t s u k i , N. Cold Spring Harbor Symp. Quant. B i o l . 33, 129 (1968). 73. Sakabe, K. and Okazaki, R. Biochim. Biophys. Acta 129, 651 (1966). 74. Okazaki, R., Okazaki, T., Sakabe, K., Sugimoto, K. Japan J . Med. S c i . B i o l . 20, 255 (1965). - 164 -75. Okazaki, R., Okazaki, T., Sakabe, K., Sugimoto, K. and Sugino, A. Proc. N a t l . Acad. S c i . U.S. 59_, 598 (1968). 76. Sadowski, P., Ginsberg, B., Yu d e l e v i c h , A., F e i n e r , L. and Hurwitz, J . Cold Spring Harbor Symp. Quant. B i o l . 33, 165 (1968). 77. Y u d e l e v i c h , A., Ginsberg, B. and Hurwitz, J . Proc. N a t l . Acad. S c i . U.S. 61, 1129 (1968). 78. O i s h i , M. Proc. N a t l . Acad. S c i . U.S. 60, 329 (1968). 79. O i s h i , M. Proc. N a t l . Acad. S c i . U.S. 60, 691 (1968). 80. O i s h i , M. Proc. N a t l . Acad. S c i . U.S. 60, 1000 (1968). 81. Tsukada, K., Moriyama, T., Lynch, W.E. and Lieberman, I . Nature 220, 162 (1968). 82. Berger, H. and I r v i n , J.L. Proc. N a t l . Acad. S c i . U.S. 65, 152 (1970). 83. T a y l o r , J.H. J . Mol. B i o l . 31, 579 (1968). 84. T a y l o r , J.H. and Miner, P. Cancer Res. 28, 1810 (1968). 85. Schandl, E.K. and T a y l o r , J.H. Biochem. Biophys. Res. Commun. 34, 291 (1969). 86. Hyodo, M. , Koyama, H. and Ono, T. Biochem. Biophys. Res. Commun. 38, 513 (1970). 87. P a o l e t t i , C. , D u t h e i l l e t - L a m o n t h e z i e , N., Jeanteur, P. and Obrenovitch, A. Biochim. Biophys. Acta 149, 435 (1967). 88. Sato, S., Tenaka, M. and Sugimura, T. Biochim. Biophys Acta 209, 43 (1970). - 165 -89. L e v i s , A.G., Kpsmanovic, V., M i l l e r - F a u r e r , A. and E r r e r a , M. Eur. J . Biochem. 3, 57 (1967). 90. Habener, J.F., Bynum, B.S. and Shack, J . Biochim. Biophys. Acta 195, 484 (1969). 91. P a i n t e r , R.B. and Schaefer, A. Nature 221, 1215 (1969). 92. Habener, J.F., Bynum, B.S. and Shack, J . J . Mol. B i o l . 49, 159 (1970). 93. Nuzzo, F., Brega, A. and F a l a s c h i , A. Proc. N a t l . Acad. S c i . U.S. 65, 1017 (1970). 94. K i d w e l l , W.R. and M u e l l e r , G.C. Biochem. Biophys. Res. Commun. 36.. 7 5 6 (1969). 95. Richardson, C.C., L i v e , T.R., Jacquemin-Sablon, A., Weiss, B. and Fareed, G.C. Cold Spring Harbor Symp. Quant. B i o l . 33, 151 (1968). 96. K o z i n s k i , A.W. Cold Spring Harbor Symp. Quant. B i o l . 33, 375 (1968). 97. Masamune, Y. and Richardson, C C . Proc. N a t l . Acad. S c i . U.S. 61, 1328 (1968). 98. Berger, H. and K o z i n s k i , A.W. Proc. N a t l . Acad. S c i . U.S. 64, 897 (1969). 99. Kram, J.D. Biochem. Biophys. Res. Commun. 37_, 416 (1969). 100. Chan, V.L., Shugan, S. and E b i s u z a k i , K. V i r o l o g y 40, 403 (1970). 101. Hosoda, J . and Mathews, E. J . Mol. B i o l . 55, 155 (1971). - 166 -102. K o z i n s k i , A.W. and M i t c h e l l , M. J . V i r o l . 4, 823 (1969). 103. I w a t s u k i , N. and Okazaki, R. J . Mol. B i o l . 52, 37 (1970). 104. G e l l e r t , M. and B u l l o c k , M.L. Proc. N a t l . Acad. S c i . U.S. 67, 1580 (1970). 105. Okazaki, T. and Okazaki, R. Proc. N a t l . Acad. S c i . U.S. 64, 1242 (1969). 106. P a u l i n g , C. and Hamm, L. Proc. N a t l . Acad. S c i . U.S. 64, 1195 (1969) 107. P a u l i n g , C. and Hamm, L. Biochem. Biophys. Res. Commun. 37, 1015 (1969). 108. Richardson, C C . Ann. Rev. Biochem. 38_, 795 (1969). 109. Sugimoto, K., Okazaki, T. and Okazaki, R. Proc. N a t l . Acad. S c i . U.S. 6_0, 1356 (1968). 110. Newman, J . and Hanawalt, P.C. J . Mol. B i o l . 35, 639 (1968). 111. G e l l e r t , M. Proc. N a t l . Acad. S c i . U.S. 57, 148 (1967). 112. G e f t e r , M.L.A., Becker, A. and Hurwitz, J . Proc. N a t l . Acad. S c i . U.S. 58, 240 (1967). 113. G e l l e r t , M.W., L i t t l e , J.W., Oshinsky, C.K. and Zimmerman, S.B. Cold Spring Harbor Symp. Quant. B i o l . 33_, 21 (1968). 114. O l i v e r a , B.M., H a l l , Z.W., Anraku, Y., Chien, J.R. and Lehman, I.R. Cold Spring Harbor Symp. Quant. B i o l . 33, 27 (1968). 115. O l i v e r a , B.M., H a l l , Z.W., and Lehman, I.R. Proc. N a t l . Acad. S c i . U.S. 61, 237 (1968). - 167 -116. Becker, A., Lyn, G., G e f t e r , M. and Hurwitz, J . Proc. N a t l . Acad. S c i . U.S. 58, 1996 (1967). 117. Weiss, B. and Richardson, C C . Proc. N a t l . Acad. S c i . U.S. 57, 1021 (1967). 118. L i n d a h l , T. and Edelman, G.M. Proc. N a t l . Acad. S c i . U.S. 61, 680 (1968). 119. C o z z a r e l l i , N.R., Melechen, N.E., J o v i n , T.M. and Romberg, A. Biochem. Biophys. Res. Commun. 28_, 578 (1968). 120. L i t t l e , J.W., Zimmerman, S.B., Oshinsky, CR. and G e l l e r t , M. Proc. N a t l . Acad. S c i . U.S. 58, 2004 (1967). 121. O l i v e r a , B.M. and Lehman, I.R. Proc. N a t l . Acad. S c i . U.S. 57, 1426 (1967). 122. Romberg, A. S c i . Am. October 1968 123. Ryter, A. B a c t e r i o l . Rev. 32, 35 (1968). 124. Comings, D.E. Amer. J . Human Genet. 20, 440 (1968). 125. Levine, A.J. and Sinsheimer, R.L. J . Mol. B i o l . 39_, 619 (1969). 126. S a l i v a r , W.O. and Sinsheimer, R.L. J . Mol. B i o l . 41_, 39 (1969). 127. H a l l i c k , L., Boyee, R.P. and E c h o l s , H. Nature 232, 1 2 3 9 (1969). 128. S a l i v a r , W.O. and G a r d i n i e r , J . V i r o l o g y 41, 38 (1970). 129. B o t s t e i n , D. and Levine, M. Cold Spring Harbor Symp. Quant. B i o l . 33, 659 (1968). 130. F r a n k e l , F.R., Majumdar, C , Weintraub, S. and F r a n k e l , D.M. Cold Spring Harbor Symp. Quant. B i o l . 33, 495 (1968). - 168 -131. E a r h a r t , C.F., Tremblay, G.Y., D a n i e l s , M.J. and Schaechter, M. Cold Spring Harbor Symp. Quant. B i o l . 33, 707 (1968). 132. Altman, S. and Lerman, L..S. J . Mol. B i o l . 50, 235 (1970). 133. E a r h a r t , C F . V i r o l o g y 42, 429 (1970). 134. Sueoka, N. and Quinn, W.G. Cold Spring Harbor Symp. Quant. B i o l . 33> 695 (1968). 135. Snyder, R.W. and Young, F.E. Biochem. Biophys. Res. Commun. 35, 354 (1969). 136. Ryter, A., H i r o t a , Y. and Jacob, F. Cold Spring Harbor Symp. Quant. B i o l . 6 6 9 (1968). 137. Tremblay, G.Y., D a n i e l s , M.J. and Schaechter, M. J . Mol. B i o l . 40, 65 (1969). 138. F i e l d i n g , P. and Fox, C F . Biochem. Biophys.'Res. Commun. 41, 157 (1970). 139. Fuchs, E. and Hanawalt, P.C J . Mol. B i o l . 52, 301 (1970). 140. Chambon, P., DuPraw, W.J. and Kornberg, A. J . B i o l . Chem. 243, 5101 (1968). 141. F i r s h e i n , W. and G i l l m o r , R.G. Science 169_, 66 (1970). 142. Friedman, D.L. and M u e l l e r , G.C. Biochim. Biophys. Acta 174, 253 (1969). 143. O'Brien, R.L., Sanyal, A.B. and Stanton, R.H. E x p t l . C e l l Res. 70, 106 (1972). - 169 -144. Comings, D.E. and Kakefuda, T. J . Mol. B i o l . 33,' 225 (1968). 145. Jackson, V., Earnhardt, J . and Cha l k l e y , R. Biochem. Biophys. Res. Commun. _33, 253 (1968). 146. Nass, M.M.K. J . Mol. B i o l . 42, 521 (1969). 147. Laurent, M. and S t e i n e r t , M. Proc. N a t l . Acad. S c i . U.S. 66, 419 (1970). 148. Jacob, F., Brenner, A. and Cuzin, F. Cold Spring Harbor Symp. Quant. B i o l . 28, 329 (1963). 149. Jacob, F., Ryt e r , A. and Cuzin, F. Proc. Roy. Soc. Ser. B. 164, 267 (1966). 150. Ganeson, A.T. and Lederberg, J . Biochem. Biophys. Res. Commun. 18, 824 (1965). 151. Smith, D.W. and Hanawalt, P.C. Biochim. Biophys. Acta 149, 519 (1967). 152. Lark, C. and Lark, K.G. J . Mol. B i o l . 10, 120 (1964). 153. Marvin, D.A. Nature 219, 485 (1968). 154. Smith, D.W., S h a l l e r , H.E. and Bonhoeffer, F.J. Nature 226, 711 (1970). 155. Knippers, R. and S t r a t l i n g , W. Nature 226, 713 (1970). 156. Knippers, R. Nature 228, 1050 (1970). 157. Kornberg, T. and G e f t e r , M.L. Biochem. Biophys. Res. Commun. 40, 1348 (1970). 158. Moses, R.E. and Richardson, C C . Biochem. Biophys. Res. Commun. 41, 1557 (1970). - 170 -159. Moses, R.E. and Richardson, C C . Biochem. Biophys. Res. Commun. 41, 1565 (1970). 160. Loeb, L.A., S l a t e r , J.P., Ewald, J.L. and Agarwal, S.S. Biochem. Biophys. Res. Commun. 42_, 147 (1971). 161. Bleecken, S. J . Theor. B i o l . 32, 81 (1971). 162. S i c c a r d i , A.G., Shapiro, B.M., H i r o t a , Y. and Jacob, F. J . Mol. B i o l . 56, 473 (1971). 163. Inouye, M. and G u t h r i e , J.P. Proc. N a t l . Acad. S c i . U.S. 64, 957 (1969). 164. Shapiro, B., S i c c a r d i , A.G., H i r o t a , Y. and Jacob, F. J . Mol. B i o l . 52_, 75 (1970). 165. Pawlowski, P.J. and B e r l o w i t z , L. E x p t l . C e l l Res. 56, 154 (1969). 166. Kay, R.R., Haines, M.E. and Johnston, I.R. FEBS L e t t e r s 16, 233 (1971). 167. Hanaoka, F. and Yamada, M. Biochem. Biophys. Res. Commun. 42, 647 (1971). 168. Yoshida, S., Moda, M.J. and Y a g i , K. Biochem. Biophys. Res. Commun. 43, 1408 (1971). 169. Mizuno, N.S., Stoops, C E . and Sinha, A.A. Nature New Bi o l o g y 229, 22 (1971). 170. Yoshikawa-Fukada, M. and Ebert, J.D. Biochem. Biophys. Res. Commun. 43, 133 (1971). 171. Loeb, L.A. Nature 226, 448 (1970). - 171 -172. DuPaw, E.J. Proc. N a t l . Acad. S c i . U.S. 53, 161 (1965). 173. Fawett, D.W. Amer. J . Anat. 119, 129 (1966). 174. Woolam, D.H.M., M i l l e n , J.W. and Ford, D.H.R. Nature 213, 298 (1967). 175. Comings, D.E. and Okada, T.A. E x p t l . C e l l Res. 62_, 293 (1970). 176. Comings, D.E. E x p t l . C e l l Res. 63, 62 (1970). 177. Comings, D.E. E x p t l . C e l l Res. 63^, 471 (1970). 178. B a r i l , E.F., J e n k i n s , M.D., Brown, O.E. and L a s z l o , J . Science 169, 87 (19 70). 179. Hecht, N.B. E x p t l . C e l l Res. 70, 248 (1972). 180. Yoshikawa, H. Proc. N a t l . Acad. S c i . U.S. 58, 312 (1967). 181. C a i r n s , J . J . Mol. B i o l . (6, 208 (1963). 182. G i l b e r t , W. and D r e s s i e r , D. Cold Spring Harbor Symp. Quant. B i o l . 33, 473 (1968). 183. E i s e n , H., P e r e i r a da S i l v a , L. and Jacob, F. Cold Spring Harbor Symp. Quant. B i o l . 33, 755 (1968). 184. J a e n i s c h , R., Mayer, A. and Levine, A. Nature New Bi o l o g y 233, 72 (1971). 185. Champoux, J . J . and Dulbecco, R. Proc. N a t l . Acad. S c i . U.S. 69, 143 (1972). 186. Meselson, M. Nature 219, 17 (1968). 187. Deutscher, M.P. and Kornberg, A. J . B i o l . Chem. 244, 3019 (1969). - 172 -188. Kornberg, A. i n "Regulation of N u c l e i c A c i d and P r o t e i n S y n t h e s i s " , p. 22, Amsterdam, E l s e v i e r (1967). 189. Kornberg, A. Science 162, 1 4 1 0 (1969). 190. Bonhoeffer, F. and S c h a l l e r , W. Biochem. Biophys. Res. Commun. 20_» 93 (1965). 191. Fangman, W.L. and Novick, A. Genetics 60, 1 (1968). 192. Gross, J.D., Karamata, D. and Hempstead, P.G. Cold Spring Harbor Symp. Quant. B i o l . 2 2 , 307 (1968). 193. Kohiyama, M. Cold Spring Harbor Symp. Quant. B i o l . 33, 317 (1968). 194. Mendelson, N.H. Cold Spring Harbor Symp. Quant. B i o l . 2 3 , 313 (1968). 195. H i r o t a , Y., Ryt e r , A. and Jacob, F. Cold Spring Harbor Symp. Quant. B i o l . 2 1 . 677 (1968). 196. G o u l i a n , M. Ann. Rev. Biochem. 40, 855 (1971). 197. G o u l i a n , M. Cold Spring Harbor Symp. Quant. B i o l . 33, 11 (1968). 198. De L u c i a , P. and C a i r n s , J . Nature 224, 1164 (1969). 199. Gross, J . and Gross, M. Nature 224, 1166 (1969). 200. Kornberg, T. and G e f t e r , M. Proc. N a t l . Acad. S c i . U.S. 68, 761 (1971). 201. K e l l e y , W.S. and W h i t f i e l d , H.J. Nature 230, 33 (1971). 202. G e f t e r , M.L., H i r o t a , Y., Kornberg, T. and Wechsler, J.A. Proc. N a t l . Acad. S c i . U.S. 68_, 3150 (1971). - 173 -203. N u s s l e i n , V., Otto, B., Bonhoeffer, F. and S c h a l l e r , H. Nature 234, 285 (1971). 204. K e l l y , R.B., A t k i n s o n , M.R., Huberman, J.A. and Kornberg, A. Nature 224, 495 (1969). 205. K e l l y , R.B., C o z z a r e l l i , N.R., Deutscher, M.P., Lehman, I.R. and Kornberg, A. J . B i o l . Chem. 245, 39 (1970). 206. Smith, S.M., Symonds, N. and White, P. J . Mol. B i o l . 54_, 391 (1970). 207. Englund, P.T. J . B i o l . Chem. 246, 5684 (1971). 208. Monk, M. , Peacey, M. and Gross, J.D. J . Mol. B i o l . 58, 623 (1971). 209. Gross, J.D., Grunstein, J . and W i t k i n , E.M. J . Mol. B i o l . 58, 631 (1971). 210. Wicknen, R.B., Ginsberg, B., Berkower, I . and-Hurwitz, J . J . B i o l . Chem. 247, 489 (1972). 211. Wicknen, R.B., Ginsberg, B. and Hurwitz, J . J . B i o l . Chem. 247, 498 (19 72). 212. Okazaki, R., Sugimoto, K., Okazaki, T., Imac, Y. and Sugino, A, Nature 228, 223 (1970). 213. Moses, R.E. and Richardson, C C . Proc. N a t l . Acad. S c i . U.S. 67, 674 (1970). 214. Mordoh, J . , H i r o t a , Y. and Jacob, F. Proc. N a t l . Acad. S c i . 67, 773 (1970). - 174 -215. Burger, R.M. Proc. N a t l . Acad. S c i . U.S. 68, 2124 (1971). 216. M a t s u s h i t a , T., White, K.P. and Sueoka, N. Nature New Bi o l o g y 222, H I (1971). 217. S c h a l l e r , H., Otto, B., N u s s l e i n , V., Huf, J . , Herrmann, R. and Bonhoeffer, F. J . Mol. B i o l . 63, 183 (1972). 218. O l i v e r a , B.M. and Bonhoeffer, F. Proc. N a t l . Acad. S c i . U.S. 69, 25 (1972). 219. Yoshida, S. and C a v a l i e r i , L.F. Proc. N a t l . Acad. S c i . U.S. 68, 200 (1971). 220. S m e l l i e , R.M.S. and Eason, R. Biochem. J . 80, 39P (1961). 221. Behki, R.M. and Schneider, W.C. Biochim. Biophys. A c t a 68, 34 (1963). 222. S i e b e r t , G. Biochem. Z. 334, 369 (1961). 223. Main, R.K. and Cole, L . J . Nature 203, 646 (1964). 224. B i r n i e , G.D. and Fox, S.M. Biochem. J . 101, 33P (1966). 225. Granick, S. and G i b o r , A. Progr. N.A. Res. Mol. B i o l . 6_, 143 (1967). 226. Roodyn, D.B. and W i l k i e , D. i n "The Biogenesis of M i t o c h o n d r i a " , Methuen, London (1968). 227. Nass, M.M.K. Science 165, 25 (1969). 228. Reich, E. and Luck, D.J.L. Proc. N a t l . Acad. S c i . U.S. 55, 1600 (1966). 229. K a r o l , M.H. and Simpson, M. Science 162, 470 (1968). - 175 -230. Smith, D., Tauro, P., Schweizer, E. and Halvorsen, H.O. Proc. N a t l . Acad. S c i . U.S. 60, 936 (1968). 231. Wintersberger, E. Biochem. Biophys. Res. Commun. 25_, 1 (1966). 232. Parson, P. and Simpson, M.V. Science 155, 91 (1967). 233. K a l f , G.F. and Chih, J . J . J . B i o l . Chem. 243, 4904 (1968). 234. Meyer, R.R. and Simpson, M.V. Biochem. Biophys. Res. Commun. 34, 238 (1969). 235. Meyer, R.R. and Simpson, M.V. J . B i o l . Chem. 245, 3426 (1970). 236. Wallace, P.G., Hewish, D.R., Venning, M.M. and Burgoyne, L.A. Biochem. J . 125, 47 (1971). 237. B e l l a i r , J.T. Biochim. Biophys. Acta 161, 119 (1968). 238. Chiu, J.F. and Sung, S.C. Biochim. Biophys. Acta 209, 34 (1970). 239. Chiu, J.F. and Sung, S.C. Biochim. Biophys. Acta 246, 44 (1971). 240. Mantsavinos, R. and Munson, B. J . B i o l . Chem. 241, 2840 (1966). 241. Iwamura, Y., Ono, T. and M o r r i s , H.P. Cancer Res. 28, 2466 (1968). 242. Ove, P., L a z l o , J . , J e n k i n s , M.D. and M o r r i s , H.P. Cancer Res. 29, 1557 (1969). 243. Ove, P., J e n k i n s , M.D. and L a z l o , J . Cancer Res. 30, 535 (1970). 244. Bollum, F.J. J . B i o l . Chem. 235, P.C. 18 (1960). 245. Kato, K., Goncalves, J.M., Houts, G.E. and Bollum, F.J. J . B i o l . Chem. 242, 2780 (1967). 246. Leung, F.Y.T. and Zbarsky, S.H. Can. J . Biochem. 48, 537 (1970). - 176 -247. Dubuy, H.G., Mattern, C.F.T. and R i l e y , L. Biochim. Biophys. Acta 123, 298 (1966). 248. Kissane, J.M. and Robins, E. J . B i o l . Chem. 233, 184 (1958). 249. Mandel, P., Rein, H., Harth-Edel, S. and M a r d e l l , R., i n "Comparative Neurochemistry", p. 149. Pergamon P r e s s , New York (1964). 250. Rappoport, D.A., F r i t z , R.R. and Myers, J.L. i n L a j t h a , A. "Handbook of Neurochemistry", V o l . 1, p. 101, Plenum Press (1969). 251. Adams, D.H. Biochem. J . 98, 636 (1966). 252. Sung, S.C. Can. J . Biochem. 47, 47 (1969). 253. Altman, J . i n L a j t h a , A. "Handbook of Neurochemistry", V o l . 2, p. 137, Plenum Press (1969). 254. M a r g o l i s , F.L. J . Neurochem. 16, 447 (1969).. 255. B r a s e l , J.A., Ehrenkranz, R.A. and Winick, M. Develop. B i o l . 23, 424 (1970). 256. Bharucha, A.D. and Murthy, M.R.V. Can. J . Biochem. 49, 978 (1971). 257. Murthy, M.R.V. and Bharucha, A.D. Can. J . Biochem. 49, 1285 (1971). 258. Murthy, M.R.V. and Bharucha, A.D. Can. J . Biochem. 50, 186 (1972). 259. Bessman, M.J., Lehman, I.R., Simms, E.S. and Kornberg, A. J . B i o l . Chem. 233, 171 (1958). - 177 -260. Reichard, P., C a n e l l a k i s , Z.N. and C a n e l l a k i s , E.S. J . B i o l . Chem. 236, 2514 (1961). 261. Gray, E.D., Weissman, S.M., Ric h a r d s , J . , B e l l , D., K e i r , H.M., S m e l l i e , R.M.S. and Davidson, J.N. Biochim. Biophys. Acta 45_, 111 (1960). 262. Roth, J.S. i n B i a n c h i , C P . and H i l f , R. " P r o t e i n Metabolism and B i o l o g i c a l F u n c t i o n , p. 141, Rutgers U n i v e r s i t y Press (1970). 263. Sung, S.C B r a i n Res. 35, 268 (1971). 264. Altman, J . J . Comp. Neurol. 128, 431 (1966). 265. Gurdon, J.B. and Woodland, H.R. B i o l . Rev. 43_, 233 (1968). 266. H a r r i s , H. J . C e l l S c i . 2, 23 (1967). 267. Thompson, L.R. and McCarthy, B.J. Biochem. Biophys. Res. Commun. 30, 166 (1968). 268. Maaloe, 0. and Hanawalt, P.C J . Mol. B i o l . 3, 144 (1961). 269. Schaechter, M. Cold Spring Harbor Symp. Quant. B i o l . 26, 53 (1961). 270. Lark, K.G., Repko, T. and Hoffman, E.J. Biochim. Biophys. Acta 76, 9 (1963). 271. L i t t l e f i e l d , J.W. and Jacobs, P.S. Biochim. Biophys. Acta 108, 652 (1965). 272. S a l a s , J . and Green, H. Nature New B i o l o g y 229. 165 (1971). 273. A l b e r t s , B.M., Amodio, F . J . , J e n k i n s , M., Gutmann, E.D. and F e r r i s , F.L. Cold Spring Harbor Quant. B i o l . 33, 289 (1968). - 178 -274. Masker, W.E. and E b e r l e , H. Proc. N a t l . Acad. S c i . U.S. 68, 2549 (1971). 275. Szent-Gyorgyi, A. Proc. N a t l . Acad. S c i . U.S. 57_, 1642 (1967). 276. Kasakura, S. and Lowenstein, L. Nature 208, 794 (1965). 277. Erhan, S., R e i s h e r , S., Franko, E.A., Kamath, S.A. and Rutman, R.J. Nature 225, 340 (1970). 278. Erhan, S. Nature 219, 160 (1968). 279. A l b e r t s , B.M. and Frey, L. Nature 227_, 1313 (1970). 280. Kosaganov, Y.N. , Zarudnaja, M.I., L a z u r k i n , Y.S. and Frank-Kamenetskii, M.D. Nature New B i o l o g y 231, 212 (1971). 281. Tabor, H. and Tabor, C.W. Ann. Rev. Pharmacol. 16, 247 (1964). 282. Bacharach, U. Ann. Rev. M i c r o b i o l . 24, 109 (1970). 283. Stevens, L. B i o l . Rev. 45, 1 (1970). 284. Dykstra, W.G. and Herbst, E.J. Science 149, 428 (1965). 285. R u s s e l l , D.H. and McVicker, T.A. Biochim. Biophys. Acta 244, 85 (1971). 286. Raina, A. A c t a p h y s i o l . scand. 60 (Suppl. 1) 218 (1963). 287. R u s s e l l , D.H. and Levy, C.C. Cancer Res. 31, 248 (1971). 288. R u s s e l l , D.H. Nature New B i o l o g y 233, 144 (1971). 289. Ham, R.G. Biochem. Biophys. Res. Commun. 14, 34 (1964). 290. B e r t o s s i , F. , Bagni, N. , M o r u z z i , G. and C a l d a r e r a , CM. E x p e r i e n t i a 21, 80 (1965). - 179 -291. Dion, A.S. and Herbst, E.J. Proc. N a t l . Acad. S c i . U.S. 58, 2367 (1967). 292. Rosenthal, S.M. and Tabor, CW. J . Pharmacol. 116, 131 (1956). 293. P e r r y , T.L. , Hansen, S., Foulks, J.G. and L i n g , CM. J . Neurochem. 12, 397 (1965). 294. C a l d a r e r a , CM. , M o r u z z i , M.S., Rosseni, C. and B a r b i r o l i , B. J . Neurochem. 16, 309 (1969). 295. Shimizu, H., Kakimoto, Y. and Sano, I . Nature 207, 1196 (1965). 296. C a l d a r e r a , CM., B a r b i r o l i , B. and M o r u z z i , G. Biochem. J . 97, 84 (1965). 297. Pearce, L.A. and Schanberg, S.M. Science 166, 130 (1969). 298. M o r u z z i , G. , B a r b i r o l i , B. and C a l d a r e r a , CM. Biochem. J . 107, 609 (1968). 299. R u s s e l l , D.H., Medina, V.J. and Snyder, S.H. J . B i o l . Chem. 245, 6732 (1970). 300. Dion, A.S. and Cohen, S.S. Proc. N a t l . Acad. S c i . U.S. 69_, 213 (1972). 301. Winick, M. and Noble, A. Develop. B i o l . 12., 451 (1965). 302. Peterson, E.A. and Sober, H.A. A n a l . Chem. 31, 857 (1959). 303. Sung, S.C. J . Neurochem. 15, 477 (1968). 304. B a r i l , E., Brown, 0. and L a s z l o , J . Biochem. Biophys. Res. Commun. 43, 754 (1971). 305. Marmur, J . J . Mol. B i o l . 3_, 208 (1961). - 180 -306. Sinsheimer, R.L. and Koerner, J.F. J . B i o l . Chem. 198, 293 (1952). 307. F i s k e , C H . and Subbarow, Y. J . B i o l . Chem. 66_, 375 (1925). 308. Mandel, P., Dr a v i d , A.R. and Pete, N. J . Neurochem. 14, 301 (1967). 309. Noda, L. and Kuby, S.A. J . B i o l . Chem. 226, 541 (1957). 310. M a r t i n , R.G. and Ames, B.N. J . B i o l . Chem. 236, 1372 (1961). 311. Umbreit, W.W., B u r r i s , R.H. and S t a u f f e r , J.F. Manometric Techniques, 4th ed., Burgess P u b l . Co., Minneapolis (1964). 312. Lowry, O.H., Rosebrough, N.J., F a r r , A.L. and R a n d a l l , R.J. J . B i o l . Chem. 193, 265 (1951). 313. Bollum, F.J. i n Davidson, J.N. and Cohen, W.E. "Progress i n N u c l e i c A c i d Research", V o l . 1, p. 1. Academic Press New York (1963). 314. Otsuka, H. and Terayama, H. Biochim. Biophys. Acta 123, 274 (1966). 315. Sasada, M. and Terayama, H. Biochim. Biophys. Acta 190, 73 (1969). 316. Miyamoto, M. and Terayama, H. Biochim. Biophys. Acta 228, 324 (1971). 317. W i l l i a m s , R.J.P. i n Boyer, P.D., Lardy, H. and Hyrback, K. "The Enzymes", 2nd ed., V o l . 1, p. 391, New York, Academic Press (1959). - 181 -318. Reich, E. and Goldberg, I.H. i n Davidson, J.N. and Cohen, W.E. "Progress i n N u c l e i c A c i d Research and Molecular B i o l o g y " , V o l . 3, p. 183, Academic P r e s s , New York (1964). 319. Wang, T.Y. Proc. Soc. E x p t l . B i o l . Med. 129, 469 (1968). 320. M c l l w a i n , H. i n "Biochemistry and the C e n t r a l Nervous System", p. 270, J . & A. C h u r c h i l l L t d . (1966). 321. Long, G.L. and Garren, L.D. Biochem. Biophys. Res. Commun. 46, 1228 (1972). 322. Brewer, E.N. and Rusch, H.P. Biochem. Biophys. Res. Commun. 25, 579 (1966). 323. O'Brien, R.L., O l e n i c k , J.G. and Hahn, F.E. Proc. N a t l . Acad. S c i . U.S. 55, 1511 (1960). 324. Mandel, M. J . Mol. B i o l . _5, 435 (1962). 325. Wyatt, G.R. and Cohen, S.S. Biochem. J . 55, 774 (1953). 326. Sinsheimer, R.L. Science 120, 551 (1954). 327. L i q u o r i , A.M., Co s t a n t i n o , L., C r e s e n z i , V., E l i a , V., G i g l i o , E., P u l i t i , R., De S a n t i s Savino, M. and V i t a g l i a n o , V. J . Mol. B i o l . 24, 113 (1967). 328. Takeda, Y. J . Biochem. 66, 345 (1969). 329. Takeda, Y. and I g a r a s h i , K. Biochem. Biophys. Res. Commun. 37, 917 (1969). 330. Schwimmer, S. Biochim. Biophys. Acta 166, 251 (1968). 331. T i s s i e r e s , A., Watson, J.D., S c h l e s s i n g e r , D. and H o l l i n g w o r t h , B.R. J . Mol. B i o l . 1, 221 (1959). 

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