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DNA polymerases from nuclei of rat intestinal mucosa Krasny, Jiri Ladislav 1973

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DNA POLYMERASES FROM NUCLEI OF RAT.INTESTINAL MUCOSA BY JIRI LADISLAV KRASNY M.D., Charles University, Prague, Czechoslovakia, 1964 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n the Department of BIOCHEMISTRY We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA May, 1973 In 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 requirements f o r an advanced degree at the U n i v e r s i t y o f B r i t i s h Columbia, 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 study. 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 purposes may be granted by the Head of my Department or by h i s r e p r e s e n t a t i v e s . I t i s understood t h a t copying or p u b l i c a t i o n of 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 not be allowed without 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 i -ABSTRACT DNA polymerase a c t i v i t y associated with p u r i f i e d n u c l e i of rat i n t e s t i n a l mucosa was studied. Two DNA polymerase a c t i v i t i e s have been i s o l a t e d , p a r t i a l l y p u r i f i e d and characterized. One of the enzymes was extracted from p u r i f i e d n u c l e i with 10 mM Tris - H C l , pH 8.0, containing 5 mM d i t h i o t h r e i t o l while the second enzyme, which was associated with the nuclear deoxyribonucleoprotein complex, was extracted only i n a high i o n i c strength medium containing 1 M NaCl i n 0.1 M Tris-HCl, pH 8.0 and 5 mM d i t h i o t h r e i t o l . The molecular weights of these nuclear DNA polymerases were estimated by g e l f i l t r a t i o n on Sephadex G-150. Two peaks of DNA polymerase a c t i v i t y were detected when the T r i s - s o l u b l e extract was chromatographed. The molecular weights of these peaks of a c t i v i t y were calculated to be 266,000 and 104,000. I t was concluded that the f i r s t peak of a c t i v i t y represented an aggregate of the second. A s i n g l e peak of DNA polymerase a c t i v i t y was obtained when the NaCl-soluble nuclear extract was chromatographed on Sephadex G-150. I t corresponded to a molecular weight of approximately 40,000. Chromatography on DEAE-cellulose indicated that the two enzymes d i f f e r e d i n i o n i c charge. The bulk of the T r i s - s o l u b l e DNA polymerase a c t i v i t y eluted with 0.045-0.055 M KC1, while the NaCl-soluble enzyme had a higher a f f i n i t y f o r the anion exchange r e s i n and was not eluted u n t i l the KC1 concentration was 0.165-0.21 M. The p a r t i a l l y p u r i f i e d enzymes were very l a b i l e . Storage at 4°C, 0°C or -20°C did not increase enzyme s t a b i l i t y . The presence of g l y c e r o l , which had no e f f e c t on - i i i -enzyme a c t i v i t y , helped maintain the s t a b i l i t y of both enzymes for at lea s t 1 month at -20°C. The enzymic properties of the nuclear DNA polymerases d i f f e r e d . In T r i s - H C l buffer, a pH of 7.5 was optimal for the polymerase reaction catalyzed by eit h e r enzyme, but i n phosphate buffer the pH optima were 7.2 and 6.0 f o r the T r i s - s o l u b l e and NaCl-soluble enzymes, res p e c t i v e l y , 2+ The presence of DNA, a l l 4 deoxynucleoside 5'-triphosphates and Mg ions was required f o r the a c t i v i t y of both crude and p a r t i a l l y p u r i f i e d 2+ 2+ forms of the nuclear DNA polymerases. Substi t u t i o n of Mn or Ca 2+ fo r Mg resulted i n lower enzymic a c t i v i t y . The addition of d i t h i o -t h r e i t o l greatly enhanced the a c t i v i t y of both enzymes, e s p e c i a l l y the p u r i f i e d preparations. The presence of t h i o l reagents, p_-hydroxy-mercuribenzoate and N-ethylmaleimide, i n h i b i t e d both of the nuclear DNA polymerase a c t i v i t i e s . In the presence of 1 mM n a l i d i x i c acid the a c t i v i t y of the T r i s - s o l u b l e enzyme was abolished whereas the NaCl-soluble DNA polymerase a c t i v i t y was greatly enhanced. The a c t i v i t i e s of the nuclear DNA polymerases were also affected d i f f e r e n t l y by monovalent cations. The addition of NH^+, K + or Na + to the assay mixture i n h i b i t e d the a c t i v i t y of the T r i s - s o l u b l e enzyme but stimulated by 30-170% the a c t i v i t y of the NaCl-soluble DNA polymerase. The two enzymes also d i f f e r e d i n template preference. The crude T r i s - s o l u b l e DNA polymerase functioned equally w e l l with ei t h e r heat-denatured or native DNA, while the p u r i f i e d form showed a s l i g h t preference f o r native over heat-denatured DNA. The p u r i f i e d NaCl-soluble DNA polymerase, which i n crude extracts c o n s i s t e n t l y preferred native DNA as template, showed a s t r i c t dependence on native DNA. - i v -Differences between the T r i s - s o l u b l e and NaCl-soluble DNA polymerases i n e x t r a c t a b i l i t y from p u r i f i e d n u c l e i , molecular vweight, i o n i c charge and i n t h e i r enzymic properties c l e a r l y i n d i c a t e that two d i s t i n c t DNA polymerase a c t i v i t i e s are associated with p u r i f i e d n u c l e i prepared from rat i n t e s t i n a l mucosa c e l l s . The close a s s o c i a t i o n between the NaCl-soluble DNA polymerase and the deoxyribonucleoprotein complex and i t s absolute dependence on native DNA template support the conclusion that i t i s a repair enzyme i n vivo. The r o l e of the T r i s -soluble enzyme i s less c e r t a i n . - v -TABLE OF CONTENTS Page INTRODUCTION 1 B a c t e r i a l DNA Polymerases ^ Mammalian DNA Polymerases 4 A. I n t r a c e l l u l a r l o c a t i o n 4 B. Characterization 6 The Present Investivation 8 MATERIALS AND METHODS 10 Materials 10 Methods 11 A. Preparation of crude enzyme extracts 11 1. Preparation of nuclear f r a c t i o n 11 2. Extraction of DNA polymerase from p u r i f i e d n u c l e i 12 B. Enzyme assays 16 1. DNA polymerase assay 16 2. Terminal deoxyribonucleotidyltransferase assay 17 3. DNase I assay 17 4. Triphosphatase assay 18 5. Protein determination 18 6. Heat-denatured DNA 19 C. Column chromatography on DEAE-cellulose 19 1. Preparation of DEAE-cellulose column 19 2. Procedure f o r DEAE-cellulose chromatography 19 - v i -Page D. Gel f i l t r a t i o n on Sephadex G-150 2 0 1. Preparation of Sephadex G-150 column 21 2. C a l i b r a t i o n of Sephadex G-150 column 21 3. Procedures for gel f i l t r a t i o n on Sephadex G-150 22 4. C a l c u l a t i o n of molecular weight by g e l f i l t r a t i o n 23 RESULTS AND DISCUSSION A. Properties of crude nuclear DNA polymerases .. 24 B. P a r t i a l p u r i f i c a t i o n of crude enzymes 28 1. Ammonium s u l f a t e f r a c t i o n a t i o n 28 2. Chromatography on DEAE-cellulose 30 3. Chromatography on phosphocellulose 34 C. P a r t i a l l y p u r i f i e d nuclear DNA polymerases — 36 1. Properties of p a r t i a l l y p u r i f i e d nuclear DNA polymerases 36 2. Time-course of the rea c t i o n 40 3. E f f e c t of increasing amounts of protein on the incorporation of [ 3H]dTTP 40 4. E f f e c t of template concentration on the incorporation of [ 3H]dTTP 43 5. Substrate concentration curves 45 6. E f f e c t of pH 47 7. E f f e c t of divalent cations 49 8. E f f e c t of monovalent cations 52 9. E f f e c t of i n h i b i t o r s 5 8 - v i i -Page 10. S t a b i l i t y of nuclear DNA polymerases 63 a. S t a b i l i t y upon storage at various temperatures 63 b. S t a b i l i t y at d i f f e r e n t pH values 67 D. Molecular weight determination by gel f i l t r a t i o n 68 1. NaGl-soluble DNA polymerase 68 2. T r i s - s o l u b l e DNA polymerase 69 CONCLUSIONS 79 BIBLIOGRAPHY 84 - v i i i -LIST OF TABLES Table Page I D i s t r i b u t i o n of DNA polymerase a c t i v i t y i n p u r i f i e d n u c l e i i s o l a t e d from rat i n t e s t i n a l mucosa 24 II Properties of the nuclear DNA polymerases 25 I I I Reaction requirements of the nuclear DNA polymerases 27 IV Ammonium s u l f a t e f r a c t i o n a t i o n 29 V Chromatography of crude T r i s - s o l u b l e and NaCl-soluble DNA polymerases on DEAE-cellulose 32 VI Reaction requirements of the T r i s - s o l u b l e DNA polymerase 37 VII Reaction requirements of the NaCl-soluble DNA polymerase 38 VIII E f f e c t of t h i o l reagents on nuclear DNA polymerases. 60 IX E f f e c t of n a l i d i x i c acid on nuclear DNA polymerases. 62 X Gel f i l t r a t i o n on Sephadex G-150 of T r i s - s o l u b l e and NaCl-soluble DNA polymerases 78 - i x -LIST OF FIGURES Figure Page 1 Electron micrograph of p u r i f i e d n u c l e i i s o l a t e d from rat i n t e s t i n a l mucosa c e l l s ^ 2 Chromatography of T r i s - s o l u b l e nuclear extract on DEAE-cellulose 3 1 3 Chromatography of NaCl-soluble nuclear extract on DEAE-cellulose 3 1 4 Time-course of the reaction f o r the p a r t i a l l y p u r i f i e d T r i s - s o l u b l e DNA polymerase 41 5 Time-course of the reaction of the p a r t i a l l y p u r i f i e d NaCl-soluble DNA polymerase 41 6 E f f e c t of increasing amounts of T r i s - s o l u b l e p rotein on the incorporation of [ 3H]dTTP 42 7 E f f e c t of increasing amounts of NaCl-soluble protein on the incorporation of [ 3H]dTTP 42 8 E f f e c t of template concentration on the incorporation of [ 3H]dTTP 44 A. T r i s - s o l u b l e DNA polymerase B. NaCl-soluble DNA polymerase 9 Substrate concentration curves 46 A. T r i s - s o l u b l e DNA polymerase B. NaCl-soluble DNA polymerase 10 E f f e c t of pH on p a r t i a l l y p u r i f i e d nuclear DNA polymerases 48 A. T r i s - s o l u b l e DNA polymerase B. NaCl-soluble DNA polymerase 11. E f f e c t of divalent cations on nuclear DNA 50 A. T r i s - s o l u b l e DNA polymerase B. NaCl-soluble DNA polymerase 12 E f f e c t of monovalent cations on the a c t i v i t y of the T r i s - s o l u b l e DNA polymerase , 53 - x -Figure Page 13 E f f e c t of monovalent cations on the a c t i v i t y of NaCl-soluble DNA polymerase 54 14 E f f e c t of Na acetate on the a c t i v i t y of nuclear DNA polymerases 56 15 S t a b i l i t y upon storage at various temperatures of the T r i s - s o l u b l e DNA polymerase 64 16 S t a b i l i t y upon storage at various temperatures of the NaCl-soluble DNA polymerase 66 17 C a l i b r a t i o n curve f o r the estimation of the molecular weight of the NaCl-soluble DNA polymerase. ?0 18 Gel f i l t r a t i o n of the NaCl-soluble DNA polymerase on Sephadex G-150 71 19 C a l i b r a t i o n curve f o r the estimation of the molecular weight of the T r i s - s o l u b l e DNA polymerase. 73 20 Gel f i l t r a t i o n of the T r i s - s o l u b l e DNA polymerase on Sephadex G-150 1 L r - x i -ACKNOWLEDGEMENTS I would l i k e to express my thanks to Dr. S.H. Zbarsky for h i s understanding and c r i t i c i s m throughout the course of both the research and w r i t i n g of th i s t h e s i s . To my colleague, Dr. Rozanne Poulson, I extend my sincerest thanks for her invaluable help and encouragement during the past two years. Thanks are also due to Dr. Ivo Hynie for h i s many h e l p f u l suggestions, to Mr. Peter Flanagan f o r h i s advice and help with enzyme i s o l a t i o n and to Miss Ester Lo for kin d l y performing electron~micrographs. To my wife Elena and my three c h i l d r e n go my warmest thanks for t h e i r patience and my apologies f o r spending more time with DNA polymerase than with them. It i s a pleasure to acknowledge the personal support of the Medical Research Council i n the form of Fellowship. - 1 -INTRODUCTION The primary genetic material of most c e l l s i s deoxyribonucleic acid (DNA). I t functions by coding f o r the amino acid sequences of proteins thereby determining the biochemical s p e c i f i c i t y of the c e l l . This genetic information i s responsible for the perpetuation of heredity since i t passes from parent to progeny on c e l l d i v i s i o n . DNA biosynthesis, e s p e c i a l l y the processes involved i n the exact r e p l i c a t i o n of DNA molecules during chromosome d u p l i c a t i o n , i s therefore of great i n t e r e s t and has been extensively studied. The e l u c i d a t i o n of the complementary double h e l i c a l structure of DNA molecules by Watson and C r i c k 1 provided the f i r s t i n s i g h t of the mechanism by which a p a r t i c u l a r pattern of nucleotides may be r e p l i c a t e d . Each chain of the DNA molecule serves as a template for the synthesis of a complementary chain, so that two r e p l i c a s of the o r i g i n a l double stranded structure are produced. This was confirmed by Meselson and 2 Stahl who demonstrated that a "semi-conservative" type of r e p l i c a t i o n occurs i n vivo. The complexity of DNA synthesis i s evident from the many postulated mechanisms of DNA synthesis i n b a c t e r i a and mammalian ti s s u e s . B a c t e r i a l DNA Polymerases A large number of compounds was studied as possible DNA precursors i n vivo, but the enzymatic mechanism involved i n DNA synthesis remained unknown u n t i l the experiments of Arthur Romberg and h i s 3 c o l l a b o r a t o r s . Using extracts of E_. c o l i , these investigators - 2 -demonstrated the presence of an enzyme which converted deoxynucleoside polyphosphates into polymeric material. The enzyme catalyzing t h i s r e a c t i o n was termed DNA polymerase (EC 2.7.7.7. Deoxynucleoside-4 triphosphate:DNA deoxynucleotidyltransferase). The o v e r a l l reaction may be i l l u s t r a t e d as follows: ndATP ndGTP ndCTP ndTTP + DNA enzyme DNA + Mg 2+ dAMP dGMP dCMP dTMP + 4nPPi n DNA polymerase i s now known to catalyze the addi t i o n of mono-nucleotide units to the 3'-hydroxyl terminus of a template DNA chain with synthesis proceeding i n the 5' to 3' d i r e c t i o n . T h e synthesized material i s DNA with p h y s i c a l c h a r a c t e r i s t i c s resembling those of the template. E. c o l i DNA polymerase, sometimes re f e r r e d to as "the Romberg enzyme" or DNA polymerase I, has been p u r i f i e d to apparent homogeneity and extensively studied. This DNA polymerase was found to possess several c a t a l y t i c properties. However, the findings that the enzyme does not bind to i n t a c t double-stranded DNA and that DNA polymerization occurs only i n the 5' to 3' d i r e c t i o n , raised some doubts concerning the p h y s i o l o g i c a l r o l e of the Romberg enzyme i n DNA r e p l i c a t i o n . Examination of d i v i d i n g b a c t e r i a by autoradiography^ or by gene d u p l i c a t i o n had indicated that there was simultaneous sequential r e p l i c a t i o n of both strands. Several schemes were proposed, - 3 -therefore, which attempted to accommodate these c o n f l i c t i n g f i n d i n g s . g Okazaki and coworkers reported the i s o l a t i o n of small pieces of DNA at the nascent r e p l i c a t i n g region. They suggested that the r e p l i c a t i o n i n 5' to 3' d i r e c t i o n proceeds f or some distance and then switches to the complementary strand to form a fork, which i s then cleaved by endonuclease. Repetition of t h i s process r e s u l t s i n small pieces of DNA near the r e p l i c a t i n g fork which are linked together by l i g a s e . This hypothesis was not e n t i r e l y s a t i s f a c t o r y as the r e p l i c a t i o n i s staggered, a l t e r n a t i n g from one strand to the other, and i s , therefore, i n disagreement with the findings of Cairns and Davern and Sueoka et a l . 7 mentioned above. 9 These observations together with the f i n d i n g that the 5' to 3' nuclease a c t i v i t y of E. c o l i DNA polymerase I i s able to remove mismatched bases and thymine dimers suggested a repair function f o r t h i s enzyme i n vivo. This idea was strengthened by the discovery of mutants of E_. c o l i defective i n DNA polymerase I . 1 ^ These mutants have e s s e n t i a l l y normal growth c h a r a c t e r i s t i c s but are s e n s i t i v e to u l t r a v i o l e t i r r a d i a t i o n . A membrane-bound enzyme which i s apparently involved i n r e p l i c a t i o n i n DNA polymerase I defective mutants was 11 i s o l a t e d by Knippers and was termed DNA polymerase I I . Unlike the DNA~polymerase I (Romberg enzyme), t h i s enzyme i s strongly i n h i b i t e d by s u l f h y d r y l reagents and i s r e s i s t a n t to an antiserum which i n h i b i t s the Romberg enzyme. While studying DNA polymerase II from E_. c o l i , Romberg and Gefter found another DNA-synthesizing enzyme, present i n low amounts i n DNA polymerase I defective mutants, which was temperature - 4 -s e n s i t i v e and d i f f e r e d from both DNA polymerase I and II on the basis 80 of i t s chromatographic behavior and s e n s i t i v i t y to s a l t s . Similar 13 findings were reported by other i n v e s t i g a t o r s . Recently, mutants 14 15 defective i n DNA polymerase II have been i s o l a t e d . ' Experiments with these mutants and with the thermosensitive mutants defective i n 16 DNA r e p l i c a t i o n suggest that DNA polymerase III enzyme i s required for DNA r e p l i c a t i o n i n E_. c o l i . These and numerous other observations c l e a r l y show the complexity of DNA r e p l i c a t i o n and emphasize the d i f f i c u l t y of c o r r e l a t i n g r e s u l t s obtained i n i n v i t r o studies with the events taking place i n vivo. Mammalian DNA Polymerases A. I n t r a c e l l u l a r Location 3 The discovery of DNA polymerase a c t i v i t y i n E. c o l i was quickly extended to a wide v a r i e t y of mammalian tissue s . Mammalian DNA polymerase a c t i v i t y was f i r s t observed i n soluble cytoplasmic f r a c t i o n s of ti s s u e homogenates. 1 7 ^ The discrepancy between the l o c a -t i o n of DNA polymerase i n the cytoplasm and the b e l i e f that DNA should be synthesized i n the nucleus was resolved p a r t i a l l y when n u c l e i , prepared e i t h e r i n non-aqueous medium or i n aqueous medium containing 2+ 2+ Ca or Mg ions, were found to possess DNA polymerase a c t i v i t y . In a l l these studies, however, considerable DNA polymerase a c t i v i t y 21-23 was also detected i n the cytoplasmic f r a c t i o n . - 5 -Later a second DNA polymerase a c t i v i t y was observed associated 24 with the chromatin extracted from n u c l e i with buffered 1 M NaCl. Thus ex t r a c t i o n of n u c l e i with b u f f e r of low and high i o n i c 25 26 strength y i e l d s d i s t i n c t DNA polymerase a c t i v i t i e s . ' In general, i t has been found that one of the nuclear polymerases c l o s e l y resembles the cytoplasmic enzyme whereas the other nuclear enzyme appears to 25 27 possess unique properties. ' However, the precise r e l a t i o n s h i p between the cytoplasmic and nuclear enzymes has not been established although i t seems l i k e l y that they are i d e n t i c a l . Of the cytoplasmic DNA polymerases, only the enzyme located i n 28—30 the mitochondria has been extensively characterized. B a r i l 31 et a l . found DNA polymerase a c t i v i t y associated with ribosomes and smooth membranes prepared from r a t l i v e r homogenates. However, 32 experiments i n our laboratory showed remarkable s i m i l a r i t y among the properties of the DNA polymerase a c t i v i t i e s associated with the cytoplasmic, ribosomal and T r i s - s o l u b l e nuclear f r a c t i o n s . Since electron micrographs indicated damage to the outer membrane of some of our p u r i f i e d n u c l e i , the presence of DNA polymerase a c t i v i t y i n the ribosomal and cytoplasmic f r a c t i o n s may have been due to leakage of the T r i s - s o l u b l e nuclear enzyme during t i s s u e d i s r u p t i o n . Alterna-t i v e l y , t h i s enzyme may o r i g i n a t e i n the cytoplasm and be transferred to the nucleus. - 6 -B. Characterization Very few mammalian DNA polymerases have been s u f f i c i e n t l y p u r i f i e d to permit comparison with the b a c t e r i a l and phage enzymes. Procedures i n v o l v i n g aci d p r e c i p i t 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 , chromatography on DEAE-cellulose, phosphocellulose, hydroxyapatite and Sephadex and i n some cases i s o e l e c t r i c f o c u s i n g have been used to 33 p u r i f y 150- to 400-fold the DNA polymerases from c a l f thymus gland, 34 25 29 E h r l i c h a s c i t e s tumor c e l l s , KB c e l l s , rat l i v e r mitochondria, 35 and mitochondria from rat i n t e s t i n a l mucosa c e l l s . Despite these successes, the s p e c i f i c a c t i v i t y of the p u r i f i e d enzymes i s considerably lower (30-100 times) than that of the most highly p u r i f i e d E. c o l i DNA polymerase. Most of the mammalian DNA polymerases have s i m i l a r reaction requirements, but differences have been observed which may r e f l e c t e i t h e r species s p e c i f i c i t y or f u n c t i o n a l d i f f e r e n c e s . A l l mammalian 2+ DNA polymerases require the presence of Mg ion for maximum a c t i v i t y , but the optimum concentration varies according to the enzyme source. 2+ Substi t u t i o n of another divalent cation for Mg i n v a r i a b l y r e s u l t s i n lower enzymic a c t i v i t y . ^ 7 Few mammalian DNA polymerases display a s t r i c t requirement for 37 38 a l l four deoxynucleoside triphosphates. ' Many of the enzyme preparations when presented with only three triphosphates can synthesize at 25-50% of the rate observed i n the presence of a l l four deoxy-nucleoside triphosphates and appreciable a c t i v i t y i s often obtained 25 39 with only one deoxynucleoside triphosphate present. ' . The l a t t e r - 7 -observation may be due to contamination with terminal deoxynucleotidyl-38 25 transferase. Sedwick et a l . however, measured the number of av a i l a b l e 3'-hydroxyl termini i n DNA template, and demonstrated that the amount of si n g l e nucleotide incorporated was always s u b s t a n t i a l l y l e s s than the number of i n i t i a t i o n s i t e s a v a i l a b l e f or s i n g l e base addition. This indicates that mammalian DNA polymerase preparations are 40 not n e c e s s a r i l y contaminated by terminal transferase a c t i v i t y . The presence of DNA i s necessary for the polymerase reaction. 33 41 42 Some polymerase enzymes prefer denatured over native DNA as template, ' ' while others function equally w e l l with ei t h e r native or denatured DNA 34 template. The largest group of enzymes, however, prefers native DNA 35 39 43 44 as template. > » > j n m a n y cases native DNA activated by b r i e f 25 27 treatment with DNase.'I i s the most e f f i c i e n t template. ' Treatment with DNase I increases the number of 3'-hydroxyl termini, and also 45 generates a large number of small sin g l e stranded breaks. The question of template preference i s quite complex and no d e f i n i t e conclusions have been reached. Since d i f f e r e n t enzymes from the 42 same tiss u e show s i m i l a r template requirements Wang suggested that template s p e c i f i c i t y may be re l a t e d to tissue s p e c i f i c i t y rather than to the i n t r a c e l l u l a r f r a c t i o n . The molecular weights of mammalian DNA polymerases from various 40 sources have been reviewed recently. This survey shows that most enzymes found i n high speed supernatants of mammalian tissue homogenates sediment at 6 to 8S on sucrose densityligradients, corresponding to a molecular weight of around 100,000. On the other hand, a molecular - 8 -weight value of 40,000 to 50,000 was reported f o r the DNAi-polymerase i s o l a t e d from the deoxynucleoprotein complex of n u c l e i prepared from 46 46 rabbit bone marrow. Chang and Bollum reported the presence of a low molecular weight species of DNA polymerase i n some 40 d i f f e r e n t mammalian t i s s u e s . The Present Investigation The present i n v e s t i g a t i o n extends the work i n i t i a t e d by Leung and 47 Zbarsky. The i n t e s t i n a l t i s s u e was chosen because of i t s high 48 49 m i t o t i c rate. ' DNA polymerase a c t i v i t y of i n t e s t i n a l mucosa tissu e i s also very high and i s comparable with that of regenerating r a t l i v e r . The presence of DNA polymerase a c t i v i t y i n both n u c l e i and cytoplasm of mucosal c e l l s has been established.^7,51,52 The present work involves a p a r t i a l p u r i f i c a t i o n and chara c t e r i z a t i o n of nuclear DNA polymerase a c t i v i t y . The DNA polymerase, previously 47 described by Leung and Zbarsky, was studied and the presence of another nuclear DNA polymerase was demonstrated. P u r i f i c a t i o n and char a c t e r i z a t i o n of the nuclear enzymes f a c i l i t a t e d a comparison with the cytoplasmic and mitochondrial enzymes from the mucosal t i s s u e . Highly p u r i f i e d n u c l e i were obtained by a combination of the 53 54 procedures described by Clark and Porteous, and P e r r i s . DNA polymerase was i n i t i a l l y extracted from the p u r i f i e d n u c l e i according 39 to the method described by Mantsavinos and then a second extraction 24 was c a r r i e d out using a method described by Patel et a l . - 9 -The p a r t i a l l y p u r i f i e d enzymes showed many s i m i l a r properties 2+ (absolute requirement f o r Mg and DNA template, a l l four deoxy-nucleoside triphosphates necessary for maximum a c t i v i t y , the same optimum pH for both p u r i f i e d polymerases i n Tris-HCl b u f f e r ) ; however, a number of differences were also noted (preference for native or heat-denatured DNA, e f f e c t of monovalent cations, pH optima i n phosphate buffer, s e n s i t i v i t y to n a l i d i x i c acid, behavior on DEAE-cellulose and molecular weight values). The enzyme which i s r e a d i l y extracted from n u c l e i with Tris-HCl b u f f e r resembles the cytoplasmic enzyme, whereas the enzyme t i g h t l y bound to the deoxynucleoprotein complex and extracted only with 1 M NaCl possesses unique properties. The function of the two d i s t i n c t DNA polymerase enzymes present i n p u r i f i e d nuclear preparations of r a t i n t e s t i n a l mucosa c e l l s i s discussed i n r e l a t i o n to the properties of the enzymes. - 10 -MATERIALS AND METHODS MATERIALS Mat e r i a l Whatman microgranular DEAE-c e l l u l o s e (DE 32) Phosphocellulose, Cellex P Sephadex G-150 Ca l i b r a t i o n K i t for mol. wt. determination D i t h i o t h r e i t o l Unlabeled deoxynucleoside 5'-triphosphates [Me- H] Thymidine 5'-triphosphate [ ( t e t r a l i t h i u m s a l t ) (17.5 C/mmole)] Calf thymus DNA C r y s t a l l i n e bovine albumium (Fraction V) Tris(hydroxymethyl)aminomethane p_-Hydroxymercuribenzoate, Na s a l t N a l i d i x i c acid N-Ethyl maleimide B a c t e r i a l a l k a l i n e phosphatase A-Nitrophenylphosphate, disodium 1.4- bis-2-(4-Methyl-5-phenyloxazolyl)-benzene (dimethyl POPOP) 2.5- Diphenyloxazol (PPO) Deoxyribonuclease I from Bovine Pancrease Supplied by Reeve Angel Bio-Rad Laboratories Pharmacia Fine Chemicals Pharmacia Fine Chemicals Calbiochem P-L Biochemicals Schwarz-Mann Sigma Chemical Company Metrix (Div. of Armour Pharma-c e u t i c a l Company) Sigma Chemical Company Calbiochem Sigma Chemical Company Sigma Chemical Company Worthington Biochem. Corp. Raylo Packard Instrument Co. Kent Laboratories Sigma Chemical Company - 11 -A l l other chemicals and reagents used were purchased from Fisher S c i e n t i f i c Company. Male Wistar r a t s , weighing 190-210 g each, were obtained from the University of B r i t i s h Columbia, Animal Unit. METHODS A. Preparation of Crude Enzyme Extracts 1. Preparation of Nuclear F r a c t i o n A l l operations were performed between 0° and 4°C. Ten to twenty male Wister r a t s , weighing 190 to 210 g each, were k i l l e d by a blow to the head and immediately decapitated. The small i n t e s t i n e s were removed r a p i d l y and t h e i r contents flushed out with 0.154 M NaCI. The e n t i r e length of each i n t e s t i n e was everted and f i l l e d with 54 0.154 M NaCI as described by P e r r i s . Each i n t e s t i n e was washed by s w i r l i n g gently for 2 1/2 min i n 500 ml of i c e - c o l d s a l i n e and then twice i n the same volume of Krebs-Ringer phosphate buffer, pH 7.4, containing 6% (w/v) dextran and f i n a l l y i n s a l i n e again. Mucosal scrapings were obtained by stroking the mucosa gently with the edge of a glass s l i d e . The mucosal scrapings were suspended i n 3 volumes of 0.32 M sucrose i n TKM buffer (50 mM T r i s - H C l , pH 7.5, 25 mM KC1, 5 mM MgC^)"^ containing 10 mM 2-mercaptoethanol and homogenized i n a Potter-Elvehjem type homogenizer with 5 complete passes of the Teflon pestle r o t a t i n g at 650 rev/min. The homogenate was f i l t e r e d through two layers of nylon mesh and the f i l t r a t e centrifuged at 700 x g for 10 min. The supernatant was discarded and the p e l l e t was re-homogenized 4 times i n a S e r v a l l omnimixer using 4 volumes of 0.32 M sucrose-TKM - 12 -buf f e r each time. The sequence of homogenizations was: (1) 3 min at 350 rev/min, (2) 2 min at 350 rev/min followed by 2 min at 400 rev/min, (3) 3 min at 350 rev/min and (4) 2 min at 350 rev/min. Af t e r each homogenization the homogenate was centrifuged for 10 min at 700 x g. To p u r i f y the n u c l e i further the p e l l e t was resuspended i n 5 volumes of 0.32 M sucrose-TKM buffer and the suspension centrifuged f o r 10 min at 300 x g. This procedure was repeated 3-4 times. P u r i f i c a t i o n of n u c l e i was monitored under a l i g h t microscope f i t t e d with a phase contrast attachment. The p u r i f i e d nuclear f r a c t i o n was generally contaminated with le s s than 30-50 whole c e l l s per 1000 n u c l e i . However, the preparations did contain large numbers of ribosomes but only a few mitochondria and small amounts of c e l l u l a r debris. Electron micrographs (Fig. 1) showed that the majority of n u c l e i were i n t a c t , however, s l i g h t damage to the outer membrane of some had occurred. 2. Extraction of DNA Polymerase from P u r i f i e d Nuclei The p u r i f i e d nuclear p e l l e t was resuspended i n 3 volumes of 10 mM Tris-HCl, pH 8.0, containing 5 mM d i t h i o t h r e i t o l and homogenized i n a S e r v a l l omnimixer for 3 min at 375 rev/min. The homogenate was s t i r r e d on a magnetic s t i r r e r f o r 10 min at 0°C p r i o r to c e n t r i f u g a t i o n at 20,000 x g for 10 min. The supernatant was recentrifuged at 33,000 rev/min for 1 h i n a Spinco 50.1 rotor. The r e s u l t i n g supernatant was designated the T r i s - s o l u b l e nuclear extract. - 13 -Figure 1. Electron micrograph of p u r i f i e d n u c l e i i s o l a t e d from rat i n t e s t i n a l mucosa c e l l s . Magnification 15,000 times. - 14 -The 20,000 x g p e l l e t was re-extracted i n 8 volumes of 0.1 M Tr i s - H C l , pH 8.0, 1 M NaCl, and 5 mM d i t h i o t h r e i t o l and homogenized i n an ommimixer at 350 rev/min for 10 min followed by 3 min at 400 rev/min. The homogenate was s t i r r e d on a magnetic s t i r r e r at 0°C f o r 1 hour p r i o r to ce n t r i f u g a t i o n at 33,000 rev/min for 1 hour i n a Spinco 50.1 rotor. The supernatant was dialyzed for 4-6 hours at 4°C against 40 volumes of 20 mM Tris-HCl buffer, pH 7.5 and the p r e c i p i t a t e 5 removed by cent r i f u g a t i o n at 20,000 x g for 20 min. According to Wang th i s p r e c i p i t a t e i s probably a reconstituted DNA-histone complex which was d i s s o c i a t e d e a r l i e r with 1 M NaCl. The supernatant was designated the NaCl-soluble nuclear extract. Most mammalian DNA polymerases have been extracted from whole c e l l s or s u b c e l l u l a r p a r t i c l e s i n aqueous media generally consisting of 0.01-0.1 M T r i s - H C l buffer which sometimes contained 0.01-0.15 M NaCl or K C l . 2 ^ ' ^ ' " ^ ' " ^ Preliminary studies using various extraction procedures indicated that DNA polymerase was extracted from rat i n t e s t i n a l mucosa with T r i s - H C l buffer, pH 8.0, over a concentration range from 10 mM to 100 mM. When s i m i l a r concentrations of NaCl or KC1 were added to the T r i s - H C l buffer the amount of DNA polymerase a c t i v i t y obtained was almost i d e n t i c a l . However, the y i e l d increased when the s a l t concentration was increased to 0.14-0.15 M even though the deoxyribonucleoprotein complex was insolu b l e i n t h i s s a l t 24 concentration. Experiments indicated that 0.15 M NaCl i n combination - 15 -with mechanical shearing f a c i l i t a t e d the extraction of some DNA polymerase a c t i v i t y bound to chromatin. An i n i t i a l e x t r a c t i o n was therefore performed using 0.01 M Tris-HCl buffer, pH 8.0, to separate the " f r e e " nuclear DNA polymerase a c t i v i t y from the chromatin-bound DNA polymerase enzyme. The second extraction procedure using 1 M NaCl to d i s s o c i a t e the chromatin-bound DNA polymerase was e s s e n t i a l l y the same as described by Wang"'*' f o r the i s o l a t i o n of chromatin a c i d i c (non-histone) proteins from r a t l i v e r . Nuclei, previously extracted with 10 mM T r i s - H C l , pH 8.0, were re-extracted with increasing concentrations of NaCl i n 0.1 M T r i s - H C l , pH 8.0. Maximum y i e l d of DNA polymerase a c t i v i t y was achieved with 1 M NaCl. Salt concentrations above 1 M, however, resul t e d i n a loss of enzyme a c t i v i t y . Extraction with 1 M phosphate 26 buffer, pH 7.4 was less successful, since only 50% of the DNA polymerase a c t i v i t y was extracted as compared with the 1 M NaCl extr a c t i o n . On the basis of these findings buffered 1 M NaCl was used as the 2nd extraction medium of the 2-step i s o l a t i o n procedure. The 2-step i s o l a t i o n procedure e f f e c t s a preliminary f r a c t i o n a t i o n of the two enzymes as w e l l as permitting the extraction of each enzyme under optimum conditions e s s e n t i a l for maximum y i e l d . A s i n g l e step e x t r a c t i o n of the n u c l e i with 1 M NaCl resulted i n a considerable loss of the enzyme a c t i v i t y which i s extractable with low i o n i c strength T r i s - H C l buffer, probably because the a c t i v i t y of t h i s enzyme i s d r a s t i c a l l y i n h i b i t e d by monovalent cations. - 16 -B. Enzyme Assays 1. DNA Polymerase Assay The a c t i v i t i e s of the T r i s - s o l u b l e and NaCl-soluble DNA polymerases were determined by measuring the incorporation of a radioactive deoxyribonucleoside triphosphate into an acid-insoluble product. Since the p a r t i a l l y p u r i f i e d enzymes from the p u r i f i e d n u c l e i showed s i m i l a r optimal assay requirements one assay mixture was used. The re a c t i o n mixture contained i n a t o t a l volume of 0.35 ml the following: 20 ymoles T r i s - H C l , pH 7.5, 2 ymoles MgC^, 2 ymoles of d i t h i o t h r e i t o l , 20 nmoles each of dATP, dGTP and dCTP with 115 pmoles [ H]dTTP 100 yg c a l f thymus DNA and 0.05-0.2 ml of enzyme s o l u t i o n . The T r i s - s o l u b l e extract contained 30-120 yg protein and the NaCl-soluble extract contained 35-115 yg protein. The reaction mixtures were incubated i n t e s t tubes at 37°C for 30 min and the reactions terminated by c h i l l i n g . Three mg of bovine serum albumin were added to the mixture followed by 2 ml of 20% (w/v) t r i c h l o r o a c e t i c a c i d . The mixture was s t i r r e d and, a f t e r 10 min, the a c i d - p r e c i p i t a b l e material 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 washed 3 times with 5 ml of 5% (w/v) t r i c h l o r o a c e t i c acid. The washed p r e c i p i t a t e was dissolved i n 0.5 ml of 1 M hyamine hydroxide i n methanol and mixed with 10 ml of toluene based s c i n t i l l a t i o n s o l u t i o n (15 g of 2,5-diphenyloxazole, 150 mg of 1,4-bis-(5-phenyloxazolyl-2)-benzene, and 240 g of naphthalene i n 1 1. each of toluene, dioxane 59 and 95% ethanol) and the mixture counted i n a Packard Li q u i d S c i n t i l l a t i o n spectrometer. - 17 -One unit of DNA polymerase a c t i v i t y i s defined as the amount required to convert 1 pmole of labeled deoxynucleotide into the a c i d -i n s o l u b l e product i n 30 min i n the assay described above. The reaction 3 rates of both enzymes, as measured by the incorporation of [ H]dTTP into a c i d - p r e c i p i t a b l e material, were l i n e a r f or at l e a s t 75 min under these assay conditions. S p e c i f i c a c t i v i t y i s expressed as units per mg of p r o t e i n . 2. Terminal Deoxyribonucleotidyltransferase Assay 23 A modification of the procedure described by Krakow et a l . was used to determine terminal deoxyribonucleotidyltransferase a c t i v i t y . The incubation mixture was i d e n t i c a l with the mixture used for the DNA polymerase assay except for the omission of dATP, dGTP and dCTP. Af t e r incubation, the samples were processed as described for the DNA polymerase assays. One: unit of terminal transferase a c t i v i t y i s 3 defined as the enzyme required to convert 1 pmole of [ H]dTTP into the a c i d - i n s o l u b l e product i n 30 min under the assay conditions. 3. DNase I Assay The presence of contaminating DNase I i n nuclear preparations was determined by the d i f f u s i o n s l i d e technique developed by J a r v i s and Lawrence.^ The assay was performed under conditions s i m i l a r to those i n which DNA polymerase was assayed, except that triphosphates were omitted. - 18 -A hot s o l u t i o n of agar (2% w/v) containing 2 mg/ml c a l f thymus DNA was mixed with an equal volume of hot 0.1 M T riS'-HCl b u f f e r , pH 7.8 and MnC^ was added to a f i n a l concentration of 0.01 M. One ml of hot mixture was spread over a 1 i n . by 2 i n . area on a microscopic s l i d e outlined by c e l l u l o s e tape. A 2.7 mm diameter well was made i n the center of agar with a t h i n s t e e l tube, and 0.004 ml of the enzyme preparation was placed i n the w e l l . The s l i d e s were placed i n a p l a s t i c box containing moist b l o t t i n g paper and incubated at 37°C for 20 hours. The s l i d e s were then dipped i n 1 N HC1 for 15 sees, washed with water and the diameter of the clear zone was measured. 4. Triphosphatase Assay Nucleoside triphosphatase a c t i v i t y was measured under the conditions described for the assay of DNA polymerase except that DNA was omitted from the reaction mixture and the i n d i v i d u a l triphosphate under i n v e s t i g a t i o n was present at a concentration of 0.16 mM. A f t e r incubation at 37°C for 45 min the reaction was terminated by the addition of 10% (v/v) p e r c h l o r i c a c i d . Acid soluble inorganic phosphate released from the i n d i v i d u a l triphosphates was estimated by the method of Ames.*'1 5. Protein Determination 62 Protein was estimated according to the method Lowry et a l . with c r y s t a l l i n e bovine serum albumin (Fraction V) as a standard. - 19 -6. Heat-Denatured DNA DNA solutions (5 mg c a l f thymus DNA/ml of 0.1 M NaCl) were heated at 100°C f o r 10 min and quickly cooled i n i c e . The heat-denatured DNA had an absorbance at 260 nm which was 15-20% higher than that of native DNA. C. Column Chromatography on DEAE-Cellulose 1. Preparation of DEAE-Cellulose Column DEAE-cellulose (diethylaminoethyl-cellulose, Whatman DE 32) was prepared for use according to the manufacturer's i n s t r u c t i o n s . DEAE-cellulose was washed successively with 15 volumes of 0.5 N HC1 and 0.5 N NaOH. It was then e q u i l i b r a t e d by washing several times with 0.2 M potassium phosphate buffer, pH 7.5. A f t e r the fin e s had been removed the s l u r r y was de-aerated under vacuum. The s l u r r y was then packed under gravity i n a 2.0 cm diameter column to a height of 10 cm. The column was washed with 20 mM potassium phosphate bu f f e r , pH 7.5, containing 5 mM d i t h i o t h r e i t o l or 5 mM 2-mercaptoethanol ( s t a r t i n g buffer) u n t i l the conductivity and pH of the e f f l u e n t were i d e n t i c a l with the s t a r t i n g buffer. 2. Procedure for DEAE-Cellulose Chromatography The two nuclear extracts were dialyzed at A°C f o r 8-10 hours against 50 volumes of s t a r t i n g b u f f e r . A f r e s h l y dialyzed NaCl-soluble or T r i s - s o l u b l e nuclear extract was applied to a DEAE-cellulose - 20 -column which had been previously e q u i l i b r a t e d with the same bu f f e r . The sample contained 120-150 mg and 60-90 mg protein of the NaCl-soluble and T r i s - s o l u b l e nuclear extracts, r e s p e c t i v e l y . Each column was washed with 60 ml of 20 mM potassium phosphate, pH 7.5, containing 5 mM d i t h i o t h r e i t o l or 5 mM 2-mercaptoethanol and then eluted with a 250 ml l i n e a r gradient of KC1 from 0 to 0.5 M i n the same buffer. The flow rate was about 30 ml/hour and 5 ml f r a c t i o n s were c o l l e c t e d . The gradient p r o f i l e was determined by measuring the conductivity of each f r a c t i o n . The p r o t e i n e l u t i o n pattern was measured eit h e r by 62 absorbance at 280 nm or by the method of Lowry et_ a l . using c r y s t a l l i n e bovine albumin (Fraction V) as standard. Fractions from the DEAE-cellulose columns were dialyzed for 8 hours at 4°C against 20 mM Tris-HCl, pH 7.5, containing 5 mM d i t h i o t h r e i t o l and immediately assayed f o r DNA polymerase a c t i v i t y . For each eluted peak of a c t i v i t y the f r a c t i o n s with the highest s p e c i f i c a c t i v i t i e s were pooled and stored e i t h e r at 0°C or at - 20°C i n the presence of 20% (v/v) g l y c e r o l . These p a r t i a l l y p u r i f i e d f r a c t i o n s were used for comparative studies of the two nuclear DNA polymerases. D. Gel F i l t r a t i o n on Sephadex G-150 Since preliminary r e s u l t s indicated that the two enzymes behaved d i f f e r e n t l y on Sephadex G-150 i t was necessary to devise techniques appropriate f o r each. For t h i s reason two buffer systems were used, namely, TKM (50 mM T r i s - H C l , pH 7,5, 25 mM KC1 and 5 mM MgCl 2) f o r the T r i s - s o l u b l e enzyme and TS (0.1 M T r i s - H C l , pH 7.5 with 1 M NaCI) for the NaCl-soluble enzyme. - 21 -1. Preparation of Sephadex G-150 Column Sephadex G-150 beads (Pharmacia) were allowed to swell i n e i t h e r TKM buffer or TS buffer f o r 4 days at room temperature. Af t e r removal of the f i n e s the s l u r r y was de-aerated under vacuum. A 2 column (4.9 cm x 45 cm, K 25/45 Sephadex) equipped with upward flow adaptors was prepared according to the i n s t r u c t i o n s from Pharmacia. The prepared column was coated with p r o t e i n by upward flow e l u t i o n with a s o l u t i o n of 30 mg of bovine serum albumin (Fraction V) i n 15 ml of appropriate buffer. The column was then washed with 5-7 l i t e r s of the TKM or TS buffer u n t i l absorbance at 280 nm was zero. Unless the Sephadex beads were coated with bovine serum albumin the recovery of polymerase a c t i v i t y was only 5-10% due to nonspecific i r r e v e r s i b l e 63 adsorption of the enzyme to the column. 2. C a l i b r a t i o n of Sephadex G-150 Column The void volume (V ) was determined with Blue Dextran 2000 v o (M.W. 2000,000; Pharmacia). The t o t a l bed volume (Vfc) was determined at the end of the experiment by weighing the volume of d i s t i l l e d water which f i l l e d the column to the same l e v e l as the Sephadex g e l . The column was c a l i b r a t e d with standard p r o t e i n markers, namely, ribonuclease A (M.W. 13,700; 15 mg/2 ml), ehymotrypsinogen A (M.W. 25,000; 10 mg/2 ml), ovalbumin (M.W. 45,000; 15 mg/2 ml), aldolase (M.W. 158,000; 15 mg/2 ml) ( C a l i b r a t i o n K i t for molecular weight determination, Pharmacia) and b a c t e r i a l a l k a l i n e phosphatase (M.W. 80,000; 10 U' (units)/2 ml) (Worthington Biochemical Corporation). Protein - 22 -markers were run i n groups of three to permit optimum r e s o l u t i o n . Sample 1 - aldolase, a l k a l i n e phosphatase and chymotrypsinogen A. Sample 2 - a l k a l i n e phosphatase, ovalbumin and ribonuclease A. 3. Procedures for Gel F i l t r a t i o n on Sephadex G-150  Method A This method was employed to determine the molecular weight of the T r i s - s o l u b l e nuclear enzyme. A 2.0 ml sample of the T r i s - s o l u b l e enzyme (10-15 mg protein/2ml) which had been dialyzed f o r 3-4 hours at 4°C against 2 l i t e r s of TKM buffer, was applied to a c a l i b r a t e d Sephadex G-150 column, which had been thoroughly washed with TKM buffer containing 5 mM 2-mercaptoethanol. The sample was eluted i n the upward d i r e c t i o n with the same buffer system. The flow rate was maintained at approximately 14 ml per hour and 2.0 ml f r a c t i o n s were c o l l e c t e d . The absorbance at 280 nm and DNA polymerase a c t i v i t y of each f r a c t i o n were determined. Method B Method B was used to determine the molecular weight of the NaCl-soluble DNA polymerase. I t was s i m i l a r to Method A except that 0.1 M Tris-HCl buffer, pH 7,5, containing 1 M NaCl was substituted for the TKM buffer f or c a l i b r a t i o n of the column and for determination of the molecular weight. The NaCl-soluble enzyme was f i r s t chromato-graphed on DEAE-cellulose and then concentrated against Sephadex G-25 powder f o r 5-7 hours. The sample applied to the column contained between 5 to 9 mg protein i n 2.0 ml. - 23 -4. C a l c u l a t i o n of Molecular Weight by Gel F i l t r a t i o n The molecular weights of the T r i s - s o l u b l e and NaCl-soluble DNA polymerases were calculated according to the procedure of Laurent 64 and K i l l a n d e r . This method i s based on the observation that for a homologous serie s of compounds, a l i n e a r r e l a t i o n s h i p e x i s t s between th e i r e l u t i o n volumes and the logarithm of t h e i r molecular weights. If the e l u t i o n volumes of several globular proteins of known molecular weight are measured, a constant, K , can be calculated for each v - v a v 6 O according to the equation — - — = K (where V = e l u t i o n volume V - V av e t o for the protein; V = e l u t i o n volume f o r Blue Dextran 2000 and o V = t o t a l bed volume). A p l o t of K values versus the logarithm t r av of the molecular weight of each substance i s c a l l e d a s e l e c t i v i t y curve and i s l i n e a r within the f r a c t i o n a t i o n range of the Sephadex gel employed. Consequently, i f the K value for a protein of av unknown molecular weight i s determined, i t s molecular weight can be estimated d i r e c t l y from the s e l e c t i v i t y curve. - 24 -RESULTS AND DISCUSSION A. Properties of Crude Nuclear DNA Polymerases Two DNA polymerase activities were isolated from partially purified nuclei by the extraction procedures described previously. The data obtained using the 2-step extraction procedure are summarized in Table I. The amount of DNA polymerase activity extracted with 10 mM Tris-HCl buffer, pH 8.0, was usually only 10-15% of the activity obtained by extraction with 1 M NaCI in 0.1 M Tris-HCl, pH 8.0. The specific activity of the Tris extractable enzyme was also considerably lower than that of the NaCl-soluble enzyme. Table I. Distribution of DNA Polymerase Activity in Purified Nuclei Isolated from Rat Intestinal Mucosa. Purified nuclei were prepared from 50 g of mucosa as described in Methods. Appropriate reaction mixture was .used, to assay 50 yg of protein of each fraction. Fraction Total Activity Specific Activity units units/mg protein Tris-soluble NaCl-soluble 262 2312 6.8 28 - 25 -The crude enzymes exhibited maximum a c t i v i t y at an a l k a l i n e pH, the pH optimum of the T r i s - s o l u b l e DNA polymerase being s l i g h t l y higher than that of the NaCl-soluble enzyme (Table I I ) . A magnesium concentration of 3 mM was optimal f o r the a c t i v a t i o n of the T r i s -soluble DNA polymerase a c t i v i t y and a sharp decline i n a c t i v i t y was observed at both lower and higher concentrations. In contrast, the NaCl-soluble enzyme was activated to the same extent by a range of concentrations of magnesium (4-10 mM). The T r i s - s o l u b l e enzyme exhibited no preference between native and heat-denatured DNA as template, however, a very s l i g h t preference f o r native DNA was observed on occasions. On the other hand, the NaCl-soluble enzyme con s i s t e n t l y preferred native DNA. Table I I . Properties of the Nuclear DNA Polymerases. The DNA polymerases were assayed using the appropriate re a c t i o n mixture as described i n Methods. Fracti o n pH Optimum Optimum concentration Preference f o r native (50 mM of magnesium (mM) DNA Tris-HCl) (native/denatured) T r i s - s o l u b l e 7.9-8.2 3 1.0-2.0 NaCl-soluble 7.5 4-10 2.0-3.0 - 26 -The reaction rates of both enzymes, as measured by the incorpora-t i o n of l a b e l l e d deoxynucleoside triphosphate into a c i d - i n s o l u b l e product, were l i n e a r f o r at le a s t 90 min. A l i n e a r r e l a t i o n s h i p was also found between the incorporation of [ 3H]dTTP and increasing amounts of p r o t e i n from 0 to 150 mg per assay. The T r i s - s o l u b l e DNA polymerase a c t i v i t y was i n h i b i t e d by mono-valent cations whereas the a c t i v i t y of the NaCl-soluble enzyme increased i n the presence of monovalent cations, the optimum concentration was approximately 60-110 mM. The requirements of the crude T r i s - s o l u b l e and NaCl-soluble DNA polymerases are summarized i n Table I I I . The data i l l u s t r a t e that both enzymes require a l l four deoxynucleoside 5'-triphosphates, magnesium, DNA and presence of a t h i o l protecting reagent for"maximum a c t i v i t y . - 27 -Table I I I . Reaction Requirements of the Nuclear DNA Polymerases. The enzymes were assayed using the appropriate assay mixture as de t a i l e d i n Methods. Reaction mixture complete DNA polymerase a c t i v i t y (%) T r i s - s o l u b l e NaCl-soluble 100 100 42 1.1 40 42 57 50 29 15.5 21 4.7 45 -native DNA + heat- 93 denatured DNA -MgCl 2 2 -MgCl 2 + 1 mM MnCl 2 16 -dATP 70 -dGTP 83 -dCTP 61 -dGTP and dCTP 33 -dATP, dGTP and dCTP 18 - d i t h i o t h r e i t o l 78 -DNA 0.7 -Tr i s - H C l , pH 8.0 or 7.5 + 13 potassium phosphate, pH 6.0 - 28 -B. P a r t i a l P u r i f i c a t i o n of Crude Enzymes 1. Ammonium Sulfate 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 by a number of inve s t i g a t o r s as a p u r i f i c a t i o n step i n the preparation of mammalian 31 33 63 31 DNA polymerases. ' ' Leung attempted to use th i s procedure to obtain a preliminary p u r i f i c a t i o n of the DNA polymerase i s o l a t e d from the i n t e s t i n a l mucosa of r a t . However, up to 60% of the o r i g i n a l enzymic a c t i v i t y was l o s t when the a c t i v i t y was recovered i n the 60% ammonium s u l f a t e f r a c t i o n . The p o s s i b i l i t y of using ammonium s u l f a t e f r a c t i o n a t i o n to separate the two nuclear DNA polymerases was investigated. Table IV shows that most of the DNA polymerase a c t i v i t y i n the T r i s and the NaCl extracts was recovered i n the 60% and 75% ammonium s u l f a t e f r a c t i o n s . The p u r i f i c a t i o n achieved was n e g l i g i b l e i n both cases. Recovery of the T r i s - s o l u b l e DNA polymerase a c t i v i t y was frequently below 40% and only about 60% of the NaCl-soluble DNA polymerase a c t i v i t y was recovered. The reasons f o r the loss of the a c t i v i t y are probably quite complex. Since the T r i s - s o l u b l e DNA polymerase was strongly i n h i b i t e d by ammonium ions t h i s may account, i n part, f o r the lower recovery of th i s enzyme. Another p o s s i b i l i t y i s that high s a l t concentrations may a l t e r the s t r u c t u r a l conformation of the mammalian 37 DNA polymerases molecule, as suggested by Keir. Ammonium s u l f a t e f r a c t i o n a t i o n was not used r o u t i n e l y as a p u r i f i c a t i o n step i n the present study as higher y i e l d s and a greater p u r i f i c a t i o n were achieved employing other procedures. Table IV. Ammonium Sulfate Fractionation. (NH 4) 2S0 4 saturation T r i s - s o l u b l e DNA polymerase NaCl-soluble DNA polymerase Tot a l a c t i v i t y (units) Total protein (mg) S p e c i f i c a c t i v i t y (units/mg) Y i e l d % P u r i f i c a -t i o n Total a c t i v i t y (units) T o t a l p r o t e i n (mg) S p e c i f i c a c t i v i t y (units/mg) Y i e l d P u r i f i c a -% t i o n Crude enzyme 2150 256.0 8.4 100 1.0 1693 188.5 9.0 100 1.0 20% 3.6 1.4 2.6 0.2 - 12.6 6.3 2.0 0.75 40% 50 14.3 3.5 2.3 - 120 48 2.5 7.1 60% 589 131.0 4.5 27.0 - 560 70 8.0 33.0 i M 75% 705 77.5 9.1 32.8 1.1 708 47.2 15.0 42.0 1.7 VO 1 75 (Supern.) 45 9.0 5.0 2.1 - 12.9 5.6 2.3 0.76 - 30 -2. Chromatography on DEAE-Cellulose E l u t i o n p r o f i l e s obtained when the T r i s - s o l u b l e DNA polymerase and the NaCl-soluble DNA polymerase were chromatographed on DEAE-c e l l u l o s e are shown i n Figure 2 and Figure 3. A major peak of DNA polymerase a c t i v i t y from the T r i s - s o l u b l e nuclear extract eluted with 0.045-0.055 M KC1 and a minor peak, c o n s t i t u t i n g 5-15% of the t o t a l a c t i v i t y , eluted with 0.175-0.18 M KC1 (Fig. 2). DNA polymerase a c t i v i t y from the NaCl-soluble nuclear extract also showed two peaks -a major peak e l u t i n g with 0.165-0.21 M KC1 and a minor peak, containing 15% of the t o t a l a c t i v i t y , e l u t i n g with 0.045-0.055 M KC1 (Fig. 3). The minor peak from the T r i s - s o l u b l e f r a c t i o n eluted i n the same p o s i t i o n as the major a c t i v i t y i n the NaCl-soluble f r a c t i o n . Other parameters-stimulation by monovalent cations, preference f o r native DNA also ind i c a t e that t h i s a c t i v i t y i s probably i d e n t i c a l to the NaCl-soluble enzyme. Conversely, a minor component of the DNA polymerase a c t i v i t y i n the NaCl-soluble nuclear f r a c t i o n had properties 32 i d e n t i c a l to the p r i n c i p a l a c t i v i t y i n the T r i s - s o l u b l e f r a c t i o n . In a l l experiments 80-90% of the protein and 80-95% of the DNA polymerase a c t i v i t y was recovered. Data obtained from DEAE-cellulose chromatography of the T r i s -soluble and the NaCl-soluble DNA polymerases are summarized i n Table V. Under the conditions used for chromatography on DEAE-cellulose the bulk of DNase I a c t i v i t y eluted with the unadsorbed proteins i n the column wash. Peak I from the T r i s - s o l u b l e extract contained approximately 15% of the t o t a l DNase I a c t i v i t y o r i g i n a l l y present i n CD "3 O Q. s_ O u c D-r — "D « j J) O FRACTION Figure 2. Chromatography of Tri s - s o l u b l e nuclear extract on DEAE-cellulose. E l u t i o n with 20 mM potassium phosphate buffer, pH 7.5, containing 5 mM d i t h i o t h r e i t o l , with l i n e a r gradient of KC1. FRACTION Figure 3. Chromatography of NaC-soluble nuclear extract on DEAE-cellulose. E l u t i o n with 20 mM potassium phosphate b u f f e r , pH 7.5, containing 5 mM d i t h i o t h r e i t o l , with l i n e a r gradient of KC1. - 32 -Table V. Chromatography of Crude T r i s - S o l u b l e and NaCl-Soluble DNA Polymerases on DEAE-Cellulose. E l u t i o n with 20 mM potassium buffer, pH 7.5 , with l i n e a r gradient of KC1. Protein T o t a l Y i e l d S p e c i f i c P u r i f i - Ratio (mg) a c t i v i t y (units) % a c t i v i t y cation (units/mg protein) Act. with native DNA Act. with denat. DNA T r i s - s o l u b l e DNA polymerase Crude 135.5 542 100 4 1.0 2.6 enzyme Peak I 22.0 429 79 19.5 4.9 3.2 Peak II 13.0 70 13 5.4 1.35 5.9 To t a l 35.0 499 92 NaGl-soluble DNA polymerase Crude 225 2137 100 9.5 1.0 3.0 enzyme Peak I 16.0 336 15.7 21 2.2 3.7 Peak II 45.0 1507 70.5 33.5 3.5 6.6 Tot a l 61.0 1843 86.2 - 33 -the crude extract, whereas Peak II from the NaCl-soluble extract possessed only a trace of DNase I a c t i v i t y . Studies i n t h i s laboratory using the same conditions as described for the DEAE-cellulose chromatography of nuclear extracts have demonstrated three basic chromatographic patterns which account for a l l the DNA polymerase a c t i v i t y found i n the s u b c e l l u l a r f r a c t i o n s obtained from rat' i n t e s t i n a l mucosa. One pattern of DNA polymerase a c t i v i t y , which was s i m i l a r to the T r i s - s o l u b l e nuclear polymerase a c t i v i t y , was obtained when the soluble cytoplasmic and ribosomal f r a c t i o n s were chromatographed on DEAE-cellulose, except that the minor peak obtained from the ribosomal extract amounted to only 1-2% of the t o t a l a c t i v i t y . Chromatography of the mitochondrial extract demonstrated a second pattern of DNA polymerase a c t i v i t y . A s i n g l e peak of DNA polymerase a c t i v i t y was observed which had a high a f f i n i t y for DEAE-cellulose and was not eluted u n t i l the concentration of KC1 reached 0.28 M. The smooth membrane f r a c t i o n appeared to contain several DNA polymerase species with the predominant a c t i v i t y e l u t i n g , l i k e the mitochondrial enzyme, with 0.28 M KC1. The t h i r d chromatographic pattern was shown by the NaCl-soluble nuclear DNA polymerase, which exhibited an intermediate a f f i n i t y f o r DEAE-cellulose. It seems, therefore, that while the NaCl-soluble DNA polymerase has unique chromatographic properties on DEAE-cellulose, the chromatograph - 34 -properties of the T r i s - s o l u b l e DNA polymerase are i n d i s t i n g u i s h a b l e from those of the ribosomal and soluble cytoplasmic f r a c t i o n s . I t i s extremely d i f f i c u l t to compare the e l u t i o n p r o f i l e s obtained i n the present work with those of other i n v e s t i g a t o r s because of the d i f f e r e n t conditions employed. I t i s apparent, however, that most mammalian DNA polymerases extracted i n aqueous media of low i o n i c strength have a very low a f f i n i t y f o r DEAE-cellulose and are 34 52 ei t h e r not adsorbed or elute at the front of the gradient. ' On the other hand, DNA polymerases extracted from s u b c e l l u l a r p a r t i c l e s by s a l t s at high concentrations exhibit a strong a f f i n i t y f o r DEAE-25 29 30 35 43 44 c e l l u l o s e . » » > » > These r e s u l t s suggest that the DNA polymerase enzyme associated with non-histone chromosomal proteins has a higher anionic charge than the cytoplasmic and nuclear DNA polymerases which are not t i g h t l y bound to s u b c e l l u l a r structures. However, no general conclusions can be drawn on the basis of these DEAE-cellulose chromatography r e s u l t s since the f r a c t i o n a t i o n conditions employed here d i f f e r from those used i n other studies. 3. Chromatography on Phosphocellulose The d i f f e r e n t a f f i n i t i e s displayed by the two nuclear DNA polymerases f o r the anion-exchanger DEAE-cellulose suggested that chromatography on a cation-exchanger, such as phosphocellulose would provide a d d i t i o n a l p u r i f i c a t i o n of the two enzymes. The T r i s - s o l u b l e and NaCl-soluble enzymes, which had previously been chromatographed on DEAE-cellulose, were therefore subjected to - 35 -phosphocellulose chromatography. The T r i s - s o l u b l e f r a c t i o n (15-30 mg of protein) and the NaCl-soluble f r a c t i o n (20-40 mg of protein) were f i r s t dialyzed against 40 volumes of 20 mM potassium phosphate buffer, pH 6.5, with 5 mM d i t h i o t h r e i t o l at 4°C for 4 hours, and then added to 3-7 g of phosphocellulose which had been e q u i l i b r a t e d with the same buffer. The suspension was s t i r r e d overnight at 4°C, transferred to a column (2.0 cm x 15 cm) and washed with 50 ml of 20 mM potassium phosphate buffer, pH 6.5, containing 5 mM d i t h i o t h r e i t o l and then eluted with a l i n e a r gradient of 0.02 to 0.5 M potassium phosphate, pH 7.2, containing 5 mM d i t h i o t h r e i t o l . The flow rate was maintained at about 15 ml/hour and 5 ml f r a c t i o n s were c o l l e c t e d , dialyzed and assayed as described for the f r a c t i o n s from the DEAE-cellulose columns. Neither enzyme was retained by the phosphocellulose. While t h i s r e s u l t i s consistent with the behavior of the NaCl-soluble DNA polymerase on DEAE-cellulose, i t was expected that the T r i s - s o l u b l e enzyme would bind to the phosphocellulose under these conditions. The a f f i n i t y of the T r i s - s o l u b l e DNA polymerase f o r phosphocellulose was increased by lowering pH, but t h i s was associated with a dramatic decrease i n y i e l d , p o ssibly because the enzyme was very unstable at a pH of 6.0 or below. Phosphocellulose chromatography was not employed as a routine p u r i f i c a t i o n procedure i n the present study because the y i e l d s were very low (20-30% for both enzymes) and e s s e n t i a l l y no p u r i f i c a t i o n was achieved. - 36 -The T r i s - s o l u b l e and NaCl-soluble enzyme a c t i v i t i e s were i n h i b i t e d by phosphate ion at concentrations as low as 0.1 M and they were i n h i b i t e d almost completely at concentrations of 0.2 M. D i a l y s i s of the f r a c t i o n s against 20 mM Tris-HCl buffer, pH 7.5 f a i l e d to restore enzyme a c t i v i t y . The recovery of protein and DNA polymerase a c t i v i t y from phosphocellulose was only s l i g h t l y improved by s u b s t i t u t i n g a l i n e a r gradient of KC1 i n 20 mM potassium phosphate buffer f or the phosphate gradient. This suggests that i n t e r a c t i o n s between the DNA polymerase molecules and the phosphonic acid exchange groups of phosphocellulose are probably more important than the concentration of phosphate. C. P a r t i a l l y P u r i f i e d Nuclear DNA Polymerases 1. Properties of P a r t i a l l y P u r i f i e d Nuclear DNA Polymerases The requirements of the p u r i f i e d enzymes do not d i f f e r s i g n i f i -cantly from those of the crude enzyme extracts (cf. Table III and Tables VI and VII). Both of the DNA polymerases required the presence of DNA, magnesium ions and a l l four deoxynucleoside 5'-triphosphates f o r maximum a c t i v i t y . In general, the p u r i f i e d T r i s - s o l u b l e DNA polymerase showed a preference for native over heat-denatured c a l f thymus DNA; however, the r a t i o of preference varied depending on the preparation used and sometimes only a s l i g h t preference for native DNA was observed. On the other hand, the p u r i f i e d NaCl-soluble DNA polymerase, which i n - 37 -Table VI. Reaction Requirements of the Tris-^Soluble DNA Polymerase. The components of the complete system and the d e t a i l s of the assay are described i n Methods. DNA polymerase p a r t i a l l y p u r i f i e d on DEAE-cellulose was used as the enzyme source. Reaction mixture T r i s - s o l u b l e DNA polymerase units x 10/mg protein % a c t i v i t y Complete 390.0 100.0 -Enzyme 0.0 0.0 -MgCl 2 2.3 0.6 -MgCl 2 + 0.5 mM MnCl 2 44.1 11.3 -dATP 124.8 32.0 -dGTP 152.1 39.0 -dCTP 113.1 29.0 -dGTP and dCTP 60.8 15.6 -dATP, dGTP and dCTP 31.2 8.0 -DNA 0.0 0.0 -native DNA + heat-denatured DNA 214.5 55.0 - d i t h i o t h r e i t o l 42.5 10.9 +20% g l y c e r o l 354.9 91.3 +1.5 mM rATP 452.4 116 +50 mM potassium phosphate 91.6 23.5 +5 mM sodium pyrophosphate 0.4 0.1 +50 mg pancreatic DNase I 17.6 4.5 - 38 -Table VII. Reaction Requirements of the NaCl-Soluble DNA Polymerase. The complete system f or the reaction i s described i n Methods. DNA polymerase p a r t i a l l y p u r i f i e d on DEAE-cellulose was used as the enzyme source. Reaction mixture NaCl-soluble DNA polymerase units x 10/mg pr o t e i n % a c t i v i t y Complete 670.0 100.0 -Enzyme 0.0 0.0 -MgCl 2 5.4 0.8 -MgCl 2 + 1.5 mM MnCl 2 228.5 34.1 -dATP 107.2 .16.0 -dGTP 147.4 22.0 -dCTP 120.6 18.0 -dGTP and dCTP 70.3 10.5 -dATP, dGTP and dCTP 36.8 5.5 -DNA 0.0 0.0 -native DNA + heat-denatured DNA 100.5 15.0 - d i t h i o t h r e i t o l 79.0 11.8 +20% g l y c e r o l 556.0 83.0 +1.5 mM rATP 685.4 102.3 +50 mM potassium phosphate 100.5 15.0 +5 mM sodium pyrophosphate 13.5 2.0 +50 mg pancreatic DNase I 10.0 1.5 - 39 -crude extracts c o n s i s t e n t l y preferred native DNA as a template, showed an almost complete dependence on native DNA. The e f f e c t of divalent cations on DNA polymerase a c t i v i t y w i l l be discussed f u l l y i n a separate section. B r i e f l y , of the cations tested, magnesium was the most e f f i c i e n t a c t i v a t o r while manganese and calcium were much less e f f e c t i v e . The omission of any one of the unlabelled deoxynucleoside 5 1-triphosphates resulted i n a decrease of the T r i s - s o l u b l e and NaCI soluble DNA polymerase a c t i v i t i e s to 29^-39% and 16-22%, resp e c t i v e l y , of the con t r o l values, depending on the p a r t i c u l a r nucleotide omitted. The omission of a l l three unlabelled triphosphates 3 decreased the amount of [ H]dTMP incorporated to less than 10% of that obtained i n the complete system, i n d i c a t i n g that the preparations were not contaminated with terminal deoxyribonucleotidyltransferase enzyme. In both cases DNA polymerase a c t i v i t y was reduced considerably by the omission of d i t h i o t h r e i t o l i n d i c a t i n g that i t was necessary for maximum enzyme a c t i v i t y . The addition of 20% g l y c e r o l had no e f f e c t although a s l i g h t i n h i b i t i o n of both enzymic a c t i v i t i e s was observed i n some experiments. Neither of the p u r i f i e d DNA polymerase a c t i v i t i e s was s i g n i f i c a n t l y stimulated by the addition of ATP to the assay mixture. The polymerase reactions were i n h i b i t e d at l e a s t 75% i n the presence of 50 mM inorganic phosphate and the presence of 5 mM pyrophosphate almost completely i n a c t i v a t e d the enzymes. - 40 -2. Time-Course of the Reaction 3 The rates of incorporation of [ H]dTTP into a c i d - p r e c i p i t a b l e material by p a r t i a l l y p u r i f i e d nuclear DNA polymerases using e i t h e r native or heat-denatured DNA as templates were compared. The rate of polymerization by both the T r i s - s o l u b l e and NaCl-soluble enzymes was l i n e a r f o r at l e a s t 75 min and continued to increase gradually f o r approximately 2 hours before reaching a plateau (Figures 4 and 5). The lower amounts of l a b e l l e d deoxynucleoside triphosphate incorporated into the aci d - i n s o l u b l e products when heat-denatured DNA was used merely r e f l e c t the preference of both enzymes for native DNA as a template, since the l i n e a r i t y of the reaction was not affected. An incubation period of 30 min was, therefore, used ro u t i n e l y i n a l l subsequent experiments. 3. E f f e c t of Increasing Amounts of Protein on the Incorporation  of [ 3H]dTTP Figure 6 shows the e f f e c t of increasing amounts of T r i s - s o l u b l e 3 protein on the incorporation of [ H]dTTP into acid-insoluble material. There was a l i n e a r r e l a t i o n s h i p between the incorporation of l a b e l l e d deoxynucleotide and protein concentration over the range 30 to 125 yg of p r o t e i n per assay mixture. At concentrations of protein above 130 yg per assay the incorporation increased gradually u n t i l a pleateau was reached at between 200 to 300 yg of pro t e i n per assay. The amount of incorporation varied widely when high l e v e l s of protein were included i n the assay, possibly because such large amounts of protein w i l l possess - 41 -i r ~ — — i — — r ~ s—~—"T~~*^-~I Figure 4 Incubation Time (min) Figure 4. Time-course of the reaction f o r the p a r t i a l l y p u r i f i e d T r i s -soluble DNA polymerase. Figure 5. Time-course of the reaction f o r the p a r t i a l l y p u r i f i e d NaCl-soluble DNA polymerase. O Native DNA; A Heat-denatured DNA 42 -d) o 1-o a >_ o c o E a 300 Amount of extracted enzyme added (yg of Protein) Figure 6. E f f e c t of increasing amounts of T r i s - s o l u b l e protein on the 3 incorporation of [ H]dTTP. Figure 7. E f f e c t of increasing amounts of NaCl-soluble p r o t e i n on the 3 incorporation of [ H]dTTP» - 43 -considerable quantities of contaminants and these i n h i b i t the polymerase reaction and/or e f f e c t the r a d i o a c t i v i t y determinations by causing excessive quenching. An i d e n t i c a l experiment with the NaCl-soluble extract i s shown i n Figure 7. There was a l i n e a r r e l a t i o n s h i p between incorporation and increasing amounts of protein over the range 25 ug to 115 ug of protein per assay. The amount of incorporation i n the presence of high amounts of pr o t e i n was v a r i a b l e , e s p e c i a l l y when the amount of pro t e i n exceeded 250 ug per assay. i In both cases the r e l a t i o n s h i p was not l i n e a r when very low l e v e l s of p r o t e i n were present (0-25 yg pr o t e i n per assay). These r e s u l t s emphasize the necessity of ensuring that the protein concentration of the f r a c t i o n s to be assayed f a l l s within the range of l i n e a r i t y . 4. E f f e c t of Template Concentration on the Incorporation of  [ 3H]dTTP In the absence of an exogenous supply of DNA neither of the 3 p a r t i a l l y p u r i f i e d enzymes catalyzed the incorporation of [ H]dTTP int o a c i d - i n s o l u b l e material. The amount of radioactive deoxynucleotide incorporated into a c i d - p r e c i p i t a b l e product by both the T r i s - s o l u b l e and NaCl-soluble nuclear enzymes p a r a l l e l e d the increase i n DNA concentration u n t i l a saturation l e v e l was reached at 80 yg of eit h e r native or heat-denatured DNA per reaction mixture. DNA at concentra-tions above 200 yg per assay mixture was i n h i b i t o r y (Fig. 8A and B). c a> 2 a O) E - 44 -C o o a i_ o u Q. in _0) O E a 1 0 0 2 0 0 pg of DNA 3 0 0 1 0 0 2 0 0 yg of DNA 3 0 0 Figure 8. E f f e c t of template concentration on the incorporation of [ H]dTTP. A. T r i s - s o l u b l e DNA polymerase. B. NaCl-soluble DNA polymerase. • Native DNA -." : O Heat-denatured DNA - 45 -The standard assay therefore contained 100 ug of DNA to ensure i t s a v a i l a b i l i t y was not a rate l i m i t i n g f a c t o r . 5. Substrate Concentration Curves F i g . 9A and B show plots of pmoles of dTMP incorporated per mg of prot e i n of the T r i s - s o l u b l e and NaCl-soluble extracts, r e s p e c t i v e l y , versus an increasing concentration of substrates. The s p e c i f i c r a d i o a c t i v i t y , i . e . , the r a d i o a c t i v i t y per mole of dTTP was kept constant over the e n t i r e concentration range by maintaining the unlabelled dTTP:labelled dTTP r a t i o at 25. The incorporation of 3 [ H]dTTP by the T r i s - s o l u b l e enzyme reached a maximum with a substrate concentration of 50 uM, corresponding to 20 nmoles of each unlabelled dATP, dGTP, dCTP and dTTP and 0.75 nmole of [ 3H]dTTP. Maximum incorporation by the NaCl-soluble DNA polymerase was reached at a substrate concentration of 100 uM, corresponding to 35 nmoles of each of the unlabelled deoxynucleoside triphosphate and 1.4 nmoles of 3 [ H]dTTP. Since the amounts of l a b e l l e d deoxynucleoside triphosphate required f o r maximum a c t i v i t y were very high, an attempt was made to 3 reduce the amount of [ H]dTTP present i n the assay mixture. However, t h i s r e s u l t e d i n a sharp decrease i n incorporation of the l a b e l l e d deoxynucleotide because of i t s very low s p e c i f i c a c t i v i t y . I t was subsequently found that better r e s u l t s were obtained when unlabelled dTTP was omitted and 115-120 picomoles of l a b e l l e d dTTP used instead. 3 The concentration of [ H]dTTP i s therefore only a f r a c t i o n of the concentration of unlabelled deoxynucleoside triphosphates present. However, since the reaction rates of both enzymes were l i n e a r f o r at E 0 0-1 0-2 0-3 0 0-1 0-2 0-3 o. SUBSTRATE CONCENTRATION (mM) Figure 9. Substrate concentration curves. A. T r i s - s o l u b l e DNA polymerase. B. NaCl-soluble DNA polymerase. - 47 -l e a s t 75 min and as a d d i t i o n a l [ H]dTTP did not increase the extent of incorporation i t was concluded that the concentration of l a b e l l e d dTTP was not a rate l i m i t i n g f a c t o r . 6. pH - Dependence Both p a r t i a l l y p u r i f i e d nuclear DNA polymerases were assayed over a range of pH values from 6.0 to 9.0 i n 50 mM Tris-HCl buffer and also i n 50 mM potassium phosphate from pH 5.0 to 8.0. The T r i s - s o l u b l e DNA polymerase and NaCl-soluble DNA polymerase showed maximal a c t i v i t y at pH 7.4 and 7.5 i n Tris-HCl buffer, r e s p e c t i v e l y . In both cases pH 9.0 was more i n h i b i t o r y than pH 6.0. In 50 mM potassium phosphate buffer, the pH optima of the T r i s - s o l u b l e and NaCl-soluble enzymes were 7.2 and 6.0, re s p e c t i v e l y . In both cases enzyme a c t i v i t y was lower i n 50 mM potassium phosphate buffer than i n 50 mM T r i s - H C l buffer ( F ig. 10A and B). 47 Leung and Zbarsky reported pH optima of 8.0 i n Tri s - H C l buffer, and 7.4 i n phosphate buffer f o r the nuclear DNA polymerase i s o l a t e d from r a t ' i i n t e s t i n a l mucosa. P a r t i a l l y p u r i f i e d cytoplasmic and ribosomal DNA polymerases from the same tissue showed maximal a c t i v i t y at pH 7.4 i n 50 mM T r i s - H C l buffer. In contrast, the optimal pH for the mitochondrial and smooth membrane polymerases was between pH 8.3 and 8.4 i n 50 mM Tri s - H C l b u f f e r . 3 2 66 Chang and Bollum suggested that most mammalian DNA polymerases sedimenting between 6 and 8S on sucrose density gradients (mol. wt. of - 48 -C Figure 10. E f f e c t of pH on p a r t i a l l y p u r i f i e d nuclear DNA polymerases. A. T r i s - s o l u b l e DNA polymerase. B. NaCl-soluble DNA polymerase. • Tris-HCl buffer O phosphate buffer - 49 -around 100,000), which they claim are of cytoplasmic o r i g i n have a neutral pH optimum, whereas 3.3S polymerases (mol. wt. 35,000-45,000), pr i m a r i l y nuclear i n o r i g i n , have an a l k a l i n e pH optimum. While t h i s may hold true i n s p e c i f i c cases, the r e s u l t s of other investigators show that i t i s not u n i v e r s a l l y applicable. For example, Sedwick 25 et a l . showed that both the cytoplasmic DNA polymerase (mol. wt. 110,000) and nuclear DNA polymerase (mol. wt. 39,000) extracted from human KB c e l l s have pH optima at 9.2 i n Tris-HCl buffer. Since the pH optimum w i l l be influenced by the presence of contaminants i n the enzyme preparation, t h i s may account f o r differences i n the pH optima of DNA polymerases extracted from various mammalian t i s s u e s . However, since various tissues have d i f f e r e n t i n t r a c e l l u l a r pH values i t i s equally possible that the pH optimum of a p u r i f i e d DNA polymerase preparation i s a true r e f l e c t i o n of i t s environment i j i v ivo. 7. Divalent Cation Requirement The presence of divalent cations i s e s s e n t i a l f or nuclear DNA 2+ polymerase a c t i v i t y . Of the metal ions tested, Mg was the most e f f e c t i v e a c t i v a t o r . Both p a r t i a l l y p u r i f i e d T r i s - s o l u b l e and NaCl-soluble enzymes were almost completely i n a c t i v e i n the absence 2+ of Mg . The T r i s - s o l u b l e DNA polymerase showed maximal a c t i v i t y i n the 2+ presence of 4-5 mM Mg (Fig. 11A), the NaCl-soluble DNA polymerase 2+ showed nearly maximal a c t i v i t y with Mg between 4 and 8 mM (Fig. 11B). The NaCl-soluble enzyme was d i f f e r e n t from the T r i s - s o l u b l e enzyme i n 2+ 2+ i t s response to high concentrations of Mg . With 30 mM Mg and 50 mM 2+ Mg the former maintained 10-15% and 5% of i t s maximal a c t i v i t y , £ 0 2 6 10 2 0 5 0 0 2 6 10 2 0 Divalent cation concentration (mM) Figure 11. E f f e c t of divalent cations on nuclear DNA polymerase a c t i v i t i e s . A. T r i s - s o l u b l e DNA polymerase. B. • Mg" NaCl-soluble DNA polymerase. 2+ O Mn 2+ A Ca 2+ - 51 -re s p e c t i v e l y , whereas the T r i s - s o l u b l e enzyme was completely i n a c t i v e . 2+ At i t s optimal concentration, Mn was only 5-15% as e f f e c t i v e 2+ an a c t i v a t o r of the T r i s - s o l u b l e DNA polymerase as was Mg at i t s optimal concentration and 30-35% as e f f e c t i v e f o r the NaCl-soluble DNA 2+ polymerase. The optimal Mn concentrations f o r the T r i s - s o l u b l e and NaCl-soluble enzymes were 0.5 mM and 1.5 mM, r e s p e c t i v e l y . In the 2+ presence of Mn concentrations higher than 2.5 mM, template DNA i n the reaction mixture p r e c i p i t a t e d as the manganese s a l t . Neither of 2+ the nuclear DNA polymerases was activated by Ca ions. These r e s u l t s are i n agreement with the f i n d i n g that there i s an absolute requirement f o r a divalent metal cation f o r the polymerase 2+ 37 reaction and that Mg ions alone give the optimum response. 2+ However, reports i n d i c a t e that the optimum Mg concentration of mammalian DNA polymerases d i f f e r s according to the enzyme source. 67 2"i" Chiu and Sung reported an optimum Mg concentration of 3-5 mM for DNA polymerase from the r a t b r a i n whereas the DNA polymerase from rat 2+ 31 39 l i v e r showed maximal a c t i v i t y at a Mg concentration of 16 mM. ' Several enzymes are reported to be most ac t i v e i n the presence of 6-8 mM M 2+ 34,68 Mg . 2+ 2+ In general, Mn and Ca ions were much less e f f e c t i v e as 2+ ac t i v a t o r s than Mg ions. Using a p a r t i a l l y p u r i f i e d preparation from 39 2+ regenerating rat l i v e r , however, Mantsavinos found that although Ca 2+ ions or Mn ions alone did not promote the reaction, i n combination 2+ they were p a r t i a l l y able to replace Mg ions. This e f f e c t was more - 52 -apparent when crude preparations were used and a combination of o i 2"i" 2+ 69 Mg + Mn + Ca gave the maximum response. Mantsavinos suggested 2+ 2+ that the combined e f f e c t of Mn and Ca i n crude c e l l - f r e e extracts may have been due to the p a r t i c i p a t i o n of these cations i n a n c i l l a r y reactions e f f e c t i n g the o v e r a l l process of DNA synthesis rather than a d i r e c t p a r t i c i p a t i o n i n the incorporation of deoxyribonucleoside 5'-triphosphates into DNA. 2+ 2+ The s u b s t i t u t i o n of other divalent metal cations (Co , Sr or 2*4" 2H~ 6 3 Cu ) for Mg res u l t e d i n no detectable a c t i v i t y . 8. E f f e c t of Monovalent Cations The e f f e c t of monovalent cations on the a c t i v i t y of the T r i s -soluble and NaCl-soluble nuclear DNA polymerases was examined using a range of s a l t concentrations from 0 to 1 M. The a c t i v i t i e s of both enzymes were influenced i n d i f f e r e n t ways by the presence of mono-valent cations. The T r i s - s o l u b l e DNA polymerase a c t i v i t y was strongly i n h i b i t e d by Na +, K + and NH^+, while the same ions stimulated the a c t i v i t y of the NaCl-soluble DNA polymerase by 30-170%. NaCl, KC1 and NH^Cl were used i n a l l experiments. In the presence of 40-50 mM s a l t the T r i s - s o l u b l e DNA polymerase a c t i v i t y was i n h i b i t e d 50% while no polymerase a c t i v i t y was detected with 300 mM (Fig. 12). In contrast, the NaCl-soluble DNA polymerase was stimulated by NH.+, K + and Na +, the magnitude of stimulation increasing i n that - 53 -0 20 40 60 80 100 200 1000 Monovalent cation concentration (mM) Figure 12. E f f e c t of monovalent cations on the a c t i v i t y of the T r i s - s o l u b l e DNA polymerase. - 54 -Monovalent cation concentration (mM) Figure 13. E f f e c t of monovalent cations on the a c t i v i t y of the NaCl-soluble DNA polymerase. - 55 -order (Fig. 13). A concentration of 40 mM NH^Cl stimulated enzyme a c t i v i t y approximately 30%. This e f f e c t was not apparent when the concentration was increased to 80-90 mM. Higher concentrations of NH^+ ions were progressively more i n h i b i t o r y and complete i n h i b i t i o n was reached at concentrations above 300 mM. In the presence of 50-80 nM K + ion a maximum stimulation of 75-80% was observed. A concentration of 150 mM had no e f f e c t and enzyme a c t i v i t y was s t i l l detectable at concentrations as high as 800-900 mM. Similar r e s u l t s were obtained with Na + ion, the DNA polymerase a c t i v i t y was stimulated at l e a s t 150% at i t s optimum concentration of 55-60 mM. Enzyme a c t i v i t y decreased r a p i d l y at concentrations i n excess of 150-200 mM but i t was detectable even at 1 M. Studies with an anion other than C l were c a r r i e d out to determine whether anions also influence polymerase a c t i v i t y (Fig. 14). The r e s u l t s showed s i m i l a r basic patterns to those obtained with NaCI, but some differences were observed. The T r i s - s o l u b l e enzyme a c t i v i t y was i n h i b i t e d more i n the presence of Na acetate than NaCI. Enzyme a c t i v i t y was i n h i b i t e d 50% with 15 mM Na acetate and no a c t i v i t y was detected above 80 mM. The NaCl-soluble enzyme a c t i v i t y was stimulated 65-75% i n the presence of 20-40 mM Na acetate compared to a 150% stimulation with NaCI. The r e s u l t s suggest that DNA polymerase a c t i v i t y i s more susceptible to i n h i b i t i o n by acetate than by chloride anion. Thus, while the monovalent cations are l a r g e l y responsible f o r the observed e f f e c t s of the s a l t s on DNA polymerase a c t i v i t y , i t i s apparent that the a c t i v i t y i s also effected by anions. - 56 -N a acetate (mM) Figure 14. E f f e c t of Na acetate on the a c t i v i t y of nuclear DNA polymerase. O T r i s - s o l u b l e DNA polymerase • NaCl-soluble DNA polymerase Similar experiments with mitochondrial DNA polymerase from rat i n t e s t i n a l mucosa showed that monovalent cations stimulated the 3 35 incorporation of [ H]dTTP into DNA whereas cytoplasmic DNA polymerase from the same tissue was i n h i b i t e d . In one of t h e i r c l a s s i c a l papers describing DNA synthesis by 4 enzymes extracted from E_. c o l i , Lehman and coworkers reported an i n h i b i t o r y e f f e c t of NaCl on the a c t i v i t y of p u r i f i e d polymerase. Walwick and M a i n ^ were the f i r s t to examine the e f f e c t of monovalent cations on mammalian DNA polymerase a c t i v i t y . Using crude extracts of r a t thymus, they found that DNA synthesis was stimulated i n the presence of monovalent cations i n the order, L i + , Na +, Cs +, Rb +, NH^+ and K +. Although optimum concentration f o r each ion d i f f e r e d , the majority showed maximum response over the r e l a t i v e l y narrow range of 50-60 mM. Stimulation was maximal at the pH optimum of the enzyme (pH 7.3) and decreased when a lower or higher pH was used. Bollum^ 1 reported that p a r t i a l l y p u r i f i e d c a l f thymus DNA polymerase was i n h i b i t e d by monovalent cations. I n h i b i t i o n by monovalent cations was also 20 72 reported for DNA polymerase i s o l a t e d from rat l i v e r ' and r a t Walker 43 tumor. In contrast, the a c t i v i t i e s of the Landschutz a s c i t e s tumor DNA polymerase*'"' and the rat> l i v e r mitochondrial DNA polymerase 2^ were greatly enhanced i n the presence of monovalent cations. Chiu and 26 Sung found that DNA polymerase extracted from p u r i f i e d n u c l e i from the rat b r a i n by 0.2 M phosphate buffer was greatly stimulated by K +, while the DNA polymerase a c t i v i t y extractable with 10 mM Tris-HCl buffer was i n h i b i t e d . - 58 -The way by which monovalent cations e f f e c t DNA polymerase a c t i v i t y remains unresolved. Since assay conditions and p u r i t y of the enzymes vary comparisons of the e f f e c t s of monovalent cations on the various DNA polymerases described i n the l i t e r a t u r e are i n v a l i d . There are several p o s s i b i l i t i e s which could account for the e f f e c t s of monovalent ions on the polymerase reaction. F i r s t , mono-valent cations may function by i n f l u e n c i n g the binding of the DNA polymerase enzyme to DNA. Second, monovalent cations may e f f e c t the act i v e s i t e of the enzyme. Third, monovalent cations may impede or f a c i l i t a t e the attachment of deoxynucleoside monophosphates onto the DNA strand. Presently a v a i l a b l e information does not permit a decision amongst these and other possible modes of action. 9. E f f e c t of I n h i b i t o r s A c t i v i t i e s of both p a r t i a l l y p u r i f i e d nuclear DNA polymerases were greatly enhanced by the addition of d i t h i o t h r e i t o l (DTT), a ragent which helps to maintain -SH groups i n the reduced state. This f i n d i n g suggests that the presence of s u l f h y d r y l group(s) i s important for enzymic a c t i v i t y . This supposition was tested by studying the e f f e c t s on polymerase a c t i v i t y of compounds which react with t h i o l groups. The r e s u l t s summarized i n Table VIII.show that both the T r i s -soluble and NaCl-soluble DNA polymerases were i n h i b i t e d by N-ethyl-maleimide (NEM) and p-hydroxymercuribenzoate (PHMB). The r e s u l t s also i n d i c a t e that the i n h i b i t i o n can be p a r t i a l l y prevented by d i t h i o t h r e i t o l . - 59 -However, the presence of 5 mM d i t h i o t h r e i t o l did not prevent i n h i b i t i o n when 10 mM NEM or 2.5 mM PHMB were included i n the reaction mixture. 2-Mercaptoethanol was less e f f e c t i v e i n protecting -SH groups than d i t h i o t h r e i t o l . Enzyme a c t i v i t y was restored, however, when d i t h i o -t h r e i t o l was added back to the assay mixture. I t i s evident from these r e s u l t s that s u l f h y d r y l groups are e s s e n t i a l f o r a c t i v i t y of both nuclear DNA polymerases. Romberg observed that the s u l f h y d r y l group of _E. c o l i DNA polymerase (DNA polymerase I) can be modified with ei t h e r iodoacetate or mercuric ion to give derivatives with f u l l polymerase a c t i v i t y and he concluded, therefore, that the -SH group was not part of the 73 a c t i v e s i t e . However, Moses and Richardson and Wickner and 74 Hurwitz working with E_. c o l i mutants d e f i c i e n t i n DNA polymerase I, found that N-ethylmaleimide completely i n h i b i t e d DNA synthesis. Mammalian DNA polymerases show a wide v a r i e t y of responses towards 25 t h i o l poisons. Sedwick and coworkers found that the cytoplasmic DNA polymerase from human KB c e l l s was very s e n s i t i v e to p-hydroxymercuri-benzoate, whereas the nuclear DNA polymerase was i n s e n s i t i v e . Weissbach 27 et a l . reported s i m i l a r findings with one of the DNA polymerases i s o l a t e d from n u c l e i of HeLa c e l l s but they observed that the other nuclear enzyme as well as the cytoplasmic enzyme were i n h i b i t e d almost completely. Mitochondrial DNA polymerase from r a t i n t e s t i n a l mucosa 35 was i n h i b i t e d by both NEM and PHMB. - 60 -Table VIII. E f f e c t of T h i o l Reagents on Nuclear DNA Polymerases. Addition T r i s - s o l u b l e DNA polymerase -DTT +DTT NaCl-soluble DNA polymerase -DTT +DTT None % a c t i v i t y 100 100 % a c t i v i t y 100 100 N-E thylmaleimide 2.5 mM 5 mM 10 mM 16 15 12 83 47 13 52 51 15 91 55 22 p-Hydroxymercuribenzoate 0.5 mM 20 2.5 mM 1 5 mM 0 69 10 75 1 4 6 0 0 0.2 - 61 -An i n h i b i t o r of DNA synthesis frequently used i n studies of DNA polymerases i s n a l i d i x i c a c i d (l-ethyl-l,4-dihydro-7-methyl-4-oxo-l,8-75 naphthyridine-3-carboxylic a c i d ) . Goss et a l . found that n a l i d i x i c a c i d was l e t h a l to p r o l i f e r a t i n g cultures of IS. c o l i , and s i m i l a r 74 observations were made by Wickner and Hurwitz. Chemical analysis of c e l l u l a r constituents revealed that l i p i d , p r otein and r i b o n u c l e i c a c i d l e v e l s were of the same order of magnitude i n c o n t r o l and drug-treated c e l l s , but DNA l e v e l s were markedly lower i n the drug-treated c e l l s . They concluded, therefore, that n a l i d i x i c a c i d i n t e r f e r e d with 38 the synthesis of DNA. In a recent review, Goulian has described the possible mechanisms of a c t i o n of t h i s i n h i b i t o r . These may be summarized as: 1. destruction of the r e p l i c a t i o n points, 2. induction of premature i n i t i a t i o n , 3. r e v e r s i b l e blocking of r e p l i c a t i o n without the concomitant destruction of the r e p l i c a t i o n point, or 4. interference with normal DNA-membrane associations. Table IX indicates that the two nuclear DNA polymerases behaved d i f f e r e n t l y i n the presence of n a l i d i x i c acid. The T r i s - s o l u b l e DNA polymerase a c t i v i t y was i n h i b i t e d by 1 mM n a l i d i x i c a c i d , while that of the NaCl-soluble DNA polymerase was greatly enhanced. However, the l a t t e r was i n h i b i t e d 40% i n the presence of 2 mM n a l i d i x i c a c i d . 35 Poulson and Zbarsky found that mitochondrial DNA polymerase from rat i n t e s t i n a l mucosa was i n h i b i t e d by 1 mM n a l i d i x i c a c i d . Goss and coworkers^"* noted that n a l i d i x i c acid had l i t t l e or no e f f e c t when the c e l l u l a r growth was r e s t r i c t e d . This could i n d i c a t e that n a l i d i x i c acid influences a c t i v e synthesis of DNA more than the repair process. Since the e f f e c t s of an i n h i b i t o r on DNA synthesis - 62 -i n vivo and i n v i t r o cannot be compared d i r e c t l y , any conclusion concerning i t s mode of action must be regarded as purely speculative. Table IX. E f f e c t of N a l i d i x i c Acid on Nuclear DNA Polymerases. Addition T r i s - s o l u b l e DNA polymerase NaCl-soluble DNA polymerase % a c t i v i t y % a c t i v i t y None 100 100 N a l i d i x i c a c i d 0.25 mM 0.5 mM 1 mM 2 mM 97 72 A l 20 325 3A6 60 - 63 -10. S t a b i l i t y of Nuclear DNA Polymerases The s t a b i l i t y of the nuclear DNA polymerase a c t i v i t i e s during storage under various conditions was studied. The data presented i n t h i s section were obtained using crude enzyme preparations. In general, s i m i l a r r e s u l t s were observed with p a r t i a l l y p u r i f i e d prepara-tions and any differences between these and the crude preparations w i l l be mentioned i n the text. a. S t a b i l i t y upon storage at various temperatures Nuclear extracts were prepared as described i n Methods, dialyzed against 20 mM Tris-HCl buffer, pH 7.5, containing 5 mM d i t h i o t h r e i t o l , and Immediately assayed f o r DNA polymerase a c t i v i t y . Aliquots of the preparations were then stored f o r 1, 2, 3, 5, 7, 11, and 17 days at 4°C, 0°C or frozen at -20°C. In the l a s t case fresh aliquots were thawed for each assay and unused enzyme was discarded, as repeated freeze-thawing caused loss of enzyme a c t i v i t y . A 25% loss of the a c t i v i t y i n the T r i s - s o l u b l e extract occurred during the f i r s t 48 hours of storage at 0°C and 4°C (Fig. 15). The loss of a c t i v i t y i n the frozen sample was even more dramatic, 70% of the o r i g i n a l a c t i v i t y disappeared within 48 hours. A f t e r the i n i t i a l sharp decline when stored at e i t h e r 0°C or 4°C the a c t i v i t y then decreased gradually so that approximately 50% of the o r i g i n a l a c t i v i t y was s t i l l present a f t e r seven days of storage and 40-45% a f t e r 17 days. It i s also evident that, except for the f i r s t two days, the a c t i v i t y i n the T r i s - s o l u b l e preparation was most stable at 4°C. The a d d i t i o n of - 64 -Figure 15. S t a b i l i t y upon storage at various temperatures of the T r i s - s o l u b l e DNA polymerase. - 65 -magnesium (5 mM) or c a l f thymus DNA (1 mg/ml) did not prevent the decline i n a c t i v i t y of the crude extract. The addition of DNA to the p a r t i a l l y p u r i f i e d T r i s - s o l u b l e DNA polymerase preparation s t a b i l i z e d enzyme a c t i v i t y . Enzyme a c t i v i t y was best preserved, however, i n the presence of 20% (v/v) g l y c e r o l . I f the enzyme preparation contain-ing 20% g l y c e r o l was stored at -20°C i t did not freeze and the a c t i v i t y remained unchanged f o r at least 1 month, and i t was s t i l l detectable a f t e r storage f o r 3 months. Figure 16 shows s i m i l a r e f f e c t s of storage at d i f f e r e n t temperatures on the a c t i v i t y of the crude NaCl-soluble preparation. A 30% loss i n enzyme a c t i v i t y was observed during the f i r s t 24 hours of storage at a l l three temperatures tested. Freezing was less detrimental to the a c t i v i t y of NaCl-soluble enzyme than to that of the T r i s - s o l u b l e enzyme. The h a l f - l i f e of enzyme a c t i v i t y i n preparations stored at 0°C or 4°C was approximately 5 days. Only 25% of the o r i g i n a l a c t i v i t y remained 17 days a f t e r extraction. In contrast to the T r i s - s o l u b l e preparation, the a c t i v i t y of t h i s enzyme was more stable at 0°C than at 4°C. Frozen samples retained more a c t i v i t y than samples stored at 0°C or 4°C and t h i s e f f e c t increased with length of storage. The 2+ addition of Mg and DNA had no e f f e c t on the s t a b i l i t y of DNA polymerase a c t i v i t y . The a c t i v i t y of the p a r t i a l l y p u r i f i e d NaCl-soluble DNA polymerase, however, was more stable i n the presence of DNA. The s t a b i l i t y was increased when preparations were stored at -20°C i n the presence of 20% (v/v) g l y c e r o l . These r e s u l t s are i n agreement with the findings of other i n v e s t i -gators that mammalian DNA polymerases i n crude or p a r t i a l l y p u r i f i e d - 66 -Figure 16. S t a b i l i t y upon storage at various temperatures of the NaCl-soluble DNA polymerase. - 67 -preparations are extremely unstable. b. S t a b i l i t y at d i f f e r e n t pH values A knowledge of enzyme s t a b i l i t y at d i f f e r e n t pH values i s of great importance when devising p u r i f i c a t i o n procedures. The T r i s - s o l u b l e and NaCl-soluble DNA polymerases were extracted and dialyzed against buffers with pH values ranging from 4.0 to 10.0. The a c t i v i t y was determined immediately a f t e r d i a l y s i s , and then a f t e r 3 and 7 days. During t h i s time the enzymes were stored at 4°C. The assay mixture used was i d e n t i c a l to that described i n Methods except that the concen-t r a t i o n of the T r i s buffer, pH 7.5, was increased to 0.1 M to ensure that a l l samples were assayed at the same pH. The T r i s - s o l u b l e DNA polymerase was stable over a r e l a t i v e l y narrow range of pH values (pH 6.0-7.5) and exhibited highest a c t i v i t y and most s t a b i l i t y at pH 7.5. At t h i s pH, 30-40% of the a c t i v i t y disappeared during 7 days of storage. At extreme pH values (pH 4.0 and pH 10.0) the o r i g i n a l a c t i v i t y was very low (20% and 10% of the maximum a c t i v i t y , respectively) and no a c t i v i t y was detectable a f t e r 7 days. Approximately 40-50% of the enzyme a c t i v i t y was l o s t i n 3 days of storage at pH values between 4.0 to 6.0 or 8.0 to 10.0. The NaCl-soluble DNA polymerase was stable over a wider range of pH values than the T r i s - s o l u b l e enzyme. Approximately 30% of the a c t i v i t y was l o s t during the f i r s t 3 days at pH values from 6.0 to 10.0 and a further 10-20% during the following 4 days. In contrast to the T r i s - s o l u b l e enzyme t h i s DNA polymerase was r e l a t i v e l y stable at a l k a l i n e - 68 -pH (pH 8.0 to 10.0) but optimum conditions were between pH 7.0 and 7.5. The NaCl-soluble DNA polymerase was extremely unstable at low pH values (pH 4.0 to 5.0) and 80% of i t s o r i g i n a l a c t i v i t y was l o s t i n 3 days. D. Molecular Weight Determination by Gel F i l t r a t i o n Gel f i l t r a t i o n or "molecular sieve" chromatography was employed as a p u r i f i c a t i o n step. In addition, the method was used to estimate the molecular weights of the two enzymes. Sephadex G-150 columns were prepared and c a l i b r a t e d as described i n Methods. The procedure of Laurent and K i l l a n d e r , described i n Methods, was used to determine the molecular weights. 1. NaCl-Soluble DNA Polymerase Preliminary experiments indicated that the NaCl-soluble DNA polymerase eluted with the void volume when a 0.05 M or 0.1 M Tris-HCl buffer, pH 7.5 was employed. This e l u t i o n pattern suggested that the enzyme had a molecular weight of at l e a s t 300,000. The p o s s i b i l i t y that t h i s f i g u r e represented an aggregate of several DNA polymerase molecules was considered. The experiment was therefore repeated using a buffer of high i o n i c strength (TS buffer-0.1 M Tris-HCl buffer, pH 7.5 with 1 M NaCl) i n an attempt to prevent possible aggregation of the enzyme molecules. The column was c a l i b r a t e d as described previously using markers ribonuclease A (M.W. 13,700; V g 163.0 ml), chymotrypsinogen A (M.W. 25,000; V 147.5 ml), ovalbumin (M.W. 45,000; V 129.5 ml), b a c t e r i a l - 69 -a l k a l i n e phosphatase (M.W. 80,000; V g 112.0 ml) and aldolase (M.W. 158,000; V 95 ml). Void volume (V ) was 75.0 ml and t o t a l bed volume e o (V ) was 183.0 ml. Corresponding K values were calculated and t av p l o t t e d versus the logarithm of the molecular weight of each standard protein. This c a l i b r a t i o n ( s e l e c t i v i t y ) curve i s shown i n F i g . 17. An enzyme preparation which had previously been chromatographed on DEAE-cellulose and subsequently concentrated using Sephadex G-25 and dialyzed, was applied to the Sephadex G-150 column. The e l u t i o n p r o f i l e obtained when the high i o n i c strength buffer was used i s shown i n Figure 18. A s i n g l e peak of DNA polymerase a c t i v i t y was observed which had an e l u t i o n volume (V ) of 134.0 ml and a K value of 0.546. e av Reference to a s e l e c t i v i t y curve indicated that the molecular weight of the NaCl-soluble DNA polymerase was approximately 40,000. 2. T r i s - S o l u b l e DNA Polymerase It was shown i n the previous section (C.8) that monovalent cations strongly i n h i b i t e d the DNA polymerase a c t i v i t y of the T r i s - s o l u b l e extract. This f i n d i n g may provide an explanation for the f a i l u r e to detect DNA polymerase a c t i v i t y i n the eluate from the Sephadex G-150 column using 0.1 M Tris-HCl, pH 7.5 with 1 M NaCI. A f t e r three unsuccessful runs with both crude and p a r t i a l l y p u r i f i e d T r i s - s o l u b l e extracts using the high i o n i c strength buffer, an a l t e r n a t i v e buffer system was tested, namely, TKM buffer, pH 7.5. After e q u i l i b r a t i o n of the Sephadex G-150 column with TKM buffer i t was c a l i b r a t e d with protein markers and the following values were obtained: ribonuclease A V T T 1 1 1 I | I I 0-8 0 -6 -> o ^ 0-4 0-2 0 0 Ribonudease A Chymotrypsinogen A -M^DNA polymerase •>Pva/bum»n ,Alkaline phosphatase .Aldolase -j I I I t I i I 2 3 4 5 6 7 8 9 1 0 2 0 M o l e c u l a r weight X 10~ 4 3 0 o Figure 17. C a l i b r a t i o n ( s e l e c t i v i t y ) curve for the estimation of the molecular weight of the NaCl-soluble DNA polymerase. Figure 18. Gel f i l t r a t i o n of the NaCl-soluble DNA polymerase on Sephadex G-150. E l u t i o n with 0.1 M Tris-HCl buffer, pH 7.5 with 1 M NaCl and 5 mM d i t h i o t h r e i t o l . - 72 -(M.W. 13,700; V g 163.0), chymotrypsinogen A (M.W. 25,000; V g 147.0), ovalbumin (M.W. 45,000; V g 132.5 ml), b a c t e r i a l a l k a l i n e phosphatase (M.W. 80,000; V £ 117.0 ml) and aldolase (M.W. 158,000; V g 99.0 ml). Void volume (V ) was 78.5 ml and t o t a l bed volume (V ) was 185.0 ml. o t The s e l e c t i v i t y curve f o r t h i s column i s shown i n Figure 19. Freshly prepared extract was dialyzed against TKM buffer and a 2.0 ml sample loaded onto the column. The e l u t i o n p r o f i l e ( F ig. 20) showed two peaks of DNA polymerase a c t i v i t y . The f i r s t peak eluted immediately behind the void volume and had an e l u t i o n volume (V ) of e 85.5 ml and a K value of 0.066. The second peak eluted at V 110.0 ml av e with a K value of 0.296. The DNA polymerase which eluted at the front av was estimated to have a molecular weight between 260,000 and 270,000, whereas the enzyme e l u t i n g l a t e r had a molecular weight of 104,000-105,000. These r e s u l t s i n d i c a t e that these are at least two DNA polymerases with d i f f e r e n t molecular weights present i n p u r i f i e d n u c l e i . The DNA polymerase associated with chromatin which was extracted only by s a l t s at high concentrations had a molecular weight of approximately 40,000. The s i t u a t i o n i s more complicated with respect to the T r i s - s o l u b l e enzyme since two DNA polymerase a c t i v i t i e s with widely d i f f e r i n g molecular weights were detected i n the eluate from the Sephadex G-150 column. There are three p o s s i b i l i t i e s which could explain t h i s observation. F i r s t , two d i f f e r e n t DNA polymerases may e x i s t i n the T r i s extract; second, during g e l f i l t r a t i o n the high molecular weight - 73 -0-8 0-6 > o ^ 0-4 0-2 00 T i 1—1 I 1 I i I K Ribonuclease A Xlhymotrypsinogen A ^Ovalbumin mAlkaline phosphatase ^^DNA polymerase vA/do/crse DNA polymerase* I i I i I I L 2 3 4 5 6 7 8 910 20 M o l e c u l a r weight x 1 0 " 4 30 Figure 19. C a l i b r a t i o n ( s e l e c t i v i t y ) curve f o r the estimation of the molecular weight of the T r i s - s o l u b l e DNA polymerase. I - 74 -FRACTION NO. Figure 20. Gel f i l t r a t i o n of the T r i s - s o l u b l e DNA polymerase on Sephadex G-150. E l u t i o n with TKM b u f f e r (50 mM T r i s - H C l , pH 7.5, 25 mM KC1 and 5 mM MgCl 2) and 5 mM d i t h i o t h r e i t o l . - 75 -enzyme may be p a r t i a l l y dissociated into fragments each having a molecular weight of 100,000 and which s t i l l possess DNA polymerase a c t i v i t y ; and t h i r d , the DNA polymerase of molecular weight 100,000 may form aggregates which have molecular weights of 200,000-300,000. Of these three p o s s i b i l i t i e s the t h i r d one provides the most l i k e l y explanation since other parameters (homogeneity on DEAE-cellulose, smooth pH curve, smooth magnesium concentration curve, e f f e c t s of monovalent cations) indicated that there was only one DNE polymerase a c t i v i t y associated with the T r i s extract. I f there were two DNA polymerases i n the T r i s extract some i n d i c a t i o n could presumably have been detected, f o r example, a broad pH<-curve or perhaps a shoulder on the magnesium concentration curve etc. I t seems highly improbable that the composition of the TKM buffer (50 mM Tris-HCl, pH 7.5, 25 mM KC1 and 5 mM MgC^) would induce d i s s o c i a t i o n of large molecules into smaller ac t i v e fragments. A v a i l a b l e data i n d i c a t e that none of the buffer components at the r e l a t i v e l y low concentrations used i s l i k e l y to cause cleavage. On the contrary, a buffer of low i o n i c strength would favor aggregation. On the basis of the a v a i l a b l e information i t seems probable that the T r i s - s o l u b l e DNA polymerase i n vivo has a molecular weight of about 100,000 while the high molecular weight component which possesses DNA polymerase a c t i v i t y i s an aggregate, probably a dimer. DNA polymerase a c t i v i t y was f i r s t found i n the postmicrosomal supernatant s o l u t i o n of mammalian ti s s u e homogenates prepared i n aqueous media.^'-^ U n t i l recently t h i s f r a c t i o n was the sole source 58 76 of the crude mammalian DNA polymerase. ' A survey of mammalian tiss u e - 76 -40 extracts by L.M.S. Chang showed that most enzymes from t h i s source sedimented at 6 to 8S on sucrose density gradients, corresponding to a 52 molecular weight of around 100,000. L. Waung found that the DNA polymerase present i n the cytoplasmic preparations from rat i n t e s t i n a l mucosa had a molecular weight of 101,000. A s i m i l a r value was also determined by R. Poulson using a DNA polymerase enzyme from the soluble cytoplasmic f r a c t i o n of the same tis s u e . Other i n v e s t i g a t o r s , using both Sephadex G-100 and Sephadex G-200, have obtained s i m i l a r r e s u l t s . ^ The molecular weights of both the T r i s - s o l u b l e DNA polymerase and the DNA polymerase present i n the postmicrosomal supernatant s o l u t i o n of the r a t I n t e s t i n a l mucosa correspond c l o s e l y to t h i s group of DNA polymerases. 24 Pat e l , Howk and Wang. discovered a new method f o r the ext r a c t i o n of DNA polymerase from rat l i v e r n u c l e i . The use of an aqueous medium containing 1 M NaCl (procedure o r i g i n a l l y described by Mirsky and 78 P o l l i s t e r ) f a c i l i t a t e d the d i s s o c i a t i o n of the deoxyribonucleoprotein complex to y i e l d non-histone (ac i d i c ) chromatin proteins possessing considerable DNA polymerase a c t i v i t y . Similar extraction procedures were used to demonstrate the presence of DNA polymerase a c t i v i t y i n 43 42 pro t e i n f r a c t i o n s from rat Walker tumor, c a l f thymus, rat l i v e r n u c l e i , ribosomes and smooth membranes, 3 1 and rabbit bone marrow.^'^ 46 Chang and Bollum reported a molecular weight value of 40,000 to 50,000 for the DNA polymerase i s o l a t e d from the deoxynucleoprotein complex of n u c l e i prepared from rabbit bone marrow. These authors 46 have demonstrated the presence of a low molecular weight species of DNA polymerase i n some 40 d i f f e r e n t mammalian ti s s u e s , including several - 77 -l i n e s of t i s s u e culture c e l l s . The Sodium chloride-soluble DNA polymerase of the n u c l e i of r a t i n t e s t i n a l mucosa with a molecular weight of around 40,000 apppears s i m i l a r to the above group of DNA polymerases. Table Xvsummarizes the r e s u l t s of the gel f i l t r a t i o n on Sephadex G-150 studies. The data show that the recovery of the DNA polymerase a c t i v i t y was 25% and 40% for the T r i s - s o l u b l e and NaCl-soluble DNA polymerase enzymes, re s p e c t i v e l y . The two peaks obtained by chromatography of the T r i s extract on Sephadex G-150 were equally active with both native and denatured DNA. In contrast, the NaCl-soluble DNA polymerase was detected only when native DNA was present i n the assay mixture. The r e s u l t s of the template preference demonstrated by each enzyme have important implications, however, a discussion of these findings w i l l be deferred. - 78 -Table X. Gel F i l t r a t i o n on Sephadex G-150 of Tri s - S o l u b l e and NaCl-Soluble DNA Polymerases. Enzyme Fra c t i o n T o t a l DNA polymerase a c t i v i t y (units) S p e c i f i c DNA Molecular weight polymerase a c t i v i t y (units/mg of protein) Native DNA Denat. DNA T r i s --soluble DNA polymerase Crude enzyme applied 1150 80 Peak I 180 34 2.66 x 10 5 0.97 Peak II 108 43 1.04 x 10 5 0.92 NaCl--soluble DNA polymerase P a r t i a l l y p u r i f i e d enzyme applied 106 5 Peak 43 29 3.94 x 10 4 No a c t i v i t y detected with denatured DNA - 79 -' CONCLUSION The DNA polymerase a c t i v i t y associated with p u r i f i e d n u c l e i i s o l a t e d from r a p i d l y d i v i d i n g c e l l s of rat i n t e s t i n a l mucosa was p a r t i a l l y p u r i f i e d and characterized. The l e v e l of enzyme a c t i v i t y present i n i s o l a t e d n u c l e i prepared from mucosal c e l l s was comparable with that found i n n u c l e i of regenerating r a t l i v e r . 1. P u r i f i e d n u c l e i contained s u b s t a n t i a l DNA polymerase a c t i v i t y which was r e a d i l y extracted with low i o n i c strength buffer. Evidence was also obtained which indicated that n u c l e i which had been repeatedly extracted with 10 mM Tris-HCl buffer, pH 8.0 containing 5 mM d i t h i o -t h r e i t o l s t i l l possessed considerable polymerase a c t i v i t y . This DNA polymerase a c t i v i t y was t i g h t l y bound to chromatin, however, and was extracted only i n a high i o n i c strength medium. Experiments with media containing d i f f e r e n t concentrations of s a l t s and buffers indicated that most DNA polymerase a c t i v i t y was extracted with 1 M NaCl i n 0.1 M Tri s - H C l buffer, pH 8.0, and 5 mM d i t h i o t h r e i t o l . This enzymic a c t i v i t y was designated the NaCl-soluble DNA polymerase while the polymerase a c t i v i t y which was extracted with low i o n i c strength T r i s b u f f e r , was termed the T r i s - s o l u b l e DNA polymerase. 2. The molecular weights of the T r i s - s o l u b l e and NaCl-soluble DNA polymerases were estimated by g e l f i l t r a t i o n on Sephadex G-150. Two peaks of DNA polymerase a c t i v i t y were detected when the T r i s - s o l u b l e extract was chromatographed. The molecular weights of these peaks of a c t i v i t y were calculated to be 266,000 and 104,000. Other parameters - 80 -(homogeneity on DEAE-cellulose chromatography, smooth pH curve, e f f e c t s of monovalent cations) indicated that there was only one DNA polymerase a c t i v i t y associated with the T r i s extract. I t i s u n l i k e l y that the composition of the e l u t i o n buffer (50 mM T r i s - H C l , pH 7.5, 25 mM KC1 and 5 mM MgC^) would induce d i s s o c i a t i o n of large molecules into smaller a c t i v e fragments. On the contrary, a buffer of low i o n i c strength would favor aggregation. I t was concluded, therefore, that the high molecular weight component possessing DNA polymerase a c t i v i t y was an aggregate of T r i s - s o l u b l e DNA polymerase molecules each with a molecular weight of 104,000. A s i n g l e peak of DNA polymerase a c t i v i t y was obtained when the NaCl-soluble nuclear extract was chromatographed on Sephadex G-150. I t corresponded to a molecular weight of approximately 40,000. 3. Chromatography on DEAE-cellulose indicated that the two DNA polymerase a c t i v i t i e s d i f f e r e d i n i o n i c charge. The bulk of the T r i s -soluble DNA polymerase a c t i v i t y eluted with 0.045-0.055 M KC1, while the NaCl-soluble DNA polymerase had a higher a f f i n i t y f o r the anion exchange r e s i n and was not eluted u n t i l the KC1 concentration was 0.165-0.21 M. 4. Some enzymic properties of the two DNA polymerase a c t i v i t i e s were quire s i m i l a r while others d i f f e r e d . 2+ The presence of a DNA template and Mg ions was required for both crude and p a r t i a l l y p u r i f i e d T r i s - s o l u b l e and NaCl-soluble DNA 2+ 2+ polymerase a c t i v i t i e s . Substitution of Mn or Ca resulted i n a loss of a c t i v i t y . - 81 -For maximum a c t i v i t y , the presence of a l l four deoxynucleoside 5'-triphosphates was required. With p a r t i a l l y p u r i f i e d T r i s - s o l u b l e DNA polymerase and NaCl-soluble DNA polymerase only 29-39% and 16-22% of the enzyme a c t i v i t y , r e s p e c t i v e l y , remained when either of the three unlabelled triphosphates was omitted from the reaction mixture. When a l l three unlabelled triphosphates were omitted, the a c t i v i t y was les s than 10% of that obtained i n the complete system. The addition of d i t h i o t h r e i t o l greatly enhanced the a c t i v i t y of both nuclear extracts, e s p e c i a l l y the p u r i f i e d preparations. The presence of s u l f h y d r y l reagents, _p_-hydroxymercuribenzoate and N-ethyl-maleimide i n h i b i t e d both of the nuclear DNA polymerase a c t i v i t i e s . It i s evident from these r e s u l t s that s u l f h y d r y l groups are e s s e n t i a l f o r enzyme a c t i v i t y of both extracts. The a c t i v i t y of the T r i s - s o l u b l e DNA polymerase was strongly i n h i b i t e d i n the presence of 1 mM n a l i d i x i c a c i d whereas the NaCl-soluble DNA polymerase a c t i v i t y was greatly enhanced. The a c t i v i t i e s of the nuclear DNA polymerases were also affected d i f f e r e n t l y by mono-+ + + valent cations. In the presence of NH^ , K or Na (40-50 mM) the a c t i v i t y of the T r i s - s o l u b l e DNA polymerase was i n h i b i t e d 50% and no polymerase a c t i v i t y was detected with 300 mM. In contrast, the NaCl-soluble DNA polymerase a c t i v i t y was stimulated 30-170% i n the presence of 40-60 mM NH.+, K + or Na +. 4 In Tri s - H C l buffer, a pH of 7.5 was optimal f o r the polymerase rea c t i o n catalyzed by eit h e r the T r i s - s o l u b l e or the NaCl-soluble DNA polymerase. In phosphate b u f f e r , however, the pH optima were 7.2 and 6.0 f o r the T r i s - s o l u b l e and NaCl-soluble polymerases, respectively. - 82 -The two extracts also d i f f e r e d i n template preference. The crude T r i s - s o l u b l e DNA polymerase functioned equally well with e i t h e r heat-denatured or native DNA, while the p u r i f i e d form showed a s l i g h t preference for native over heat-denatured DNA. The most p u r i f i e d preparations of the NaCl-soluble DNA polymerase, which i n crude extracts c o n s i s t e n t l y preferred native DNA as template, showed an absolute dependence on native DNA. 5. Differences between the T r i s - s o l u b l e and NaCl-soluble DNA polymerases i n e x t r a c t a b i l i t y from p u r i f i e d n u c l e i , molecular weight, i o n i c charge and i n t h e i r enzymic properties c l e a r l y indicate that two d i s t i n c t DNA polymerase enzymes are associated with p u r i f i e d n u c l e i prepared from r a t i n t e s t i n a l mucosa c e l l s . 6. The function of each of the nuclear DNA polymerase enzymes has not been established. However, the i n a b i l i t y of the NaCl-soluble DNA polymerase to use heat-denatured DNA as template indicates that t h i s enzyme i s unable to incorporate deoxynucleotides into extensive s i n g l e stranded regions of DNA template. On the other hand, the complete dependence of the p u r i f i e d NaCl-soluble DNA polymerase on native DNA as template perhaps i s an i n d i c a t i o n that i t i s e f f e c t i v e i n i n s e r t i n g deoxynucleotides into short s i n g l e stranded regions of DNA. On the basis of i t s template preference and close a s s o c i a t i o n with the deoxynucleoprotein complex i t i s a t t r a c t i v e to speculate that the NaCl-soluble DNA polymerase i s a repair enzyme. The r o l e of the T r i s - s o l u b l e DNA polymerase enzyme i s le s s c e r t a i n . - 83 -However, the a b i l i t y of t h i s enzyme to function with s i n g l e stranded DNA may indic a t e that i t i s involved i n DNA r e p l i c a t i o n i n mammalian c e l l s . 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