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Studies on the cytoplasmic dna polymerases from the intestinal mucosa of rat Waung, Lucille Yih-Lo 1972

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STUDIES ON THE CYTOPLASMIC DNA POLYMERASES FROM THE INTESTINAL MUCOSA OF RAT hy LUCILLE YIH-LO WAUNG B.A., Smith College, U.S.A., 1 9 6 9 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in the Department of Biochemistry We accept this thesis as conforming to the required standard for the degree of Master of Science THE UNIVERSITY OF BRITISH COLUMBIA Ap r i l , 1972 In present ing th i s thes is in pa r t i a l f u l f i lmen t o f the requirements fo r an advanced degree at the Un ive rs i t y of B r i t i s h Columbia, I agree that the L ibrary sha l l make it f r ee l y ava i l ab le for reference and study. I fu r ther agree that permission for extensive copying of th is thes i s for scho la r l y purposes may be granted by the Head of my Department or by his representat ives . It is understood that copying or pub l i c a t i on of th i s thes is fo r f i nanc ia l gain sha l l not be allowed without my wr i t ten permiss ion. Department of B i o c h e m i s t r y The Univers i ty of B r i t i s h Columbia Vancouver 8, Canada May 1, 1 9 7 2 Date i ABSTRACT S i g n i f i c a n t DNA polymerase a c t i v i t y has been found i n cytoplasmic preparations from r a t i n t e s t i n a l mucosa. The present work i n v o l v e s a p a r t i a l p u r i f i c a t i o n and a study of the general p r o p e r t i e s o f t h i s cytoplasmic enzyme a c t i v i t y . Crude cytoplasmic enzyme was prepared by high-speed c e n t r i -f u g a t i o n o f the homogenate of washed mucosal s c r a p i n g s . A s t r o n g i n h i b i t o r o f DNA polymerase was sedimented by the high-speed c e n t r i -f u g a t i o n . The bulk o f the enzyme a c t i v i t y was unadsorbed on DEAE-c e l l u l o s e . However, a minor p o r t i o n of the enzyme was adsorbed, and was e l u t e d w i t h 0.1 M KC1. When crude cytoplasmic enzyme was chroma-tographed by g e l - f i l t r a t i o n on Sephadex G-150, a s i n g l e peak o f DNA polymerase a c t i v i t y was detected. By the use of p r o t e i n markers with known molecular parameters, the molecular weight o f the DNA polymerase f r a c t i o n was estimated t o be 101,000. The enzyme r e q u i r e d the presence of a DNA template and Mg++ io n s . A c t i v i t y was only s l i g h t l y enhanced by the a d d i t i o n o f d i t h i o -t h r e i t o l . For maximum a c t i v i t y , the presence o f a l l f o u r deoxy-nucleoside t r i p h o s p h a t e s were r e q u i r e d . Heat-denatured DNA was p r e f e r r e d as primer. The optimum pH f o r t h i s nzymatic a c t i v i t y was found t o be 7.2 i n potassium phosphate b u f f e r , and 8.0 i n T r i s -acetate b u f f e r . Time course s t u d i e s on the enzyme r e a c t i o n i n d i c a t e d t h a t the r e a c t i o n was l i n e a r w i t h r e s p e c t t o i n c u b a t i o n time f o r a t l e a s t 30 min. The DNA polymerase a c t i v i t y was s t a b l e up t o 13 days under temperature c o n d i t i o n s o f k C to -20 C. G l y c e r o l i n 2Q& t o i i 35% (v/v) c o n c e n t r a t i o n s was found t o have both a s t i m u l a t o r y and a s t a b i l i z i n g e f f e c t on the enzyme a c t i v i t y . Ethylene g l y c o l a t 20% (v/v) c o n c e n t r a t i o n was a l s o found t o have a s t i m u l a t o r y e f f e c t on the enzyme a c t i v i t y . The enzyme was s t r o n g l y i n h i b i t e d i n the presence o f 0,10 M phosphate ions and a c t i v i t y was d r a s t i c a l l y reduced i n phosphate i o n co n c e n t r a t i o n s of 0.20 M and above. The product of the DNA polymerase r e a c t i o n c o u l d be destroyed by DNase, i n d i c a t i n g t h a t i t was DNA i n nature. The purpose of the present work was t o determine whether the DNA polymerase a c t i v i t y i n the cytoplasmic p r e p a r a t i o n i s a c t u a l l y of cytoplasmic o r i g i n , or whether i t i s due t o n u c l e a r contamination. The above r e s u l t s were compared w i t h the r e s u l t s obtained by other workers on the nuc l e a r DNA polymerases. The evidence seems t o i n d i c a t e t h a t the cytoplasmic enzyme a c t i v i t y i s not due t o n u c l e a r contamination. The n u c l e a r p r e p a r a t i o n contained s e v e r a l DNA poly-merases, while the cytoplasmic p r e p a r a t i o n contained a s i n g l e major DNA polymerase a c t i v i t y . T h i s cytoplasmic a c t i v i t y resembled one of the nuclear a c t i v i t i e s i n many r e s p e c t s . The cytoplasmic prepara-t i o n a l s o contained a minor DNA polymerase a c t i v i t y which may be m i t o c h o n d r i a l i n o r i g i n . i i i ACKNOWLEDGEMENTS The author would like to express her sincere thanks to Dr. S. H. Zbarsky for the advice and encouragement extended to her throughout the course of this research project. Personal assistance from the Canadian Medical Research Council in the form of Studentships i s very gratefully acknow-ledged. i v TABLE OF CONTENTS Page INTRODUCTION 1 B a c t e r i a l DNA polymerases 1 Mammalian DNA polymerases 8 A. Source 8 B. P u r i f i c a t i o n 8 C. Requirements. 9 D. Terminal t r a n s f e r a s e 10 E. D i s t i n c t DNA polymerases 11 F. I n t r a c e l l u l a r l o c a t i o n 12 The present work 15 MATERIALS AND METHODS A. P r e p a r a t i o n of crude enzyme e x t r a c t 18 B. Column chromatography on DEAE-cellulose 19 C. Column chromatography on phosphocellulose.... 20 D. G e l - f i l t r a t i o n on Sephadex G-150 21 E. Enzyme assays 1, DNA polymerase assay 22 2. Terminal d e o x y r i b o n u c l e o t i d y l t r a n s f e r a s e assay.., 2k 3* DNase I assay 25 k. P r o t e i n determination 26 V Page RESULTS AND DISCUSSION A>" Preparation of crude enxyme 27 B. Partial purification of crude enzyme 32 1. Chromatography on DEAE-cellulose. 33 2. Chromatography on phosphocellulose. 39 3. Gel-f i l t r a t i o n on Sephadex G-150 41 C, General properties of the enzyme 1. Requirements.. 46 2. DNA primer preference 48 3. Effect of pH 49 4. Effect of varying amounts of radioactivity in assay 51 5. Effect of glycerol and ethylene glycol 54 6. Time-course of the reaction... 55 7. Enzyme st a b i l i t y . . 56 8. Effect of phosphate ion concentration 59 9. Effect of DNase 1 60 SUMMARY 63 CONCLUSION 66 BIBLIOGRAPHY 68 v i TABLES Page I . DNA polymerase a c t i v i t y of v a r i o u s supernatant f r a c t i o n s from r a t i n t e s t i n a l mucosal c e l l s . . . . . 28 I I . DNA polymerase a c t i v i t i e s from recombination experiments with supernatant and p e l l e t f r a c t i o n s from r a t i n t e s t i n a l mucosal c e l l s , 30 I I I . DNase I a c t i v i t i e s i n va r i o u s supernatant and p e l l e t f r a c t i o n s from r a t i n t e s t i n a l mucosal c e l l s , 31 IV. Chromatography o f crude enzyme on DEAE-cellulose. E l u t i o n w i t h T r i s - H C l b u f f e r system, with l i n e a r g radient of K C 1 33 V. Chromatography o f crude enzyme on DEAE-cellulose. E l u t i o n w i t h T r i s - H C l b u f f e r system, w i t h stepwise gradient o f K C 1 3^  V I . DNase and t e r m i n a l t r a n s f e r a s e a c t i v i t i e s from crude and p a r t i a l l y p u r i f i e d enzyme preparations J6 V I I . G e l - f i l t r a t i o n o f crude enzyme preparation o f Sephadex G-150. E l u t i o n w i t h TKM b u f f e r system 46 V I I I . Requirements f o r DNA polymerase a c t i v i t i e s from r a t i n t e s t i n a l mucosal c e l l s 47 IX. DNA polymerase a c t i v i t i e s w i t h n a t i v e or heat-denatured DNA as primer 48 X. E f f e c t o f g l y c e r o l and ethylene g l y c o l on DNA polymerase a c t i v i t y 55 v i i Page XI. Stability of DNA polymerase under different temperature conditions 58 XII. Effect of phosphate ion concentration on DNA polymerase activity 59 XIII. Effect of DNase I and RNase on DNA polymerase assay. 61 v i i i FIGURES Page 1. Direction of chain growth catalyzed by DNA polymerase 2 2. Sites in the active center of DNA polymerase 4 3. Production of a 'nick' in DNA., 5 4. Chromatography of crude DNA polymerase from rat intestinal mucosa on DEAE-cellulose. Elution with TKM buffer containing 1 mM dithiothreitol and 20% ethylene glycol, with linear gradient of KC1 34 5. Chromatography of crude DNA polymerase from rat intes-t i n a l mucosa on DEAE-cellulose. Elution with TKM buffer containing 1 mM dithiothreitol and 20% ethylene glycol, with stepwise gradient of KC1..., 35 6. Standard curve for the estimation of the molecular weights of protein sample on the basis of their elution volumes from g e l - f i l t r a t i o n on Sephadex G-150 42 7. G e l - f i l t r a t i o n of crude DNA polymerase from rat intestinal mucosa on Sephadex G-150. Elution with 0.1 M phosphate buffer, pH 7 .2, containing 1 mM dithiothreitol and 20% ethylene glycol 43 8. Gel-filtration of crude DNA polymerase from rat intestinal mucosa on Sephadex G-150. Elution with TKM buffer containing 1 mM dithiothreitol and 20% ethylene glycol.... 45 9 . The effect of pH on crude DNA polymerase from rat intestinal mucosa. 50 i x Page 10. The effect of varying the total concentration of TTP in the incubation mixture for the DNA polymerase assay, the specific radioactivity being kept constant.. 52 11. The effect of varying the specific radioactivity in the incubation mixture for the DNA polymerase assay, the total TTP concentration being kept constant. 53 12. Time-course studies on the DNA polymerase reaction..... 57 1 INTRODUCTION Deoxyribonucleic a c i d (DNA) i s b e l i e v e d t o be the c e n t r a l storehouse of genetic i n f o r m a t i o n i n most c e l l s . T h i s genetic i n f o r m a t i o n determines the biochemical s p e c i f i c i t y o f the c e l l , and i s passed i n t a c t from parent t o progeny on c e l l d i v i s i o n . The processes i n v o l v e d i n DNA b i o s y n t h e s i s , e s p e c i a l l y those of s e l f - d u p l i c a t i o n , are t h e r e f o r e of great i n t e r e s t and have been e x t e n s i v e l y s t u d i e d . On the b a s i s o f t h e i r s t r u c t u r a l model f o r complementary double-stranded DNA, Watson and C r i c k ( l ) proposed t h a t each chain of the DNA duplex serves as template f o r the sy n t h e s i s of a complementary c h a i n , so t h a t two r e p l i c a s of the o r i g i n a l double-stranded s t r u c t u r e are produced. Meselson and S t a h l (2), i n t h e i r c l a s s i c a l experiments w i t h CsCl d e n s i t y gradient c e n t r i f u g a t i o n of N^/N 1^ h y b r i d E. c o l l DNA, demonstrated t h a t t h i s 'semi-conser-v a t i v e ' type of r e p l i c a t i o n a c t u a l l y takes place i n v i v o . B a c t e r i a l DNA polymerases The f i r s t i s o l a t i o n o f an enzyme i n v o l v e d i n DNA r e p l i c a t i o n was from e x t r a c t s o f E. c o l i by A. Kornberg and h i s a s s o c i a t e s i n i960 (3). T h i s enzyme converts deoxynucleoside polyphosphates i n t o polymeric m a t e r i a l , and was termed DNA polymerase (EC 2.7.7.7 Deoxynucleoside-triphosphate» DNA d e o x y n u c l e o t i d y l t r a n s f e r a s e ) . The o v e r a l l r e a c t i o n c a t a l y z e d may be described as f o l l o w s : 2 n! dATP n 2 dGTP 113 dCTP dTTP DNA DNA dAMP dGMP dCMP dTMP .+ 2(n 1+n 2) PP 2n^+2n 2 DNA polymerase i s now known to catalyze the addition of mononucleotide units to the 3'-hydroxyl terminus of a primer DNA chain (4). Synthesis proceeds in the direction of 5' to 3' (Fig. 1). — A — G T — A — T=A s /\ C =G / P OH -7 Template 5' Primer y 5" Pig. 1. Direction of chain growth catalyzed by DNA polymerase. (A, adenine; C, cytosine; G, guanine; and T,' thymine.) A l l four deoxynucleoside triphosphates are required; the diphosphates are not polymerized. The overall reaction i s believed to be reversible, as incubation of DNA with high . concentrations of pyrophosphate results in a partial depoly-merization reaction. The reaction also requires the presence 3 of a divalent metal ion, usually magnesium, and of a DNA primer. The synthetic material resulting from the catalytic reaction i s shown to he a DNA with physical characteristics largely resembling those of the primer. Much work has been done on the DNA polymerase from E. c o l i , sometimes referred to as 'the Kornberg polymerase'. This i s found to be a versatile enzyme with many catalytic properties. These include (4) t (a) the 5' •* 3' growth of a DNA chain by the polymerization of nucleotides; (b) hydrolysis of a DNA chain in the 3' -> 5' direction; (c) hydrolysis of a DNA chain in the 5' * 3' direction; (d) pyrophosphorolysis of a DNA chain from the 3* en&t and (e) exchange of inorganic pyrophosphate with the terminal pyrophosphate group of a deoxyribonucleoside triphosphate. Kornberg has presented a picture of several major sites within the active center of the enzyme ( 4 ) . These sites specifically recognize and accommodate the template chain, the primer chain, the primer terminus, or an incoming triphosphate (Fig. 2 ) . It was proposed that the triphosphate i s bound adjacent to the 3*-terminus of the primer, and oriented so that i t can form a base pair with the template. When a correct base pair i s formed, a nucleophillc attack by the 3'-OH of the primer terminus on the innermost phosphate of the triphosphate takes place. Through movement of the entire chain relative to the enzyme, the newly added nucleotide i s shifted to the primer terminus site and i s then ready to attack another triphosphate and add the next 4 nucleotide. The specificity of DNA polymerase i s probably based on i t s demand for one of the four Watson-Crick base pairs, a l l of which contain regions of identical dimensions and geometry and are symmetrical (4). Fig, 2 , Sites in the active center of DNA polymerase Studies on DNA binding to DNA polymerase indicated that intact double-stranded DNA did not bind to the enzyme. Only nicked, denatured, or single-stranded DNA's were bound and replica-ted. This observation, together with the fact that DNA polymerization occurs only in the 5' * 3' direction, has caused some skepticism about the phusiological role of the Kornberg enzyme in replication. Various schemes have been proposed for the unidirectional replica-tion of a duplex DNA chain. Replication i s init i a t e d by the introduction of a 'nick', a single break in one of the two DNA 1 strands, at which DNA polymerase binds. Replication in the 5 ' •» 3' • 5 direction proceeds for some distance and then switches to the complementary strand a6 template to form a fork. The fork i s then cleaved by an endonuclease. Repetition of this process results in small pieces of DNA near the replicating fork which may be linked up by a ligase, Okazaki and co-workers (5) have reported the isolation of small pieces of DNA at or near the nascent replicating' region. This hypothesis i s not entirely satisfactory as the replication i s staggered, alternating from one strand to the other. Examination of dividing bacteria by autoradiography (6) or by gene duplication (7) have indicated that there i s a simultaneous sequential replication of both strands. Fig. 3. Production of a 'nick' in DNA The 5' * 3' nuclease activity associated with DNA polymerase readily removes non-base-paired segments, eg. thymine dimers, suggesting a repair function for the enzyme in vivo. An endonuclease recognises a. disordered region in the DNA, and produces a nick to the 5' side (Fig. 3). Such dimer-specific endonucleases have been 6 identified and purified ( 8 , 9 ) . DNA polymerase acts at the nick excising the damaged region, and simultaneously r e f i l l i n g the resulting gap. Closure with a ligase completes the repair process. That E, c o l i DNA polymerase may function in repair in vivo i s indicated by the UV sensitivity of mutants defective in the enzyme. DeLucia and Cairns ( 10) have isolated an amber mutant of E. c o l l containing less than 1% of the DNA polymerase activity of the parent strain. This mutant, called pol A, was discovered by assaying for the enzyme in extracts from a few thousand individual clones of mutagenized E. c o l i . It has essentially normal growth characteristics but i s sensitive to ultraviolet irradiation and methylmethane sulfonate. Altogether six pol A mutants (pol 1 - 6 ) have now been isolated ( l l ) . This discovery of these mutants have strengthened the idea that E. c o l i DNA polymerase does not participate in chromosome replication. Various attempts have been made to isolate from the Cairns mutant another enzyme which can replicate DNA, A membrane-bound enzyme i s now believed to be involved in replication in pol A l . Knippers ( 12) has succeeded in solubilizing a DNA synthesizing enzyme activity from a crude cell-free membrane fraction. The enzyme has a molecular weight of between 6 0 , 0 0 0 and 9 0 , 0 0 0 , and can synthesize DNA semi-conservatively for at least 90 minutes. Unlike the Kornberg DNA polymerase, this enzyme i s strongly inhibited by mercuri-compounds and i s resistant to an antiserum which inhibits the Kornberg enzyme. Independently, T. Kornberg and M. Gefter ( 13) reported 7 the isolation from the Cairns mutant of an enzyme which could synthesize DNA in vitro. This enzyme, which they c a l l DNA polymerase II, i s apparently the same as Knipper's enzyme. It works best with double-stranded DNA as template. It polymerizes deoxynucleoside triphosphates in a 5' to 3* direction, and requires a free 3'-hydroxyl group. The enzyme i s not inhibited by antisera against A. Romberg's polymerase I. Recent genetic experiments (14) indicate that polymerase I together with an excision function, can edit out pyrimidine dimers induced by UV irradiation. It appears that in E. c o l i there are two DNA repair mechanisms, only one of which involves polymerase I. The Cairns mutants, although lacking the repair function which involves polymerase I, can survive UV irradiation because the second repair mechanism i s functional. Gross and co-workers (15) have also found that E. c o l l c e l l s in which both repair mechanisms are inactivated are inviable. That polymerase II i s the enzyme responsible for DNA replication in vivo remains to be proven. Its discovery at least demonstrates, however, that there may be several enzymes in E. c o l i , a l l of which can polymerize DNA, although most of them are not involved in DNA duplication. 8 Mammalian DNA polymerases A, Source Shortly after the earlier reports on E. c o l l DNA poly-merase;; i t became apparent that mammalian tissues also contain an enzyme that catalyzes a similar reaction. Such enzyme ac t i v i t i e s were reported by Davidson et a l (16) from Enrlich ascites tumor, by Bollum (1?) from calf thymus gland, by Mantsavinos e_t a l (18) from regenerating rat l i v e r , and by Leung and Zbarsky from rat intestinal mucosa (19» 20). DNA polymerase activity has subsequently been discovered in a l l animal ce l l s which have been examined ( l l , 21). B. Purification Mammalian systems obviously offer a wider variety of tissues for study, with the poss i b i l i t i e s of c l i n i c a l as well as theoretical applications. However, the crude c e l l extracts or homogenates require much purification before use. Possible factors which might interfere with DNA polymerase studies include endo-genous mononucleotides and nucleic acids, deoxyribonucleases (DNases), phosphodiesterases, and triphosphatases. The enzyme from calf thymus has been purified about f i f t y - f o l d with respect to protein in the crude extract (17) by acid precipitation, ammonium sulfate fractionation, and passage through DEAE-cellulose. It has also been freed from DNase and phosphodiesterase activity by chromatography on hydroxylapatite (22). In general, the techniques of ammonium sulfate fractionation, and chromatography on DEAE-9 cellulose or hydroxylapatite are used (23, 24). However, extensive purification procedures are often not recommended as they may result in a highly purified but 'non-native' enzyme (21). Gel-f i l t r a t i o n has been found to be a favorable fractionation technique since i t does not require wide fluctuations of pH and ionic strength. G. Requirements Conditions for optimal DNA polymerase activity have been described for several mammalian systems. There i s an absolute requirement for a divalent metal cation. Mg ions alone appear to give the optimum response, although partial replacement of Mg"*"*" ions with Mn"*"*" and Captions together has been observed (25). DNA as primer or template i s also required for the reaction. In most systems, denatured DNA i s preferred over native DNA as primer (23, 26, 27), although in some cases (25, 28) the preference appears to be for native DNA, DNA activated by light treatment with DNase often produces high polymerase activity (28, 29, JO), indicating the a b i l i t y of the enzyme to replicate at nicks. Most animal c e l l DNA polymerases do not display a s t r i c t requirement for a l l four deoxynucleoside triphosphates (21). An exception i s the Ehrllch ascites enzyme (29), which resembles the bacterial and phage polymerases in that synthesis drops to very low levels i f one triphosphate i s omitted. Cultured human KB ce l l s (30) and sea urchin enzymes (28) can synthesize at 25 - 50% of the normal rate with only three triphosphates. The common explanation for these observations has been contamination 10 with terminal deoxynucleotidyltransferase. This appears not to he the case in the KB c e l l enzyme although the correct mechanism remains unknown (30). D. Terminal transferase Krakow et a l (31» 32) were the f i r s t to report an enzyme in calf thymus nuclei which catalyzed the incorporation of a single deoxyribonucleoside triphosphate into terminal positions of DNA in the absence of the other three triphosphates. This enzyme i s referred to as the 'terminal transferase', to distinguish i t from the 'replicative' DNA polymerase. It requires heated DNA primer, Mg , and cysteine. The incorporation of mononucleo-ti d y l residues i s not stimulated, and i s in fact inhibited, by the addition of the other three deoxyribonucleotides to the reaction mixture. Using non-aqueous preparations of calf thymus nuclei and cytoplasm, Smith and Keir (33i 3*0 found that the nuclei and cytoplasm each contained both replicative and terminal transferases, the latter enzyme being about l/2? as active as the former. The'rterminal transferases from calf thymus have now been further purified by Bollum (35)« The molecular weight calculated from equilibrium sedimentation i s 32»600. In SDS-polyacrylamide gels the protein dissociates into two subunits. Enzyme activity i s inhibited by low concentrations of metal chelating agents such as EDTA of o-phenanthroline, suggesting that the enzyme may 11 be a metalloprotein (36). Further studies indicate that the metal participates in the binding of oligodeoxynucleotide primer rather than in the binding of the triphosphate. E. Distinct DNA polymerases Numerous attempts have been made to isolate the DNA polymerase a c t i v i t i e s from animal c e l l s . Animal c e l l s infected with Shope fibroma virus have been found to contain a distinct polymerase activity. Infected rabbit kidney c e l l s and rabbit tumors both contain two polymerase a c t i v i t i e s which can be distinguished by their behavior on phosphocellulose and reactivity with specific antibody (371 38); the enzyme reacting with anti-body i s presumed to be induced by virus infection. A DNA polymerase activity associated with hepatomas has been found which differs from the polymerase of normal l i v e r in i t s preference for denatured over native templates (39, 40, 41). A DNA polymerase associated with mitochondria has been found,* and has been purified from rat l i v e r mitochondria (42 , 43, 44) and yeast mitochondria (45). These mitochondrial polymerases have low specific a c t i v i t i e s , and d i f f e r from the nuclear enzyme with respect to chromatographic properties, Mg requirement, template preference, solubility, and catalytic parameters. The crude mitochondrial extracts are able to use both native and denatured DNA, but the highly purified mitochondrial polymerase freed from nuclease activity, preferred denatured rather than native DNA. 12 F. Intracellular location DNA polymerases in mammalian tissues were originally found to be more readily obtained ;from soluble supernatant fractions after high-speed centrifugation of disrupted c e l l s , than from nuclei and other intracellular particles ( 1 7 , 24). This apparent cytoplasmic location of the enzyme was unexpected since DNA synthesis was believed to occur within the nucleus. Using a technique involving only non-aqueous organic solvents, Keir et a l (46) prepared nuclei and cytoplasm from regenerating rat l i v e r ; DNA polymerase was then extracted from these preparations with aqueous buffers. Much enzyme activity was found in the nuclear fraction and lower but appreciable amounts in the cytoplasm. Similar results were obtained from non-aqueous nuclei and cytoplasm from rabbit and calf-thymus. Ga + + ions appear to be an important factor in the extraction of DNA polymerase in aqueous media; i t i s known that they are necessary for isolation of nuclei in a morphologically undamaged state ( 4 7 ) . =It was found that, when extracted with an aqueous medium containing 2mM Ga ions, DNA polymerase in rat thymus tissue was evenly distributed between the nuclear and the cytoplasmic fractions (48). Similar distribution of enzyme activity has been found in mouse embryo c e l l s ( 4 9 ) . Loeb et a l (50) have recently reported their findings in the early developing sea urchin embryos. Early development of sea urchin embryos i s characterized by exponential c e l l division, accompanied by an exceptionally high level of DNA polymerase 13 activity in vitro. The majority of the polymerase activity was found in the cytoplasm of the egg. As the embryo developed, progressively more polymerase activity was found in the nuclear fraction with a concomitant loss of activity in the cytoplasm. By the time of hatching, 95% of the polymerase activity was recovered in the nuclei. The authors looked for, but could not find, evidence for selective synthesis of DNA polymerase. This indicated that the translocation of polymerase activity could not have resulted from either a breakdown in the cytoplasm and preferential synthesis of the polymerase in the nuclei, or an extreme rapid turnover in the cytoplasm with some transfer to the nuclei. They concluded then that there i s a migration of a preformed enzyme from the cytoplasm into the nucleus. Attempts to show differences between the nuclear and cytoplasmic DNA polymerases have been unsuccessful so far. Weissbach et a l (5 l ) recently reported the isolation of two separable DNA polymerase a c t i v i t i e s from the nucleus of HeLa c e l l s , and only one DNA polymerase activity in the cytoplasm. The two nuclear enzymes differ in elution patterns on DEAE-cellulose and phospho-cellulose, molecular weight estimations, optimum Mg ion concentration, optimum pH, high salt concentration inhibition, and primer activation. One of the nuclear enzymes resembled the cytoplasmic enzyme in a l l these respects, although the actual relationship between the two enzyme ac t i v i t i e s remains to be established. The relationship between the two nuclear enzymes i s also unclear; they may be distinct enzymes or they may 14 share some common structural features. An identical pattern of DNA polymerases has been found in the normal human lung diploid line WI-38, which also has two separable a c t i v i t i e s in the nucleus and only one detectable DNA polymerase activity in the cytoplasm. 15 The present work The present investigation i s a continuation of the work done by Leung and Zbarsky on rat intestinal mucosa (19, 20). The intestinal tissue was chosen for study because of i t s high mitotic rate and rapid replacement time for DNA, indicating the possibility of high DNA polymerase activity (52» 53). Both a replicative and a terminal DNA nucleotidyl-transferase were detected in extracts of nuclei. The replicative enzyme could use either native or denatured DNA as primer, while the terminal enzyme preferred heat-denatured DNA primers. Results of chromatography on DEAE-cellulose and g e l - f i l t r a t i o n , and sucrose density gradient centrifugation indicated that the DNA polymerases were heterogeneous in nature, with molecular weights between 2.5 x^M^ ofeand^3ix|i.G5. In a study of the intracellular distribution of the DNA polymerases, the amount of enzyme activity was compared in nuclei isolated in the presence or absence of Oa-*"*" ions,, known to preserve nuclei in an undamaged state. The nuclei isolated in the presence of Ca"*"*" ions retained a larger proportion of i t s protein and corresponding DNA polymerase ac t i v i t y than nuclei isolated in a medium without Ga* 4 ions (5^). This indicated that there was some leakage of enzyme from the nucleus to the cytoplasm during extraction. However, when nuclear and cytoplasmic fractions were prepared in non-aqueous solvents from rapidly frozen and lyophilized tissue, enzyme activity was detected in both fractions, although the nuclear fraction 16 contained a higher total activity than the cytoplasmic fraction (54). These observations were in agreement with the findings in other mammalian systems in that DNA polymerase activity was present in both fractions (55)' The present work involves a partial purification and examination of the properties of the enzyme act i v i t y in the cytoplasmic fraction. The ultimate goal of the investigation i s to determine whether the cytoplasmic enzyme activity i s identical with that found in the nuclei, or whether i t i s a distinct enzyme. The following properties have tjeen found for the cytoplasmic activity, and a comparison with the properties of the nuclear activity i s now awaited. The cytoplasmic DNA polymerase demonstrates an absolute requirement for Mg ions and DNA primer. Dithiothreitol appears to be dispensible. About 2J% activity remained in the absence of three of the four nucleoside triphosphates. The optimum pH for the enzyme was found to be 7.2 in phosphate buffer, and 8.0 in Tris-acetate buffer. Heated DNA i s always preferred to native DNA as primer. The product of the DNA polymerase reaction can be destroyed, by DNase, indicating that i t i s DNA in nature. The enzyme appears f a i r l y stable up to about two weeks.iEnzyrae activity i s stimulated and stabilized when 20%!to 35% glycerol or ethylene glycol i s added to the enzyme preparation. The crude enzyme has been purified about 42-fold by Sephadex gel-f i l t r a t i o n and about 5-fold by DEAE-cellulose chromatography, with the removal of about 7&% of the contaminating DNase activity. 1 7 Further purification on phospho-cellulose has not been successful, however, possibly due to the strong inhibitory effect of phosphate ions present in the elution gradients applied. 18 MATERIALS AND METHODS A/ Preparation of crude enzyme extract Generally four male Wistar rats, weighing about 185 to 200 gms each, were used per experiment. The rats were k i l l e d by a blow to the head and were immediately decapitated. The small intestine of each was rapidly removed and i t s contents flushed out with cold saline (Q.9% NaCl). The entire length of the intestine was then everted as described by Perris (56). A 'sausage' was then made by tying up one end of the everted intestine, f i l l i n g i t with saline u n t i l the intestinal walls were f u l l y extended, and tying up the other end of the intestine. The sausage was washed by a modification of the metho d of Perris (56). Swirling was done f i r s t in ice-cold saline, twice in Krebs-Ringer phosphate buffer, pH 7.4, containing 6% Dextran, and fi n a l l y in saline again, for periods of 2j min in each solution. The washed intestine was s l i t open, and the mucosa was scraped off by stroking gently with the edge of a glass slide. The scrapings were then homogenized in 9 volumes of TKM buffer (0.05 M Tri s , 0.025 M KC1, 0.005 M MgCl2i6 H20, adjusted to pH 7.4 with HCl) containing O.32IM sucrose. Homogenization was done in a glass Potter-Elvehjem homogenizer with a Teflon-tipped pestle. The pestle was run el e c t r i c a l l y at a speed of 800 rpm, and five complete passes, each consisting of an upward and a downward stroke were made. The homogenate was f i l t e r e d through two layers of nylon, and the f i l t r a t e was centrifuged 10 min at 700 x g , 15 min 19 at 20,000 x g, and 1? hrs at 105,000 x g. The f i n a l supernatant o was saved and stored at -20 G. In some experiments, as w i l l be specified, glycerol was added to the f i n a l supernatant to a concentration of 20$ or 35$ before storage. B. Column chromatography on DEAE-cellulose About 20 gms of DEAE-cellulose (diethylaminoethyl-cellulose, Whatman DE 22) were washed by decantation f i r s t with 0.5 N HC1 and therewith 0.5 N NaOH. The slurry was then washed several times in TKM buffer. Fines were removed using the same buffer. The slurry was de-aerated under vacuum in a dessicator, and was packed under gravity at 4°C, into a 1.8 cm diameter column up to a height of 20 cm. The column was washed before use with TKM buffer containing 20$ (v/v) ethylene glycol. B a r i l et a l (57) have reported that ethylene glycol stabilized the activity of crude and purified rat l i v e r DNA polymerases for at least 3 weeks at 4 C and at least 6 months at -20 C, About 30 of protein were loaded onto the column, after which the column was washed with 100 mis of TKMfbuffer containing 1 mM dithiothreitol and 20$ ethylene glycol. A stepwise gradient resulting from increasing concentrations of KG1 was applied to the column. F i f t y mis each of TKM buffer containing 1 mM dithio-threitol and 20$ ethylene glycol and the following concentrations of KC1 were used: 0.10 M, O.25 M, and 0.50 M. Three ml fractions were collected by gravity. The gradient profile was followed by measuring the conductivity of each fraction collected. The 20 protein elution pattern was examined by measuring the absorbance of each fraction at 280 nm. The DNA polymerase activity of each fraction was determined by the usual enzyme assay. In some experiments, a continuous gradient was used. A DEAE-cellulose column was washed and equilibrated with 0.005 M Tris-phosphate buffer, pH 8.0. The gradient consisted of 3Q0 mis of 0.05 M phosphate buffer, pH 8.0, containing 1 M KG1 and 2 mM dithiothreitol, into 300 mis of 0.005 M Tris-phosphate buffer, pH 8.0. Five ml fractions were collected by gravity and each fraction was examined in the manner already described. G. Column chromatography on phosphocellulose About 6 gms of phosphocellulose (Bio-Rad cation exchange cellulose, Cellex-P) were washed with glass-distilled water to remove fines. The slurry was then equilibrated with 0.05 M potassium phosphate buffer, pH 6.8, containing 1 mM dithiothreitol and 20% ethylene glycol, by washing six to seven times with the buffer. The slurry was de-aerated under vacuum in a dessicator, and was packed into a 1.2 cm diameter column up to a height of 18 cm. The column was washed with the same buffer before use. About 5 rag of protein were loaded onto the column, after which the column was washed with 75 mis of 0.05 M potassium phosphate buffer, pH 6.8, containing 1 mM dithiothreitol and 20% ethylene glycol. A stepwise gradient resulting from increasing concentrations of potassium phosphate was applied onto the column. F i f t y mis each of the following concentrations of potassium 21 phosphate buffer, pH 6.8, containing dithiothreitol and ethylene glycol were used: 0.1 M, 0.2 M, and 0.5 M. Three ml fractions were collected by gravity and each fraction was examined as previously described. Another procedure has been used in which 17.5 mg of o protein were allowed to s t i r gently at 4 C in a slurry of phospho-cellulose previously washed and equilibrated in 0.05 M phosphate buffer, pH 6.8, containing !• mM dithiothreitol and 20$ ethylene glycol. The entire slurry, with the adsorbed protein, was packed into a 1.2 cm diameter column, and was washed with 60 mis of the buffer. A linear gradient resulting from an increasing concentration of phosphate was applied: one hundred mis of 0.5Q M phosphate buffer, pH 6.8 was run into an equal volume of 0.05 M phosphate buffer, pH 6.8, both buffer systems containing 1 mM dithiothreitol and 20$ ethylene glycol. The elution was followed by a f i n a l 60 mis of the 0.50 M phosphate buffer. Three ml fractions were collected, and each fraction was examined as previously described. Every third fraction was then dialyzed overnight at 4°C against TKM buffer containing 1 mM dithiothreitol and 20$ ethylene glycol before being assayed for DNA polymerase activity. D. Gel- f i l t r a t i o n on Sephadex G-150 Forty gms of Sephadex G-150 (Pharmacia) were stirred into 2 1. of either TKM buffer or 0.1 M phosphate buffer, pH 7.2, and were allowed to swell, with occasional s t i r r i n g , for 4-8 hrs o at 4 C. The slurry was de-aerated under vacuum, and was poured 22 into a 100 cm column (2.5 cm diameter) according to the instructions from Pharmacia. The prepared column was washed by upward flow elution for 24 hrs with the original buffer containing 1 mM dithiothreitol and 20% ethylene glycol. Fractions were collected by upward flow elution, with a pressure head no greater than 30 cm. Two mis each of a solution of Blue Dextran 2000 (Pharmacia) and 3 M NaCl were run into the column for the determination of void volume and total volume respectively. The column was calibrated with the standard proteins chymotrypsinogen A (5 mg)» ^-globulin (5 mg), and bovine serum albumin (10 mg). In some experiments hemoglobin (4 mg) was used instead of bovine serum albumin. To the calibrated column, a 2.0 ml sample of the crude enzyme preparation containing 10 to 42 mg protein was applied, followed by the buffer system used, containing 1 mM dithiothreitol and 20% ethylene glycol. Fractions of 2.0 of 3.0 mis were collected. The optical density at 280 nm and the DNA polymerase activity of each fraction was determined. E/' Enzyme assays 1. DNA polymerase assay The activity of DNA polymerase was measured by the incor-poration of a radioactively labeled deoxyribonucleoside triphosphate into an acid-insoluble product. The assay system used was similar to that described by Chiu and Sung (58). 23 The incubation mixture contained, in a total volume of 0.40 mlsi potassium phosphate buffer, pH 7.2 20jumoles MgGl2 2 ;umoles dithiothreitol 2 jumoles dATP, dCTP, dGTP, TTP 16 nmoles of each LlH]-TTP (Schwarz-Mann, 17 C/mmole) 115 pmoles (2/AC) calf-thymus DNA (Armour Pharmaceu-t i c a l Co.), heat-denatured k0jj,g enzyme preparation j 0.1 to 0.3 mg protein In some experiments, 20yumoles of TKM buffer was used in place of the %Q>^umoles of potassium phosphate buffer, pH 7.2. In the earlier experiments, only 0.5/>C of [3H]-TTP was used per assay. The amount of j/H]-TTP was later increased to 2 yuC per incubation mixture, in order to increase the number of counts per minute observed. Heat-denatured DNA was prepared by heating the DNA solution at 100°C for 10 min and then cooling i t in an ice-bath. In the preliminary experiments, the assay mixtures were incubated i n a water-bath at 37°C for 60 min. It was later found that the reaction was linear with time for at least 30 min. The incubation time for the assay mixture was then cut back to 30 min. After the tubes were incubated, they were rapidly cooled to 0 C in an ice-bath. To each tube, 1 mg of bovine serum albumin was added and mixed, followed by the addition of 2.5 ml of 10$ trichloroacetic acid (TCA) to precipitate the DNA and protein. 24 After each tube was chilled for 10 min in ice, i t was centrifuged for 5 min at top speed in a c l i n i c a l centrifuge. The supernatant was decanted,land the pellet was washed by thoroughly resuspending i t in 5«0 ml of % TCA. The suspension was re-centrifuged, and the washing procedure was repeated twice. The f i n a l pellet was dissolved in 0.2 ml of a 1 M solution of hyamine hydroxylate in ethanol, and was then mixed with 5«0 ml of s c i n t i l l a t i o n solution containing 15 gm of 2,5-diphenylaxazole, 150 mg of l,4-bis-(5-phenyloxazolyl-2)benzene, and 240 gm of naphthalene in 3 1. of a l s l a l solution of toluene, dioxane, and 95% ethanol. Radioactivity was then measured in a Packard Tri-Carb li q u i d s c i n t i l l a t i o n spectrometer, model 314 AX. One unit of DNA polymerase ac t i v i t y was defined as the amount of enzyme required to convert 1 pmole of f/Hl-TTP into the acid-insoluble product in 30 min under the assay conditions described. 2. Terminal deoxyribonucleotidyltransferase assay The procedure used was a modification of that reported by Krakow et a l (31). The incubation mixture was identical with that for the DNA polymerase assay except for the omission of dATP, dCTP, and dGTP, and the addition of 2.5 /jimbles^d'f cysteine. The procedures of TCA precipitation, washing, and measuring of radioactivity were the same as those for the polymerase assay. One unit of terminal transferase activity was defined as^the enzyme required to convert 1 pmole of [^H]-TTP into the acid-insoluble product in 30 min under the assay conditions described. 25 3. DNase I assay The diffusion slide assay developed by Jarvis and Lawrence (59) was used for the quantitative determination of DNase I activity. Concentrations of DNase down to 0.005 >»g/ml could be measured with high reproducibility by this method, A hot solution of agar (2% w/v) containing 2 mg/ml calf thymus DNA (Armour Pharmaceutical Co.) was mixed with an equal volume of hot 0,1 M Tris-HCl buffer, pH 7.8. MnCl 2 was added to the agar solution to a f i n a l concentration of 0.01 M. One ml of this hot mixture was spread on a microscope slide over an area of 2 in. by 1 in., which was outlined by means of cellulose tape. A hole 2.7 mm in diameter was bored with a thin steel tube, and 0.004 ml of the enzyme preparation was added to the well. The slides were placed in a plastic box containing moist blotting paper and incubated at 37°C for 20 hrs. The slides were then dipped in 1 N HCl for 15 sees, washed with water, and the diameter of each of the zones of clear-ing was read. DNase I from bovine pancreas (Worthington Biochemical Corporation) containing 2,300 Kunitz units per mg was assayed by the method of Jarvis and Lawrence and the square of the radius of the zone of clearing was calculated. In the present work, DNase act i v i t i e s were assayed by the method of Jarvis and Lawrence and were then converted to Kunitz units. The assay method developed by Kunitz was based upon the increase in UV absorption at 260 nm observed during the course of depolymerization of DNA by DNase. One Kunitz unit i s that activity which causes an increase in absor-26 bancy of 0.001 per rain per ml under the assay conditions at 25 C (67). 4. Protein determination Protein was estimated according to the method of Lowry et a l (60) with bovine serum albumin as a standard. 27 RESULTS AND DISCUSSION A>' P r e p a r a t i o n of crude enzyme In a search f o r a cytoplasmic, or e x t r a - n u c l e a r , DNA polymerase, the f o l l o w i n g study was made. mucosal scrapings were suspended i n 10 volumes o f TKM b u f f e r c o n t a i n i n g 0.32 M sucrose, and were homogenized by 5 complete passes i n a g l a s s Potter-Elvehjem homo-genizer w i t h a T e f l o n p e s t l e . f i l t e r e d through 2 l a y e r s nylon f i l t r a t e c e n t r i f u g e d 10 min a t 700 x g supernatant c e n t r i f u g e d 15 min, 20,000 x g supernatant (S^) c e n t r i f u g e d l£ h r s , 105,000 x g supernatant p e l l e t OV) : ( P i ' ) p e l l e t resuspended i n 20 v o l . TKM b u f f e r c o n t a i n i n g 0.32 M sucrose; homo-genized by 5 complete passes i n the g l a s s Potter-Elvehjem homo-gen i z e r ; f u r t h e r homo-genized i n omni-mixer f o r Z\ min, r e s u l t i n g i n breakage o f 50$ of c e l l s ; c e n t r i f u g e d 10 min a t 700 x g supernatant ( S 2 ) c e n t r i f u g e d l£ h r s , 105,000 x.g supernatant (Sg') p e l l e t ( P j ( n u c l e i ; 28 For studies of nuclear preparations, p e l l e t P 2 could be further homogenized using a Se r v a l l Omni-mixer, and the soluble proteins could then be extracted. As the present study involves the cytoplasmic preparations, the supernatants S^, S^, and S2* were each assayed for DNA polymerase a c t i v i t y . The resu l t s are tabulated below. Table I. DNA polymerase a c t i v i t y of various supernatant fractions from r a t i n t e s t i n a l mucosal c e l l s . Enzyme preparation Total a c t i v i t y (units x 1 0 3 ) Total protein (mg) Specific a c t i v i t y (units x 1 0 3 per mg protein) S l 8 5 8 149 5.8 8286 1 3 2 6 2 . 8 s 2 7 1 0 3 2 0 2 . 2 V 1 5 8 8 1 9 0 8.4 S i g n i f i c a n t DNA polymerase a c t i v i t y was found i n the supernatants S^ and S 2. However, when S^ and S 2 were centrifuged at high-speed, the t o t a l a c t i v i t i e s were increased approximately 1 0 - f o l d and 2 - f o l d respectively (S^' and S 2 ' ) / Corresponding increases i n s p e c i f i c a c t i v i t i e s were also observed. The supernatant S^' was of par t i c u l a r interest because of the high t o t a l and sp e c i f i c a c t i v i t i e s associated with i t . Other workers ( 6 l ) have made si m i l a r observations also with r a t i n t e s t i n a l mucosa. They have found,that 29 on further centrifuging the S^' fraction at 105,000 x g for 2k hrs, there was a further increase in the total DNA polymerase activity in the supernatant obtained. It appears then that may have contained a certain factor or factors which interfered with DNA polymerase activity. I f this inhibitor was sedimentable by high-speed centrifugation, an increase in total DNA polymerase activity would be observed in the resulting supernatant '. This possibility was strengthened by the results obtained from the following experiment. Supernatant S^ was prepared as before. S^ was then centri-fuged at 105.000 x g for !•§• hrs, as before, and supernatant S^' and pellet P^' were obtained. P^' was resuspended in TKM buffer containing 0.32 M sucrose. Protein determination of each of the fractions S^, S^', and Pj_' indicated that 18% of the protein present in S^ was pelleted, and that 82% remained in the superna-tant S^'. Each preparation was then assayed for DNA polymerase activity (Table II). S^ contained a low enzyme activity. The pellet P-j^ ' was found to contain a total activity very much lower than that of S^, A large increase in both total and specific a c t i v i t i e s was again obtained in S^'. When the pellet P^' was recombined with the supernatant S^', the DNA polymerase activity of the mixture was drastically reduced to a level not much higher than that of S-^  (Table II). These observations suggested that a strong inhibitor of DNA polymerase was present in the supernatant S^, and was separated from the enzyme by sedimentation into thus allowing an increase in DNA polymerase activity detected in 30 S^'. When the inhibitor was added back to the enzyme fraction, enzyme activity was immediately inhibited again. Table II. DNA polymerase a c t i v i t i e s from recombination experiments with supernatant and pellet fractions from rat intestinal mucosal c e l l s . Enzyme preparation Total activity (units x 103) Total protein (mg) Specific a c t i v i t y (units x 103 per mg protein) s l 2,343 93.1 25 v ?88,350 77.5 1140 458 16.9 27 S l ' + v 2,922 It was possible that the observed inhibitory effect was due to interference by DNase I activity present in the preparations studied. DNase activity may have two effects on DNA polymerase assays. F i r s t l y , DNase activity may produce nicks in the DNA primer strands, thus causing an activation of DNA polymerase activity. Secondly, DNase activity may cause the degradation of the newly-synthesized DNA product of the polymerase reaction, thus inter-fering with the DNA polymerase assay. Leung (54) had reported that crude homogenate preparations of rat intestinal mucosal cells contained a high DNase I activity. He found that when this crude homogenate was centrifuged at high-speed, there was a decrease in 31 the level of DNase I activity in the supernatant fraction. Leung also observed that fractions which contained a high DNase I activity generally showed a low DNA polymerase activity, indicating the possible interference of DNase I with DNA polymerase assays (54). Each of the fractions S^, S^', and P^' were therefore assayed for DNase I activity. The results are shown in Table III. Gf the total original DNase I activity present in S^, 11% was recovered in the supernatant S^', and only 14% was sedimented with the pellet P^'. These results do not f i t in with the proposed sedimentation of the inhibitor with pellet P i ' . Moreover, as w i l l be discussed in a later section, the levels of DNase I activity detected in any of the fractions were not sufficient to cause large effects on the DNA polymerase a c t i v i t i e s present (Table III). Thus DNase I does not appear to be the cause of the inhibitory effects observed in the DNA polymerase preparations. Table III. DNase I a c t i v i t i e s in various supernatant and pellet fractions from rat intestinal mucosal c e l l s . Enzyme preparation Total DNase I ac t i v i t y (Kunitz units) 6,646 5,108 923 32 Due to limitations of time, the nature of the inhibitory factor was not further investigated. In view of the high total and specific DNA polymerase a c t i v i t i e s obtainable with the S^ * supernatant, this preparation was chosen for further study. The routine procedure for preparing this fraction i s described in the preceding section. This S^' supernatant represents the f i r s t wash of presumably intact c e l l s . Further experiments were carried out to determine whether this enzyme activity i s of nuclear origin, or whether i t has properties differing from the nuclear enzyme activity, indicating a possible cytoplasmic origin. B"1, Partial purification of crude enzyme Ammonium sulfate precipitation has been used by Bollum (17), by Shepherd and Keir (62), and by Furlong (63) to fractionate DNA polymerase from tissue extracts. This procedure has also been used by Leung (5^) in the purification of the enzyme from the small intestinal mucosa of the rat. The DNA polymerase act i v i t y was recovered in the 60% ammonium sulfate fraction, but there was a loss of up to 60% of the original enzymatic activity. This result may have been due to the detrimental effects of high salt concentration on the structural conformation of the mammalian DNA polymerases as described by Keir (21), In view of these observations, ammomium sulfate fractionation was not used in the present work. The methods of purification used included DEAE-cellulose chroma-tography, phosphocellulose chromatography, and Sephadex gel-f i l t r a t i o n . 33 1. Chromatography on DEAE-cellulose The high-speed supernatant S^*, which w i l l h e r e a f t e r "ben r e f e r r e d t o as the crude enzyme p r e p a r a t i o n , was chromatographed on a column of DEAE-cellulose. The column was e l u t e d w i t h a l i n e a r gradient i n a T r i s - H C l b u f f e r system as desc r i b e d p r e v i o u s l y , and a t y p i c a l e l u t i o n p r o f i l e obtained i s shown i n F i g . 4. A s i n g l e p r o t e i n peak c o n t a i n i n g 42$ of the t o t a l p r o t e i n was e l u t e d before the grad i e n t was a p p l i e d . T h i s p r o t e i n peak, which was apparently not adsorbed to the. anion-exchanger, contained about 92$ of the t o t a l enzyme a c t i v i t y . The remaining 4$ of the a c t i v i t y was obtained as a small peak o c c u r r i n g immediately a f t e r the s t a r t o f the grad i e n t . The l a r g e p r o t e i n peak which was e l u t e d w i t h the grad i e n t was completely devoid of enzyme a c t i v i t y . Table IV. Chromatography o f crude enzyme on DEAE-c e l l u l o s e . E l u t i o n w i t h T r i s - H C l b u f f e r system, w i t h l i n e a r gradient o f KC1. P r o t e i n T o t a l Y i e l d (mg) a c t i v i t y (%) ( u n i t s x 10-3) S p e c i f i c a c t i v i t y P u r i f i -o f peak f r a c t i o n c a t i o n ( u n i t s x 103 per mg p r o t e i n ) A p p l i e d i n 48.0 12,696 100.0 282 1.0 crude enzyme Enzyme peak I 20.9 11,638 91.7 1,239 4.4 Enzyme peak I I 0.5 529 4.2 817 2.9 T o t a l 21.4 12,16? 95-9 o oo CM < 0-6 --0-75 — 0-50 0-25 c • o c o O V O L ) Fract ion No. Fig. 4. Chromatography of crude DNA polymerase from rat intestinal mucosa on DEAE-cellulose. Elution with TKM buffer containing 1 mM dithiothreitol and 20$ ethylene glycol, with linear gradient of KC1, Dotted line represents absorption at 280 nm; solid line represents units of DNA polymerase activity; alternate dots and dashes represents elution gradient of KC1. 34 Table IV refers to the enzyme peaks labeled in Fig. 4, and indicates that the purification of the enzyme activity in peak I i s between 4- and 5-fold. These protein and activity profiles are consistently reproducible. When the crude enzyme preparation was chromatographed on DEAE-cellulose using a stepwise gradient i n a Tris-HGl buffer system, as described previously, a better protein separation was obtained. A large protein peak, with an overlapping smaller peak, was again obtained before the start of the gradient (Fig. 5) . Table V. Chromatography of crude enzyme on DEAE-cellulose. Elution with Tris-HCl buffer system, with stepwise gradient of KC1. Protein Total Yield Specific activity P u r i f i -activity {%) of peak fraction cation (units (units x 103 per x 103) mg protein) Applied in ©imii crude enzyme » 31.1 37,800 100.0 1,214 1.0 Enzyme peak I 14.0 30,186 79.9 3,367 2.8 Enzyme peak II 1.5 1,803 4 .8 1,880 1.6 Total 15.5 31,989 84.7 Several major or minor protein peaks were obtained with each stepwise increase in the gradient. About % of the enzyme act i v i t y was again detected at the start of the gradient. The other protein o CO CM < o E E o -Q C o o Fraction No. Fig. 5. Chromatography of crude DNA polymerase from'rat intestinal mucosa on DEAE-cellulose. Elution with TKM buffer containing 1 mM dithiothreitol and 20$ ethylene glycol, with stepwise gradient of KC1. Dotted line represents absorption at Jt§& 280 nm? solid line represents units of DNA polymerase activityj alternate dots and dashes represents elution gradient of KC1 (at 0.10 M, bi 0.25 M, c» 0.50 M). 36 peaks eluted with the gradient were devoid of detectable enzyme activity. As indicated in Table V, the purification of DNA polymerase activity in peak I was about 3-fold. Peak I from DEAE-cellulose chromatography of the crude enzyme was assayed for DNase I and terminal transferase a c t i v i t i e s , and the results were compared with those obtained with the crude enzyme preparation (Table V l ) . Table VI. DNase and terminal transferase a c t i v i t i e s from crude and partially purified enzyme preparations. Total activity DNase I Terminal transferase (Kunitz units) (units x 1C-3) Crude enzyme 5,814 3,312 Peak I enzyme 1,261 1,944 Yield 21.7% 58.7% When the crude enzyme preparation was chromatographed on DEAE-cellulose, the DNA polymerase peak collected in the peak of unadsorbed proteins contained only 21.7% of the total DNase activity applied onto the column. The terminal transferase activity in the DNA polymerase peak was 58.7% of that in the crude enzyme preparation applied. DEAE-cellulose chromatography therefore provided a simple and speedy procedure for partial purification of DNA polymerase. The bulk of the polymerase activity was washed through the column, leaving over 50% of the protein and about 78% of the DNase activity adsorbed onto the DEAE-cellulose. In the routine partial purification of the crude enzyme preparation, the ' supernatant was applied onto a DEAE-cellulose column and was washed through with buffer, no gradient being necessary. The optical density at 280 nm was determined for each fraction collected. Those fractions which constituted the f i r s t protein peak contained between 80% and 92% of the DNA polymerase activity, and these fractions were combined for further use. In a study of rat l i v e r DNA polymerases, B a r i l and co-workers (57) chromatographed the ammonium sulfate fractions of nuclei, mitochondria, ribosomes, and smooth membranes on DEAE-cellulose. The elution profiles of the enzymes from purified nuclei and ribosomes appeared quite similar. Neither enzyme was bound to DEAE-cellulose and a l l of the DNA polymerase act i v i t y appeared in the column wash. The column washes also contained some nuclease and terminal transferase activity. The DNA polymerases from both nuclei and ribosomes preferred native DNA as primer. Chromatography of the mitochondrial fractions produced multiple peaks of very low polymerase activity, most of which was eluted with 0.1 M and 0.25 M KC1. The elution pattern of the smooth membrane fraction was similar to that of the mitochondrial fraction, but the polymerase activity was 15 to 20 times higher than that in the latter. The elution profile obtained in the present work 38 indicates that the enzyme in the high-speed supernatant from rat intestinal mucosa was similar to those of the nuclear and ribosomal fractions from rat l i v e r . Previous studies have been made by Leung and Zbarsky ( 1 9 , 20) on the soluble fractions extracted from the nuclei of fat intestinal mucosa. Chromatography of the nuclear extracts on DEAE-cellulose produced three peaks of DNA polymerase activity. The f i r s t enzyme activity was found to be associated with a peak of unadsorbed protein and nucleic acid material. The two other enzyme peaks were eluted with approximately 0.1 and 0 . 2 M KC1 solutions. Re-chromatography of the f i r s t unadsorbed enzyme peak allowed the detection of a distinct peak of 'terminal' enzyme activity. In contrast to the three separable DNA polymerase act i v i t i e s found by Leung and Zbarsky ( 1 9 , 20) in the nuclear preparations, the cytoplasmic preparations contained only a major and a minor enzyme activity peak. This major DNA polymerase peak from the cytoplasmic preparation was similar in chromatographic properties to one of the DNA polymerase peaks from the nuclear preparations, i.e., i t was not adsorbed onto DEAE-cellulose. A similar pattern of distribution of DNA polymerases has been reported by Weissbach et a l (51 ) in HeLa ce l l s and in normal human lung diploid line WI-38, as has been discussed earlier. In each case, no separable nuclear DNA polymerase a c t i v i t i e s and only one cytoplasmic DNA polymerase activity were isolated. The two nuclear enzymes differed in chromatographic and other properties but the cytoplasmic enzyme resembled one of the nuclear enzymes in 39 a l l respects. The actual relationship between the three a c t i v i t i e s remains unclear. 2. Column chromatography on phosphocellulose Since the DNA polymerase activity under study was not adsorbed onto the anion-exchanger DEAE-cellulose, i t was hoped that i t might be adsorbed onto a cation-exchanger, such as phospho-cellulose. Fractions from the enzyme peak (peak I) from the DEAE-cellulose column were combined and the solution was dialyzed over-night at 4°C against 0.05M phosphate buffer, pH 6.8, containing 1 mM dithiothreitol and 20% ethylene glycol. The dialysate was applied to a phosphocellulose column equilibrated with the same buffer. The column was eluted with a stepwise gradient as described in the preceding section. A sharp protein peak was obtained with each change in the gradient, giving a total of 4 peaks. Each peak was assayed for DNA polymerase, but no enzyme activity could be detected. The f i n a l protein peak, that eluted with the 0.5 M phosphate buffer, showed indications of a slight amount of activity. Fractions from this peak were therefore combined and the solution was dialyzed against TKM buffer containing 1 mM dithiothreitol and 20% ethylene glycol. The dialysate was concentrated by further dialysis against ice-cold sucrose, and was re-assayed. A low level of enzyme activity was present, but the total activity yield was less than 5%. Another approach to phosphocellulose chromatography was 40 used. Fractions from the DNA polymerase peak (peakl) from the DEAE-cellulose column were combined and the solution was again dialyzed against TKM buffer containing 1 mM dithiothreitol and 20$ ethylene glycol. The dialysate was added to a prepared slurry of phospho-o cellulose and the mixture was stirred overnight at 4 G. The slurry with the adsorbed protein was packed into a column (1.2 cm diameter, 18 cm height) which was then eluted with a continuous gradient as previously described. The elution profile indicated that some protein did adhere to the column, but no sharp distinct peak was eluted. Every third fraction was dialyzed overnight against TKM buffer containing 1 mM dithiothreitol and 20$ ethylene glycol, and was then assayed for DNA polymerase activity. No enzyme activity could be detected in any of the fractions. The effect of phosphate ion concentration on DNA polymerase activity was later examined. It was found that DNA polymerase a c t i -vity was drastically inhibited at phosphate concentrations of 0.20 M and above. At 0.10 M phosphate, the enzyme act i v i t y was reduced to 27$ of that at 0.05 M phosphate. Since the phosphocellulose columns were run with gradients from 0.10 M to 0.50 M phosphate, i t i s not surprising that no enzyme act i v i t y could be detected in the fractions. Dialysis of the fractions against TKM buffer seems to be ineffective for the recovery of enzyme activity. The ethylene glycol was added to the buffer systems to prevent deterioration of enzyme activity. It does not appear to haveyahyCdetrimental effects on the DNA polymerase ac t i v i t i e s since DEAE-cellulose columns eluted in the presence of 20$ ethylene glycol 41 allowed almost total recovery of enzyme activity. In their work with rat l i v e r DNA polymerases, B a r i l et a l (57)'!re-chromatographed the enzyme peaks from DEAE-cellulose chro-matography onto phosphocellulose columns. The columns were eluted by stepwise gradients of potassium phosphate buffer in concentra-tions of 0.1 to 0.5 M, containing 1 mM dithiothreitol and 20% ethylene glycol. The collected fractions were dialyzed overnight against TKM buffer containing 20% ethylene glycol before assaying. The polymerase activity from the nuclear and ribosomal fractions eluted at 0.5 M and that of the smooth membranes at 0.2 M phosphate concentration. In the present work, the slight enzyme activity detected at 0.5 M phosphate concentration again indicates a simi-l a r i t y to the nuclear and ribosomal enzymes from rat l i v e r . 3. Gel-filtration on Sephadex G-150 Molecular-sieve chromatography, or * g e l - f i l t r a t i o n ' was used for the purification of DNA polymerase and for the estimation of i t s molecular weight. Sephadex G-150 columns were prepared and calibrated as described in the preceding section. A typical calibra-tion profile i s shown in Fig. 6. When crude enzyme preparation was chromatographed on the column as previously described, elution with 0.1 M phosphate buffer gave the elution profile shown in Fig. 7. However, no DNA polymerase activity could be detected throughout the eluate. This result may be attributed to the presence of phosphate ions in 0.1 M concentra-tion, which was later found to have a strong inhibitory effect on 42 5-5 s - . 4-9 1 1 ""S. Ghymotrypsinogen A VX V ( - M . tf. 25,000) -^s. Bovine albumin ^ S f M . w. 67i000); -"tf-GlobuliriN. (M. W. 160,000) >^ l I 0 60 120 Ve-Vo (mis) Fig, 6, Standard curve for the estimation of the molecular weights of protein sample on the basis of their elution volumes from ge l - f i l t r a t i o n on Sephadex G-150. (V g = elution volume of sample; VQ= void volume of column.) ! r : r - — - — : — i Fraction No. Fig. ?. Gel - f i l t r a t i o n of crude. DNA polymerase from rat intestinal mucosa on Sephadex G-150. Elution with 0,1 M phosphate buffer, pH 7.2, containing 1 mM dithiothreitol and 20% ethylene glysol. Dotted line represents absorption at 280 nmi solid line represents elution of NaCl, as measured by conductivityj The elution volume of NaCl was taken to be the void volume of the column. 44 the enzyme activity. The..experiment was therefore repeated in which the column was equilibrated and eluted with TKM buffer instead of phosphate buffer. In addition, the crude enzyme preparation was concentrated 4- to 5 -fold against polyethylene glycol before loading onto the column. This concentration procedure led to a 55% decrease in specific activity. The elution profile obtained i s shown in Fig. 8. A single DNA polymerase peak was observed (elution volume V e 90.65 ml). The same column was calibrated using the markers -globulin ( M.W. 160,000; V e 78.75 mis), hemoglobin (M.W. 65,000; V e 101.25 mis), and chyraotrypsinogen-A (M.W. 25,000; V e 111.25 mis). The DNA poly-merase detected was thus estimated to have a molecular weight of 101,000. Previous investigators (19, 20) have obtained multiple peaks of DNA polymerase activity from Sephadex g e l - f i l t r a t i o n of nuclear preparations of rat intestinal mucosal extracts, and have obtained molecular weight values from 25,000 to 300,000. Bollum et a l (64), using both a Sephadex G-100 and G-200 column, estimated the molecular sizes of the calf thymus enzymes to be 110,000 for the replicative and 37»000 for the terminal DNA nucleotidyl transferase. The specific activity of the DNA polymerase in the peak fraction was 42-times greater than that of the original crude extract loaded onto the column (Table VII). The yield of total activity recovered from the column was over 12 times that loaded onto the column. The cause of this large increase in total activity i s unclear. An obvious possibility i s the removal of a strong inhibi-tory factor which differed in size from the DNA polymerase. As has 0-3 - 0-2 c 3 - 0:1 Fraction No. Fig. 8. Ge l - f i l t r a t i o n of crude DNA polymerase from rat intestinal mucosa oh Sephadex G-150. Eluti6n>awith TKM buffer containing 1 mM dithiothreitol and 20% ethylene glycol; Dotted line represents absorption at 280 nm; solid line represents units of DNA polymerase activity. 46 been described earlier, the crude enzyme homogenate did contain a strong inhibitory factor which was sedimentable by high-speed centri-fugation. This indicated that the inhibitor differed in size from the DNA polymerase. It i s possible that a portion of the inhibitory Table VII. Ge l - f i l t r a t i o n of crude enzyme preparation on Sephadex G-150. Elution with TKM buffer system. Enzyme fraction Total DNA poly- Yield Specific DNA poly- Puri-merase activity merase activity f i c a -(units x lo3) (units x 103 per tion mg protein ) Crude enzyme 4,730 112 applied Peak enzyme 57,680 12-fold 4,719 42-fold eluted factor did not di f f e r sufficiently from the polymerase to be pelleted, but did diff e r sufficiently to be separated from the polymerase by Sephadex g e l - f i l t r a t i o n . Removal of the strong inhibitor would result in a large increase in both total and specific DNA polymerase a c t i v i t i e s . C. General properties of the enzyme 1. Requirements The requirements for the enzymatic reaction catalyzed by DNA polymerase were studied. 'Partially purified' DNA polymerase, 47 hereafter referring to the enzyme peak (peak i ) from DEAE-cellulose chromatography, was dialyzed against 0,05 M Tris-HCl buffer, pH 7 ,6, Table VIII. Requirements for DNA polymerase a c t i v i t i e s from rat intestinal mucosal c e l l s . Crude units x 103 enzyme ^ac t i v i t y Partially ] units x 10-surified enzyme ' %a c t i v i t y Complete system 178 100.0 148 100.0 - DNA 15 8.3 0 0.0 - dATP, dCTP, dGTP 31 17.2 28 18.9 - dATP 57 32.1 38 25.7 - dCTP 86 48.1 59 39.9 - dCTP 70 39.2 49 33.1 - MgCl 2 15 8.3 8 5.4 - dithiothreitol 154 86.7 125 84.5 - enzyme 0 0.0 0 0.0 heated enzyme 1 0.6 - -containing 0.025 M KC1. The referring to the high-speed 'crude' DNA polymerase preparation, supernatant S^ * of the homogenate, was assayed without further purification. As shown in Table VIII, both enzyme a c t i v i t i e s required the presence of a DNA template and Mg iShs. Activity was only slightly enhanced by the addition of dithio-threitol. Omission of one of the three unlabeled deoxynucleoside 48 triphosphates led to a reduction of the enzyme activity to between 25% to 48$, depending on the particular nucleotide omitted. With the omission of a l l three unlabeled triphosphates, the incorpora-tion of C%]-TTP was less than 20$ of that obtained in the complete o system. Heating the enzyme at 100 C for 10 min resulted ini'drastic inactivation of the enzyme. 2. DNA primer preference DNA polymerase activity with heat-denatured DNA and native DNA as primer was studied. Heat-denatured material was obtained by heating a solution of calf thymus DNA at 100°C for 10 min, and o immediately cooling i t to 0 G in an ice-bath. The DNA polymerase act i v i t i e s from the crude and the partially purified preparations were a l l found to be several-fold higher with heated rather than with native DNA as primer (Table IX). As has been discussed previously, DEAE-cellulose chromatography of the crude cytoplasmic extracts produced one large peak (I) containing 92$ of the enzyme Table IX. DNA polymerase ac t i v i t i e s with native or heat-* denatured DNA as primer. Enzyme preparation Units activity x 103 Native DNA primer Heat-denatured DNA primer Crude enzyme DEAE-cellulose peak I DEAE-cellulose peak II 85 726 183 234 2,571 503 49 a c t i v i t y , and a small peak ( I I ) c o n t a i n i n g 4% of the enzyme a c t i v i t y . These two peaks showed no d i f f e r e n c e s i n primer preference (Table I X ) . Heat-denatured DNA was t h e r e f o r e used i n a l l subsequent assays. T h i s preference f o r denatured DNA as primer has a l s o been reported by Bollum (55) i n c a l f thymus DNA polymerase, and by Shepherd and K e i r (62) i n Landschutz a s c i t e s tumor c e l l s . Leung and Zbarsky (19, 20) observed t h a t nuclear DNA polymerase preparations from r a t i n t e s t i n a l mucosa demonstrated v a r i a t i o n s i n t h e i r preference f o r denatured or n a t i v e DNA as primer, depending upon the enzymic f r a c t i o n t e s t e d . 3. E f f e c t o f pH The e f f e c t o f pH on DNA polymerase a c t i v i t y was examined. Crude enzyme pr e p a r a t i o n was assayed i n 0,05 M potassium phosphate b u f f e r s from pH 6.0 t o 8.0, and a l s o i n 0.05 M T r i s - a c e t a t e b u f f e r s from pH 5.0 to 9.5. The optimum pH values f o r the enzyme r e a c t i o n was found to be 7.2 i n the phosphate b u f f e r , and 8.0 i n the T r i s -acetate b u f f e r (Fig:'. 9)' Leung and Zbarsky (19) have reported pH optima o f 7.4 i n phosphate b u f f e r , and 8.0 i n T r i s - H C l b u f f e r f o r nuclear DNA p o l y -merase i n r a t i n t e s t i n a l mucosa. S i m i l a r optimal pH values have been r e p o r t e d by Zimmerman i n b a c t e r i a l t i s s u e s (65) and by Mantsavinos i n mammalian t i s s u e s (25) under s i m i l a r c o n d i t i o n s . In the present Bwork, potassium phosphate b u f f e r , pH 7.2, to a c o n c e n t r a t i o n o f 0.05 M, was used i n the r o u t i n e assay proce-dures. 50 Fig. 9. The effect of pH on crude DNA y. polymerase from rat intestinal mucosa. 3 Dotted line represents enzyme activity when assayed in 0.05 M Tris-acetate buffers from' pH 5.0 to 9.5. Solid line represents enzyme activity when assayed in 0.05 M potassium phosphate buffers from pH 6.0 to 8.0. 51 4. Effect of varying amounts of radioactivity in assay,:-The effect of varying amounts of radioactivity on DNA polymerase was examined in an effort to determine the optimal proportions of unlabeled and t r i t i a t e d TTP to be used in the assay mixtures. The effect of varying the total concentration of TTP in the incubation mixture i s shown in Fig. 10. Tritiated and unlabeled TTP were mixed to give a 1.8 mM solution of L^lO-TTP containing 9 o f radioactivity per ml. Varying amounts of this solution were used in the incubation mixtures ?for DNA polymerase assay. The specific radioactivity, i.e., the radioactivity per mole of TTP, was therefore constant for a l l incubation mixtures. The incorporation of -TTP into DNA increased with increasing amounts of the -TTP solution in the assay mixture, approaching a maximum level at about 81 nmoles [%3-TTP per assay (Fig. 10). Another experiment was carried out in which the specific radioactivity in the assay mixture was varied, but the total amount of TTP present i n each was kept constant. Each incuba-tion mixture contained 16 nmoles TTP and varying amounts of radioactivity ranging from 0.2 to 2.0/A.C. The incorporation of l 3H] -TTP into DNA increased linearly with increasing amounts of radioactivity (Fig. 11). No tapering off of the increase in activity was observed in the range studied. In subsequent assays, 16 nmoles of a mixture of t r i t i a t e d and unlabeled TTP, containing 2juG of radioactivity, were used per incubation mixture, as a suitably high level of DNA polymerase 52 25 50 75 Volume of 3H-TTP (X) Fig. 10. The effect of varying the total concentration of TTP in the incubation mixture for the DNA poly-merase assay,. the specific-. 1 ?• radio-activity being, kept constant. Unlabeled and tritiated'TTP were omitted from the usual incubation mixture and were replaced with varying amounts of a.1,8 mM ^H-TTP solution containing 9 ;uC per ml. 53 T ~ I I I [ I I i I 1 Fig. 1 1 . The effect of varying the specific radioactivity in the incubation mixture for .the DNA polymerase assay, the total TTP concentration being kept constant. Varying amounts of a 3H-TTP solution of 1 0 jaC/ml and 57 pmoles/uC were added to the incubation mixture. The amounts of 3H -TTP present were negligible when compared with the 1 6 nmoles unlabeled TTP present in the incubation mixture. 54 activity could be obtained with these amounts. 5. Effect of glycerol and ethylene glycol Glycerol i s commonly used in the storage of enzyme preparations to reduce deterioration of enzyme activity. There-fore when crude DNA polymerase preparations were stored, glycerol was added to them in concentrations of 20% or 35%» The enzyme solutions containing glycerol did not solidify even at tempera-tures of -20°G. B a r i l et a l (57) have reported that ethylene glycol stabilized the activity of crude and purified rat l i v e r DNA poly-o o merases for at least 3 weeks at 4 C and at least 6 months at -20 C. In the present work, 20% ethylene glycol was therefore added to the buffer systems used in the elution of chromatographic columns. The effects of glycerol and ethylene glycol on DNA poly-merase activity were therefore investigated. Surprisingly, i t was found that the presence of either glycerol or ethylene glycol in the enzyme preparations led to an activation of the enzyme activity observed (Table X). When 20% glycerol was added to an enzyme preparation immediately before assaying for DNA polymerase activity, a 1.5-fold increase in the incorporation of radioactivity was observed. Over 10-fold activation has been observed in certain preparations. The level of increase in activity was the same in 20% or 35% glycerol. An increased level of activity could be observed in enzyme preparations containing glycerol after at least 17 days of storage at -20°C. In the case of ethylene glycol, a 2-fold 55 Table X. Effect of glycerol and ethylene glycol on DNA polymerase activity. Enzyme preparation cpm Control preparation cpm 3,364 V ,1 67 + 20% glycerol 5,177 water + 20% glycerol 79 S 1' + 20% ethylene glycol 6,537 water +20% ethylene glycol 64 increase in DNA polymerase activity was observed when 20% ethylene glycol was added to the enzyme preparation immediately before assaying. Control samples were prepared in which enzyme was substi-tuted by water, water containing 20% glycerol, and water containing 20% ethylene glycol. These preparations were assayed in exactly -> the same manner as the enzyme preparations. The results show no significant differences in the counts per minute of tritium incor-porated. Similar stimulation of DNA polymerase a c t i v i t i e s in the presence of 5% glycerol have been observed by other workers in rat brain tissue ( 6 6 ) . The cause of these increases in enzyme activity i s unclear. 6, Time-course of the reaction The time-course for the incorporation of ipHl-TTP into DNA by the crude DNA polymerase extract was studied. The crude enzyme preparation was assayed both in the presence and in the absence of 56 35% glycerol. In both cases, the reaction was linear with respect to time for at least 30 min, as i s shown in Fig. 12. A maximum level of activity was reached in both cases after an incubation time of about 60 min. The maximum level of activity reached by the enzyme containing 35% glycerol was much higher than that reached by the enzyme in the absence of glycerol. In view of these results, an incubation period of 30 min was used in a l l subsequent assays for DNA polymerase activity. 7. Enzyme st a b i l i t y The s t a b i l i t y of the DNA polymerase activity upon storage under various temperature conditions was studied. Under condition A, a sample of crude enzyme preparation was stored at 4 C, A l i -quots were withdrawn from i t for each assay. Under condiHon B, a o sample of crude enzyme preparation was stored frozen at -20 C. This sample was thawed and aliquots were withdrawn from i t for each assay, after which the sample was re-frozen and stored for further use. Under condition G, several aliquots of crude enzyme preparation 0 were stored frozen at -20 C. Fresh aliquots were thawed for use in each assay. Any remaining thawed enzyme preparation was discarded and was hot re-frozen for further use. Enzyme preparations stored under each of the three conditions were assayed at 1, 6, and 13 days after extraction. The results are presented in Table XI. The DNA polymerase a c t i v i t i e s appeared to be stable for at least 13 days. An increase in enzyme activity was observed on day 6 under a l l three temperature conditions, the act i v i t y leveling 57 0-8 ? 0 - 4 | -• _ i i I 1/ . . . A — — ® — - — 50 100 150 linutes Fig. 12. Time-course studies on the DNA polymerase reaction. Circles represent the time-curve obtained when crude DNA polymerase was assayed in the absence of glycerol. Triangles" represent the time-curve obtained when crude DNA polymerase was assayed in "the presence of 35$ (v/v) glycerol. Abscissa represents the incubation time in minutes. 58 Table XI. Stability of DNA polymerase under different temperature conditions. (See text). Number of days Units activity x io3 per ml enzyme after extraction Condition A Condition B Condition C 1 60.0 60.0 60.0 6 97.0 94.5 93.5 13 67.O 106.5 80.0 off after 13 days, but remaining higher than the act i v i t y on day 1. Under condition B, where the enzyme preparation was frozen and thawed for use each time, the activity continued to increase even after 13 days. The observed increased in enzyme activity on storage were repeatable, and were found to occur as early as day 3« A possible explanation for these results may be the degra-dation of an inhibitory factor present in the enzyme preparation. As has been discussed earlier, a strong inhibitor of DNA polymerase has been found to be present in the crude c e l l homogenate. The nature of the inhibitor i s not known, but i t was found to be sedimentable by high-speed centrifugation. It i s probable that some of the inhibitor remained in the supernatant from the high-speed centrifugation, i.e., in the crude enzyme preparation. l£ this inhibitory factor i s unstable and i s degraded upon storage, an apparent increase in DNA polymerase activity would be observed. In the present work, crude enzyme preparations were used 59 within one week after extraction. 8. Effect of phosphate ion concentration An examination of the effect of phosphate ion concentra-tion on DNA polymerase activity was prompted by the observation that elution of chromatographic columns with phosphate buffers always resulted in a total loss of enzyme activity in the eluate.. Table XII. Effect of phosphate ion concentration on DNA polymerase activity. Phosphate ion concentration of incubation mixture Units activity x 103 (average of two experiments) 0.05 M 972 0.10 M 262 0.15 M 116 0.20 H 13 0.25 M 2 It was suspected that phosphate ions might exert an inhibitory effect on the enzyme. Crude enzyme extract was assayed in the presence of increasing concentrations of phosphate ions, and the results are shown in Table XII. It can be seen that enzyme activity was drastically inhibited at phosphate concentrations of 0.20 M and above. At 60 0.10 M phosphate, the enzyme activity was reduced to 27% of that at 0.05 M phosphate. This inhibitory effect of phosphate ions on DNA polymerase activity i s believed to be the principal cause of the failure to detect enzyme activity in the eluates of phospho-cellulose and Sephadex columns eluted with phosphate buffers at concentrations of 0.10 M or above. 9. Effect of DNase I A study of the effect of DNase I on the DNA polymerase assay was made for two reasons. F i r s t l y , Leung (5^) had observed that fractions which contained a high DNase I activity generally showed a low DNA polymerase activity. He suggested the possible interference of DNase I with DNA polymerase assays. Secondly, the study was made i n order to determine the nature of the product formed from the DNA polymerase reaction. Crude enzyme preparation was measured in the presence or absence of added DNase I. The DNase I was added either at the beginning of the incubation period, i.e., at zero time, or after 30 min of incubation, after which the assay mixture was further incubated for 30 min. The effect of RNase on the DNA polymerase assay was also b r i e f l y studied. The results are summarized in Table XIII. In the f i r s t case, crude enzyme was assayed, in the absence of added DNase I, with an incubation time of JO min. When 10 jug of DNase I was added to the assay mixture at zero time, and the mixture incubated for 30 min, a slight increase in DNA polymerase 61 Table XIII. Effect of DNase I and RNase on DNA polymerase assay. Enzyme RNase DNase I (2,300 Kunitz Incubation DNA polymerase units per mg) time activity (min) cpm units x 103 - - 30 1,799 181 V 10 Mg, added at 0 time 30 1,912 194 50 jug, added at 0 time 30 1,480 147 s l ' 50 >Ag, added after 30 min 60 580 52 S i ' - - 60 2,202 224 - - - 30 84 0 - - - 60 84 0 10 >»g, added - 30 2,061 209 at 0 time activity was observed. This increase was probably due to nicking of the DNA primer by the added DNase I. When the level of DNase I addedsat zero time was increased to 50 >»g and the assay mixture was incubated for 30 min, a 17% decrease in the DNA polymerase activity was observed. This indicated that the level of DNase I present was probable high enough to cause breakdown of the DNA product formed from the polymerase reaction. In the next case, the DNA polymerase assay mixture was incubated for 30 min in the absence of DNase I. At this time, a significant level of [3H]-TTP has presumably been incorporated 62 into the newly-formed DNA (see f i r s t case, 1799 cpm). F i f t y jag of DNase I was added at this point and the assay mixture was further incubated for 30 min. The DNA polymerase activity observed was great-l y reduced, indicating that a large portion of the pre-formed product of the polymerase reaction had been degraded by the DNase I. This clearly demonstrated that the product formed by the DNA poly-merase reaction was DNA i n nature. When the crude enzyme was incu-bated for 60 min without added DNase I, the DNA polymerase activity was slightly higher than that after JO min of incubation, confirming that the loss in activity in the previous case was not due to the increase in incubation time. Blank samples containing no enzyme gave identical results for both 30 and 60 min incubation periods. A brief study of the effect of RNase on DNA polymerase activity was made. Ten jug RNase was added to the assay mixture at zero time and the mixture was incubated for 30 min. The level of DNA polymerase activity obtained was slightly higher than that in the absence of RNase under similar conditions. This indicated that this level of RNase did not have any significant effect on the DNA polymerase activity. 63 SUMMARY Previous studies have been made by Leung and Zbarsky (1-9, .20) on the nuclear DNA polymerases from the small intestinal mucosa of the rat. Significant DNA polymerase act i v i t y was also found in the cytoplasmic fractions even under non-aqueous separation conditions where leakage from the nuclei into the cyto-plasm was minimized. The present work involves a partial p u r i f i -cation and a study of the general properties of this cytoplasmic DNA polymerase activity. The experiments done and the observations made can be summarized as followsi 1. The crude cytoplasmic enzyme fraction studied was prepared by high-speed centrifugation of the homogenate of washed mucosal scrapings. 2. A factor which caused strong inhibition of DNA polymerase activity was sedimented by the high-speed centrifugation of the c e l l homogenate. The nature of this inhibitory factor remains unclear. 3. On chromatography of the crude cytoplasmic extract on DEAE-cellulose, over 96$ of the DNA polymerase activity was found in the fractions representing unadsorbed material. Nevertheless, four-fold purification of the DNA polymerase was achieved because 78$ of the contaminating DNase I activity was adsorbed onto the DEAE-cellulose. 4. A minor peak containing h% of the total DNA polymerase a c t i -vity was eluted when a gradient of KG1 was applied to the DEAE-64 cellulose column. This enzyme activity was similar to that in the major DNA polymerase peak in that hoth a c t i v i t i e s preferred denatured DNA as primer. 5. The unadsorbed protein peak containing the bulkAofyftheiB DNA polymerase activity from the DEAE-cellulose column was re-chromatographed on a phosphocellulose column. No enzyme activity could be detected in the eluate. The absence of detectable DNA polymerase activity may have been due to the later discovered strong inhibitory effect of phosphate ions on the enzyme. However, no enzyme activity could be detected even after each fraction was dialyzed overnight against a Tris-HGl buffer system. 6. Crude cytoplasmic enzyme was also chroraatographed by gel-f i l t r a t i o n on Sephadex G-150 columns. Several protein peaks were eluted using a Tris-HCl buffer system, but only a single peak of DNA polymerase activity was detected. The specific enzyme activity was increased 42-fold and the total activity was increased 12-fold. The reason for this latter increase in total activity i s unclear. The possible removal of certain inhibitory factors has been suggested. 7. By the use of protein markers with known molecular parameters, the molecular weight of the DNA polymerase fraction was estimated to be 101,000. 8. The activity of DNA polymerase was measured by the incorpo-ration of [3H]-TTP into an acid-insoluble product, under suitable assay conditions. The enzyme required the presence of a DNA template and Mg ions. Activity was only slightly enhanced by the addition of dithiothreitol. For maximum activity, the presence of a l l four 65 deoxynucleoside triphosphates were required. Only 25$ to 48$ of the enzyme activity remained when either of the three unlabeled deoxy-nucleoside triphosphates were omitted from the incubation mixture. When a l l three unlabeled triphosphates were omitted, the activity was less than 20$ of that obtained in the complete system, indicating primarily replicative rather than terminal addition activity. Heat-denatured DNA was preferred as primer. The optimum pH for this enzymatic activity was found to be 7.2 in potassium phosphate buffer, and 8.0 in Tris-acetate buffer. 9. Time-course studies on the enzyme reaction indicated that the reaction was linear with respect to incubation time for at least 30 min; 10. The DNA polymerase activity was stable up to 13 days under temperature conditions of 4°C to -20°G. 11. Glycerol in 20$ to 35$ (v/v) concentrations was found to have both a stimulatory and a stabilizing effect on the enzyme activity. 12. Ethylene glycol at 20$ (v/v) concentration was also found to have a stimulatory effect on the enzyme activity. 13. The enzyme was strongly inhibited in the presence of 0.10 M phosphate ions and activity was drastically reduced in phosphate ion concentrations of 0.20 M and above. 14. The product of the DNA polymerase reaction could be destroyed by DNase, indicating that i t was DNA in nature. 66 CONCLUSION The purpose of the present work was to determine whether the DNA polymerase activity in the cytoplasmic preparation i s actually of cytoplasmic origin, or whether i t i s due to nuclear contamination. Work done by Leung and Zbarsky (19, 20) showed that the nuclear preparations contained several DNA polymerase a c t i v i t i e s with mole-cular weights ranging from 25,000 to 300,000. These enzyme ac t i v i t i e s demonstrated similar requirements and pH optima, but differed in chromatographic properties and DNA primer preferences. The present results indicate&that^theocytoplasmic preparation contained a single major DNA polymerase activity with a molecular weight of about 101,000. This activity resembled one of the nuclear a c t i v i t i e s in many respects, i.e., requirements, pH optima, chromatographic properties on DEAE-cellulose, DNA primer preference. As has been discussed earlier, Weissbach et a l (5l) have reported a similar pattern of DNA polymerases in HeLa ce l l s and i n normal human lung diploid line WI-38. In both these cases, two separable DNA polymerases were found in the nucleus and only a single activity was detected in the cytoplasm, the cytoplasmic enzyme resembling one of the nuclear enzymes in a l l respects tested. The evidence indicates that the cytoplasmic enzyme activity i s not due to nuclear contamination. F i r s t l y , the amount of enzyme activity present in the cytoplasmic fraction i s high compared with that in the nuclear fraction, and appears d i f f i c u l t to be accounted for by nuclear contamination alone. Secondly, the nuclear preparation 67 contained several distinct polymerase a c t i v i t i e s . 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