Fidelity of DNA Polymerases Isolated from Regenerating Liver ...

Download PDF
2 downloads 0 Views 2MB Size Report
Comment
Jan 25, 2016 - Joseph Gottstein Memorial Cancer Research Laboratory, Department of Pathology ... pancreatic deoxyribonuclese as described by Loeb (10).
THEJOURNAL OF BIOLOGICAL CHEMISTRY

Vol. 260, No. 2, Issue of January 25, pp. 1304-1310,1385 Printed in U.S.A.

Fidelity ofDNA Polymerases Isolatedfrom Regenerating Liver Chromatin of Aging Mus musculus* (Received for publication, May 2, 1984)

John R. SilberS, Michael Fry& George M. Martinll, and Lawrence A. Loebll** From the $National Center for Toxicological Research, Jefferson, Arkansas 72079, the §Unit of Biochemistry, Faculty of Medicine, Technicon-Israel Institute of Technology, Haifa 31096, Israel, and the llDivision of Genetic Pathology, and 11 The Joseph Gottstein Memorial Cancer Research Laboratory, Department of Pathology SM-30, University of Washington, Seattle, Washington 98195

DNA polymerase-a and -/3 were fractionated from the chromatin of regenerating liver of young and old mice. The DNA polymerases were resolved from each other and partially purified by DEAE-cellulose, phosphocellulose, and DNA-cellulose column chromatography. No significant age-relateddifference in the kinetics of heat inactivation was observed for either DNA polymerase. No age-dependent difference was found in the fidelity with which these enzymes copied 4x174 DNA. These results suggest that the functional properties of these DNA polymerases do not change with ageas is postulated in some theories of aging.

differences in the mechanismof aging in thesetwo situations. As a test of the protein-synthesis error-catastrophe theory of aging, we have asked if the DNApolymerases of replicating cells are altered withage. We have examined DNA polymerase-a and-@ isolated from regenerating liver of young and old Mus musculus for differences in their physical and fidelity parameters. Regenerating liver is a relevant source of these enzymes since an age-dependentdecrease in the proliferative capacity of the liver of M. musculus has been demonstrated (9). Moreover, polymerase-a activity is induced in the regenerating liver (9, 13). Hence, any error in its synthesis due to the accumulation of errors in DNA during aging should be reflected in its physical and catalytic properties. We find no significant difference in the thermolability of the polymerizing activity of DNA polymerase-a and -@ from young and old The protein-synthesiserror-catastrophetheory of aging predicts a positive-feedback mechanism of error generation animals. More significantly, we observe that the fidelity with which both DNA polymerase-a and -@ copy a biologically beginning with random mistakes made during protein syntheas mice age. The active DNA molecule does not decrease sis and culminating in the rapid accumulation of somatic mutations (1,2). DNA-replicating enzymes are central to this significance of these findings with respect to mutational mechanisms of aging is discussed. concept. If these proteins are altered during their synthesis and exhibit reduced fidelity, the rate of mutagenesis could EXPERIMENTALPROCEDURES increase during DNA replication. Random mutations would Materials-Calf thymus DNAwas obtained from Worthington. then occur in the genes coding for the DNA-replicating enzymes themselves and give rise to the appearance of stable, Maximally activated DNA was prepared by limited digestion with pancreatic deoxyribonuclese as described by Loeb (10). Lysis-defiinaccurate DNA synthesis. This cascading series of errors, cient 6x174 am3 single-stranded DNA andEscherichia coli spheroculminating in error-prone DNA replication, provides amech- plasts were prepared as described previously (11).The 15-nucleotide anism for the rapid accumulation of somatic mutations and primer (5’-d[GGA-AAG-CGA-GGG-TAT1-3’), 2’,3’-dideoxythymidine triphosphate, and T4polynucleotide kinase were obtained from the consequences they engender. If enhanced mutagenesis is a hallmark of senescence, the P-L. Biochemicals. Unlabeled deoxyribonucleoside triphosphates fidelity of DNA replication should be lower in aged organisms were purchased fromCalbiochem-Behring.Radioactivitylabeled [methyL3H]TTP and [w3’P]TTPwere the products of New England than inyounger ones. Studies with culturedcells and animals Nuclear, while [Y-~*P]ATP was supplied by Amersham Corp.DEAEhave been conducted in order t o determine if the fidelity of cellulose (DEAE-52) andphosphocellulose (P-11)were obtained from DNA polymerases changes with age. With one exception, all Whatman Ltd, Kent, UnitedKingdom. Double-stranded calf thymus studies of DNA polymerase-a, and -@, and -7 isolated from DNA cellulose,bovine serum albumin, N-ethylmaleimide, phenylfibroblasts grown in culture haveshown an age-dependent methylsulfonyl fluoride, leupeptin, and dithiothreitol were supplied decreasein their ability to copy synthetic polynucleotides by Sigma. Aphidicolin was obtained from the National Cancer Instifaithfully (3-6). Ontheotherhand,the fidelity of DNA tute. E. coli DNA polymerase I was prepared as described elsewhere (12). polymerase-a isolated from human lymphocytes and DNA musculus (male) were (A/He Tr Wo X C57 B l f S J T r Animals-”. polymerase-@isolated from mouse liver did not change with Wo)Fl hybrids(AB6F1)from an agingcolony maintained at the age (7, 8). The observation that DNA polymerases isolated Department of Pathology, University of Washington, Seattle, WA. from aging animals do not show altered fidelity while polym- The mice were reared in a defined, specific pathogen-free environerases from senescing cultured cells do may reflect inherent ment. A contemporary sample of 200 mice yielded a mean life span *This work was supported by grants to M. F. from the Chief Scientist, Israel Ministry of Health and from the Israel Academy of Sciences and Humanities, Basis Research Foundation and by Grants AG-01451, AG-00057, and CA-24845 from the National Institutesof Health. The costsof publication of this article were defrayed in part by the paymentof page charges. This article must thereforehereby be marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ** To whom correspondence should be addressed.

of 29.5 months and a maximum life span of 42.2 months. Young and old animals used in this work were 6 and 28 months of age, respectively. Partial Hepatectomy-Liver regeneration was induced by surgical removal of 60-70% of the organ. Conditions and surgical procedure for the partial hepatectomy were detailed elsewhere (9). To obtain maximum proportion of liver cells a t S phase, mice were killed a t approximately 60 and 72 h after surgery in 6- and 28-month-old mice, respectively, when hepatic DNA synthesis has been shown to reach its peak (9). A typical preparation of DNA polymerase was obtained

1304

Fidelity of DNA Polymerases in Aging Mice from 16-20 pooled regenerating livers of mice of each age group. DNA Polymerase Assays-Total DNA polymerase activity in crude extracts and DNA polymerase-a were assayed at 37 "C for 20-60 min in an assay mixture which contained in a final volume of 50 pl: 50 mM Tris-HCI, pH 7.5, 5 mM MgC12, 50 p~ each dATP, dGTP, and dCTP, 25 p~ [3H]TTP ata specific activity of 260-400 cpm/pmol, 300 pg/ml activated DNA, 1 mM DTT,' 20% glycerol,and 300 pg/ml bovine serum albumin. DNA polymerase-0 was assayed in a similar reaction mixture except that 50 mM Tris-HC1 was a t pH 8.5 and 120 mM KC1 was present. The incorporation of labeled TTP into acidinsoluble DNA was measured as described previously (17). The DNA polymerases copied activated DNA at a linear rate for at least 120 min. One unit of polymerase activity was defined as that activity which catalyzed the polymerization of 1nmol of deoxyribonucleoside monophosphate/h a t 37 'C. Assay of Fidelity of DNA Synthesis-The 6x174 DNA fidelity assay used in these experiments was essentially that described previously (11).The assay was modified, however, by using a 15-nucleotide oligomer as a primer for DNA synthesis. Viral 4x174 am3 DNA was primed by heating at 100 "C for 3 min in the presence of a 10fold molar excess of primer, in 100 mM Hepes, pH 7.4, and 300 mM KCl. The mixture was cooled in air to room temperature and then incubated at 20 "C for 30 min. The annealing reaction was terminated by lowering its temperature to 0 "C. The oligonucleotide-primed 4x174 am3 DNA served as a template for DNA synthesis. Increasing amounts of DNA polymerase were added to 25-pl reactions containing 50 mM Tris-HC1, pH 8.0, 2 mM DTT, 300 pg/ml bovine serum albumin, 20% glycerol, and 0.1 pg of primed 4x174 am3 DNA and incubated for 60 min a t 37 "C. Conditions for Me-catalyzed polymerization by DNA polymerase-a and -@ were: 8 mM MgC12,l mM dATP, dCTP, and dGTP, and0.020 mM TTP. Reactions catalyzed by DNA polymerase-0 included 120 mM KCl. Conditions for Mn2+-catalyzedsynthesis by DNA polymerase-a were: 2.5 mM MnC12, 1.5 mM dATP, 0.050 mM dCTP, dGTP, and TTP. At the end of the incubation period, synthesis was terminated by adding EDTA to a final concentration of 12.5 mM. The reaction was split into two aliquots, and each was diluted into 1.0 ml of icecold sterile 10 mM Tris-HC1, pH 7.5, prior to transfection. The protocols for transfection using E. coli spheroplasts and titering of the resultant 6x174 phage were described elsewhere (11). Incubation of the primed 6x174 am3 DNA with either DNA polymerase did not reduce its biological activity (data not shown). Analysis of Extended Oligonucleotide Primer-Free 15-nucleotide oligomer primer was 5'- end-labeled with 32P in a 25-p1 reaction containing: 30 pmol of primer, 30 pmol of [-y-32P]ATP(5200 Ci/ mmol), 50 mM Tris-HC1, pH 7.5, 10 mM MgC12, 5 mM DTT, and 25 units of T4 polynucleotide kinase. After incubation for 60 min at 37 "C, EDTA was added to 33 mM and the reaction was stored at -20 "C. Markers were prepared which were 16, 17, and 18 nucleotides long to determine the fraction of annealed primers which were extended past the middle position of the am3 site. This was done by copying 0.1 pg of 5'-32P-oligomer-primed 6x174 am3 DNA with a 100-fold molar excess of E. coli DNA polymerase I in a 25-pl reaction containing: 50 mM Tris-HC1, pH 8.0, 2 mM DTT, 5 mM MgC12,0.10 mM dCTP and TTP, and 20% glycerol. After incubation at 37 "C for 10 min, the DNA was ethanol-precipitated and prepared for electrophoresis as described below. By including only the two deoxynucleoside triphosphates in the reaction, the primer can only by extended 1, 2, or 3 nucleotides. For analysis of primer extension, a 2-fold molar excess of 5'-32Pprimer was annealed to viral 6x174 am3 DNA as described above. Various amounts of DNA polymerase were added to 75-p1 DNAsynthesizing reactions containing 0.3 pg of this template primer. The conditions for DNA synthesis were the same as described above. After incubation, 25-pl aliquots of the reactions were prepared for transfection (11). The DNA in the remainder of the reaction was ethanolprecipitated (18), and the resultant DNA pellet was dried by vacuum desiccation. The DNA wasresuspended in 4pl of a solution containing 99% deionized formamide, 10 mM Na,EDTA, 10 mM NaOH, and 0.3% bromphenol blue. The resuspended DNA was heated at 100 "C for 3 min and immediately loaded onto a 0.4 X 200 X 400-mm 12% polyacrylamide gel containing 135 mM Tris-HC1, pH 8.5,45 mM boric 'The abbreviations used are: DTT, dithiothreitol; Hepes, N-2hydroxyethylpiperazine-N'-2-ethanesulfonicacid.

1305

acid, 2.5 mM EDTA, and 7 M urea.' Electrophoresis was carried out at 1500 V for 2 h in a buffer containing 135 mM Tris-HC1,45 mM boric acid, and 2.5 mM EDTA. The gel was then soaked for 10 min in 10% acetic acid, transferred to a sheet of Whatman No. 3MM paper, and dried under vacuum at 80 "C. The location of the extended 5'-32P-primers was determined by autoradiography using Kodak X-Omat x-ray film. Each sample lane was cut into two sections between the positions corresponding to 2 and 3 nucleotides added. The sections were cut into 2-cm strips, and theamount of 3'P in each strip was determined. The fraction of primers extended 3 or more nucleotides was determined by dividing the number of 32Pcounts/min in primers extended 3 or more nucleotides by the total number of 32Pcounts/min in all primers. This value was multiplied by 2 to compensate for the fact that one-half of the primers in the DNA polymerizing reactions were not annealed to 6x174 am3 DNA templates and could not be extended. Calculation of Error Rate-The error rate for DNA polymerase-a and -0is calculated from the following expression. Error rate

(

= reversion frequency X

1 fraction copied

X

l r penetrance

(19, 20). The penetrance is the efficiency with which E. coli spheroplasts allow the expansion of the wild-type phenotype caused by misinsertions opposite the middle position of the am3 site of the 6x174 am3DNA template (19,20).For the 15-nucleotide primer, the penetrance has avalue of 0.16: DNA Polymerase Purification-Partially hepatectomized mice were killed by cervical dislocation, and their livers wereremoved rapidly into an ice-cold solution of0.3 M sucrose, 4 mM CaC1'.All subsequent steps were conducted at 4 "C. The livers werewashed thoroughly in the above solution, blotted dry, weighed, and minced. The finely minced tissues were suspended in 9 volumes of 0.3 M sucrose, 4 mMCaC12, and cells were disrupted with a Dounce homogenizer using 20 strokes of a B-typepestle. Crude nuclei were separated from cytoplasm by centrifugation at 9000 x g for 5 min and purified by centrifugation through a discontinuous gradient of 1.85 M sucrose, 1 mM CaC12to 2.05 M sucrose as described by Lynch et al. (13). Lysis of the purified nuclei and isolation of chromatin were conducted essentially according to Knopf and Weissbach (14) except that the buffers contained, in addition to phenylmethylsulfonyl fluoride, the protease inhibitor leupeptin at 10 pg/ml (9). To extract the DNA polymerases, 5 M NaCl was added to chromatin suspended in 1 mM Tris-HC1, pH 8.5, 0.1 mM EDTA, 1 mM DTT, 10 pg/ml leupeptin, 40% glycerolto a final concentration of 0.4 M and held at 4 "C for 20 min with occasional moderate stirring with a Vortex mixer. Solubilized chromatin proteins were separated from the insoluble residue by centrifugation at 30,000 rpm in a Spinco 60Ti rotor for 30 min. To remove DNA from the extract, it was passed through a DEAEcellulose column equilibrated with 0.025 M potassium phosphate buffer, pH 7.5, 0.4 M NaCl, 1 mM DTT, 1 mM EDTA, 20% glycerol. Salt wasremoved from the protein solution by dialysis overnight against 500 volumes of 25 mM potassium phosphate buffer, pH 7.5, 1 mM DTT, 1 mM EDTA, 20% glycerol. the dialyzed chromatin To resolve DNA polymerase-a and -@, extracts were loaded onto aDEAE-cellulosecolumn equilibrated with dialysis buffer at a ratio of 1.0-1.5 mg of extract protein/l.O ml of column volume. The column was washed with 1 column volume of the dialysis buffer, and a linear gradient of10 column volumes of 0.025-0.50 M potassium phosphate buffer, pH 7.5, containing 1 mM DTT, 1 mM EDTA, and 20% glycerol was applied. Fractions, each with a volume equal to 7% of the column volume,were collected throughout loading, washing, and gradient elution. Ten p1 of each fraction were assayed forDNA polymerase activity as described above. DNA polymerase-@from both young and old mouse liver was not adsorbed to the ion exchanger. DNA polymerase-a was eluted from the column at about 0.10-0.16 M salt. Fractions containing DNA polymerase-n and -@ activity, respectively, were combined to form separate pools. DNApolymerase-a was diluted with 1 mM DTT, 1 mM EDTA, 20% glycerol to yield a final concentration of0.10 M potassium phosphate buffer. Each DNA R. Monnat and L. Loeb, personal communication. J. Abbotts and L. Loeb, personal communication.

Fidelity of DNA Polymerases in Aging Mice

1306

polymerase pool was loaded onto a phosphocellulose column equilisitivity to heat inactivation been has demonstrated for several brated with 0.10 M potassium phosphate buffer, pH 8.5, 1 mM DTT, enzymes isolated from senescent cultured fibroblasts or old 1 mM EDTA, 20% glycerol at a ratio of 1.0 mg of protein/l.O ml of organisms (3-5, 22-24). The increased heat lability of these column volume. DNA polymerase activity was eluted from the colenzymes has been suggested to be due to a subpopulation of umns by applying a linear gradient of 10 column volumes of 0.100.50 M potassium phosphate buffer, pH 8.5, containing 1mM DTT, 1 altered protein molecules. In some instances, these proteins haddifferentcatalyticproperties.Withthese findings in mM EDTA, 20% glycerol. Fractions, each with a volume of 10% of the column volume, were collected, and 10-pl aliquots were assayed mind, we compared the thermolabilityof DNA polymerase-a for DNA polymerase activity as described above. DNA polymerase-fi and -@ isolated from 6- and 28-month-oldmice. from young and old mice was eluted from phosphocellulose at about As shown in Fig. lA, when heated at 48 “C for increasing 0.22-0.28 M salt. DNA polymerase-a from young and old mice was periods of time, the activityof DNA polymerase-a from both eluted at about 0.19-0.23 M salt. Fractions containingDNA polymerase-a activitywere pooled, and young and old mice decreases with first-order kinetics. This result is consistent with the premise that the population of bovine serum albumin was added to a final concentration of 300 pg/ mi. The enzymes were dialyzed overnight against 200 volumes of 50 DNA polymerase-a for both ages is homogeneous. More immM Tris-HCI, pH 7.5, 1 mM DTT, 0.1 mM EDTA, 20% glycerol and portantly, the rates of inactivation for the enzymesfrom stored at -80 “C. The phosphocellulose-purified enzyme was used for young and old animals are indistinguishable, suggesting that heat lability and fidelity studies. Phosphocellulose fractions containthe primary structure of the two does not differ sufficiently ing DNA polymerase-fi were pooled and dialyzed overnight against to produce a detectable difference inthermolability.The 200-400 volumes of 20 mM Tris-HCI, pH7.5,0.025 M NaCI, 1 mM DTT, 1 mM EDTA, 20% glycerol. The dialyzed enzyme was loaded results of an analogous experiment in which DNA polymeronto a double-stranded DNA-cellulose column at a ratio of 0.4 mg of ase-@was heated at 44 “Care shown in Fig. 1B. As for DNA protein/l.O ml of column volume. DNA polymerase-(3 was eluted from polymerase-a,theindistinguishablefirst-orderkinetics of the column with a linear gradient of 10 column volumes of 0.025-20 heat inactivation for DNA polymerase-@from young and old M NaCl in buffer containing 20 mM Tris-HC1, pH 7.5, 1 mM DTT, 1 animals indicates that the primary structure of this enzyme mM EDTA, 20% glycerol. Fractions each with a volume equal to 20% of the column volume were collected, and 5-pl aliquots of each fraction does not change withage. Effect ofAge on the Fidelity of DNA Polymerase-a-The were assayed for DNA polymerase activity. DNA polymerase-0 was eluted from DNA cellulose at about 1.0-1.4 M NaCl. 4x174 fidelity assay (11)was used to measure the accuracy Active fractions were pooled, and the enzyme was dialyzed overof DNA polymerase-a, the putative replicative enzyme (16). night against 300 volumes of 50 mM Tris-HCI, pH 8.5, 1 mM DTT, The assay utilized a 4x174 DNA molecule containing the 0.1 mM EDTA, 20% glycerol and stored at -80 “C. BothDNA am3 codon which permits the detection of misincorporation polymerases were stable at -80 “C in the presence of 300 pg/ml bovine serum albumin and the specified storage buffer for several of any nucleotide at the middle position of the amber site months. The DNA-cellulose-purified enzyme was used for thermola- (11). Mistakes made during in vitro DNAsynthesis were measured in atransfection assayas the reversion of the amber bility and fidelity experiments. To validate the complete resolution of DNA polymerase-a and $3, mutation towild type. Since eucaryotic DNA polymerases do the partiallypurified enzymes were tested for their sensitivity to notcatalyzeextensiveDNAsynthesis withlongsingleseveral selective inhibitors (see “Results”). stranded DNA templates (24), a 15-nucleotideoligomer whose

3”hydroxyl terminus is only 3 nucleotides from the middle position of the am3 site was hybridized to 4x174 am3 DNA Purification of DNA Polymerase-a and-@ from Regenerating as a primer to ensure that DNA polymerizationoccurred Liver of Young and Old Mice-Chromatin prepared from opposite the ambercodon. regenerating liver of 6- and 28-month-oldmice was extracted In order to compare the fidelity of DNA polymerase-afrom with 0.4 M NaCl to solubilize bound DNA polymerases. DNA young and old mice, the reversion frequency resulting when polymerase-a and -@ were purified from the salt extract by the maximal number of 6x174 am3 template-primers initisequential DEAE-cellulose and phosphocellulose chromatog- ated and extended past the am3 site was determined. This raphy. DNApolymerase-@ wasfurther purified bychromatog- was done by measuring mutagenesis as increasing amounts of raphy on double-stranded DNA cellulose. DNA polymerase- DNA polymerase-a activity were added to the DNA-synthe(Y was purified94to148-fold for 6- and 28-month-old animals sizing reaction. The resultsof such a determination, inwhich respectively, while DNA polymerase-@ was purified 242- and DNA was synthesized in the presence of Mg2+and a 50-fold 116-fold, respectively. The identityof each enzymewas deter- molar excess of the deoxynucleoside triphosphates which are mined with specific DNA polymerase inhibitors, and each not complementary to the middle position of the am3 site purified enzyme was free of detectable contaminating DNA (dATP, dCTP, and dGTP), are shown in Fig. 2, A and B. For polymeraseactivity. Thus, activities of polymerase-a from both ages, there is an identicalincrease in reversion frequency young and old animals were inhibited 86-96% by 4 mM N- with increasing amounts of DNA polymerase activity. More ethylmaleimide, 83-86% by 20 pg/ml aphidicolin,and 4- importantly, mutagenesis reaches a plateau for both ages a t 14% by 25 p~ 2‘,3’-dideoxythymidine 5’-tripbosphate. Activ- anidentical valuewhenmore than 1 2 X units of polymities of polymerase-@from young and old mice were inhibited erase activity are added to the DNA-synthesizing reactions. 41-42% by 4 mM N-ethylmaleimide, 11-35% by 20 bg/ml The constantreversion frequency indicates that the maximal aphidicolin, and 90-94% by 25 p~ 2’,3’-dideoxythymidine 5’- number of 4x174 am3 template-primershave been elongated triphosphate. The partial sensitivityof murine polymerase-@ past the am3 site. Assuming that the extent of initiation of to N-methylmaleimide has been reported (15). Furthermore, the 4x174 am3 template does not differ withage, these results whereas polymerase-awas inhibited by 50 M KC1, polymerase- suggested that age is not a determinant of the fidelity of mouse DNA polymerase-a as it synthesizes DNA under these @ was stimulated by 120 mM KC1, and the former was more heat-labile than the latter (see Fig. 1 below). For each age, conditions. The fidelity of DNA polymerase-a was determined again one predominant chromatographic peakof each enzyme was with the reaction conditions altered such that Mn2+ replaced observed throughoutpurification. More importantly,the chromatographic profiles of the DNApolymerases from young Mg+, and dATP was present in a 30-fold molar excess over the remaining three deoxynucleoside triphosphates. As shown and oldmice were similar. Heat Inactivationof DNA Polymerases-An enhanced sen- in Fig. 2, C and D, the reversion frequency increased identiRESULTS

Fidelity of DNA Polymerases in Mice Aging

Minutes at 48°C

6 mb

t

0

I

I

4

1

1

I

I

I

I

12 16 Minutes at 44°C

8

I

l

l

20

FIG. 1. Heat inactivation of DNA polymerase-cY and -0 from young and old mice. A , DNA polymerase-cu from 6-month-old (0) and 28-month-old (A)mice was incubated a t 48 "C.At various times during the incubation, 15-pl aliquots were transferred to 35 pl of an assay mixture and held at 0 "C. After all aliquots were collected, the samples were incubated at 37 "C for 120 min t o determine the surviving fraction of DNA polymerase activity. DNA polymerase-cu not incubated a t 48 "C had activities of 0.062 and 0.037 units for enzyme fromyoung and old mice, respectively(averageof quadruplicate determinations). These activitieswere taken as 100% activity values. B , DNA polymerase-,l3 from 6-month-old (A)and 28-month-old (0) mice was incubated a t 44 "C. Sampling was performed as described

1307

cally with increasing DNA polymerase activity for each age. units of DNA polymerase-a activity At approximately 3 X from young and old mice, similar maximalreversion frequency was attained. However, the reversionfrequencydecreased with increasing DNA polymeraseactivity, rather than remaining constant as in the Mg2+-activated reactions. The decrease in mutagenesis was not accompanied by a reduction which would in 4x174 am3 phage titer (data not shown), indicate an endonuclease contamination. Thepresence of an exonucleasewhichdegrades theextendedprimer was a more likely explanation.Sincethisstranddeterminesthe phenotype of the copied 4x174 am3 DNAtemplates,an exonuclease could decrease the reversion frequency without affecting phage titer. The congruent pattern of the change of reversion frequencywith increasing DNA polymerase activity, however, did not indicate a difference in the fidelity of DNA polymerase-a from young and old mice, assuming that the enzymes were equally competent to initiate the 4x174 am3 DNA template-primers. We have tested directly the critical assumption that DNA polymerase-a from young and old mice does not differ in its capacity to copy the 4x174 am3 DNA template-primer past the ambercodon. To test this assumption and confirm to our initial findings,we have determined by gel electrophoresis the fraction of templates that DNA polymerase-a extended to produce a given reversion frequency. From this information, each enzyme's error rate,a measure of the intrinsic fide1it.y of a DNA polymerase for a given reactioncondition,can be calculated (19, 20). The error rate provides an unequivocal comparison of the fidelity of DNA polymerase-a from young and old mice. DNA polymerase-a-catalyzed DNA-synthesizing reactions were performed using a 4x174 am3 DNA template primed with the 15-nucleotide primer labeled at its 5' end with 32P. An aliquot of the reaction was assayed to ascertain reversion frequency. The remainder was analyzed by denaturing polyacrylamide gel electrophoresis toresolve the extended primers by chain length. Fig. 3 is an autoradiographshowing the size distribution of primers elongatedby DNA polymerase-afrom young and old mice in a synthesizingreactioncontaining Mg2' and a 50-fold molar excess of dATP, dCTP, and dGTP to TTP. Fig. 3 shows an increase in the fraction of primers which are elongated asmore DNA polymerase-a is added to the copying reactions. It is also apparent from Fig. 3 that the primers are elongated a greater number of nucleotides with increasing amounts of DNA polymerase. This visual impression is confirmed when the fraction of primers extended to the middle position of the am3 site ( 3 deoxynucleotides added) or farther is calculated from the distribution of 'lP in the gel. Table I shows that the fraction of primers extended to the am3 site increases with increasing amounts of DNA polymerase. More significantly, for a given amount of DNA polymerase-a activity, the fraction of primers extendedis not greatly different between young and old animals. Table I also shows thatthe reversionfrequency increasesasthefraction of 4x174 am3 DNA template-primers copied increases. When the error ratesof the enzymes are calculated from this information, no significant difference is found in the fidelity of DNA polymerase-a from young and old mice. above. DNA polymerase-,l3 not incubated a t 44 "C had activities of 0.076 and 0.006 units for young and old mice, respectively (average curues for A and B are lines of of quadruplicate determinations). The best fit obtained by least-squares linear-regressionanalysis. The correlation of the data with theregression line was 0.98 and 0.99 for DNA polymerase-cu and 0.88 and 0.93 for DNA polymerase-@ forthe young and old animals, respectively.

Fidelity of DNA Polymerases in Aging Mice

1308

..

.

The results of a similar experiment, in which the 4 x 1 7 4 am3 template-primer was copied in the presence of Mn2+ and a 30-fold molar excess of dATP to theother three deoxynucleoside triphosphates, are also contained in Table I. As in the case of the M$+-activated reactions, the fraction of templates copied increases with increasing DNA polymerase, as does the reversion frequency. Calculating the error rates for the enzymes from this information again shows that there is no significant age-dependent change in the fidelity of DNA polymerase-a. Effect of Age on the Fidelity of DNA Polymerase-@-We next investigated if the accuracy of DNA polymerase-@,the putative repair DNA polymerase (16), is altered with age. An age-dependent decrease in the fidelity of DNA polymerase-@ could enhance mutagenesis during the gap-filling step of excision repair (25) in both replicating and quiescent cells. Considering the high incidence of spontaneously induced DNA lesions such as apurinic sites (26), as well as the great variety of exogenous agents which cause DNA damage which is repaired by the excision-repair pathway (27), any decrease in the faithfulness of DNA polymerase-@could result in a significant increase in mutagenesis. The error rate of DNA polymerase-@from young and old mice was determined as described above in a Me-activated synthesizing reaction containing a 50-fold molar excess of dATP, dCTP, and dGTP toTTP. The results of this experiment are presented in Table 11. As with DNA polymerase-a, a directcorrelation was observed between the amount of DNA polymerase-@activity, the fraction of 4 x 1 7 4 am3 DNA template-primers initiated and extended past themiddle position of the amber site, and the reversion frequency. Most important, there was no significant difference in the error rates of DNA polymerase-@from young and old mice. DISCUSSION

I

. 2

. 4

.

.

.

.

.

,

6 8 IO 12 14 16 Unlts ( x lo3)

FIG. 2. Comparison of the fidelity of DNA synthesis by DNA polymerase-&of young and old mice. #X174 am3 DNA primed

In this work, we report the results of experiments designed to detect age-dependent changes in the properties of DNA polymerases isolated from regenerating liver of the mouse, M. musculus. The motivation for our work stems from a consideration of the protein-synthesis error-catastrophe theory of aging. By this hypothesis, unfaithful macromolecular synthesis ultimately results in the accumulation of somatic mutations which cause the progressive decrease of cellular function and proliferative capacity characteristic of aging (1, 2). In addition, the theory suggests that mutations occurring in genes coding for DNA replication proteins will result in the stable expression of error-prone DNA replication. Thus, a crucial premise of the error-catastrophe theory is the appearance of unfaithful DNA replication with increasing age. In order to test the validity of the error-catastrophe theory, we examined the enzymes central to DNA replication for agedependent changes in their properties. DNA polymerase-a and -@ were purified from regenerating liver of 6- and 28month-old mice. We chose regenerating liver as a source of with the 15-nucleotide oligomer was the template for DNA polymerase-a from 6- and 28-month-old mice in DNA-synthesizing reactions containing M$+ and a 50-fold molar excess of dATP, dCTP, and dGTP to TTP( A and E ) or MnZ+and a 30-fold molar excess of dATP to the other three deoxynucleoside triphosphates (C and D).Unbalanced deoxynucleotide triphosphate pools and Mn2+have been shown to decrease the accuracy of DNA polymerases (20, 21) and were used in these experiments to enhance the sensitivity of the assay. The reversionfrequency of the copied 6x174 am3 DNA template was plotted as a function of DNA polymerase-a activity. Each symbol designates the results obtained in an independent experimental determination.

Fidelity of DNA Polymerases in Aging Mice

1309

... FIG.3. Size distribution of 5’-32Pprimers extended by DNA polymerase-a of young and old mice. @X174 am3 DNA primed with the 15nucleotide primer5’ end-labeled with32P served as a template for DNA synthesis catalyzed by DNA polymerase-awith M e and a 50-fold molar excess of dATP, dCTP, and dGTP to TTP. The lane on the farright shows the position of the primer when extended 1, 2, or 3 deoxynucleotides by E. coli DNA polymerase I. Autoradiography was for 16 h at 20 “C. Contamination of the 15-nucleotide primer by lower-molecular-weight molecules is the cause of the size heterogeneity of the primer.

-.. I

rl e d

-. c

5

.

*t 3 dNMP

e

1.8

No Polymerase

3.7

Units X polYoung

7.3

H

0.9

1.5

Units X

3.0

pol-% Old

TABLE I TABLE I1 Comparison of fidelity of DNA polymerase-a fromyoung and old Comparison of fidelity of DNA polymerase-@from young and old mice mice See “Experimental Procedures” for techniques used to determine The techniques used for determining thefraction of 15-nucleotide fraction of primers copied, reversion frequency, and error rate. Reprimers extended past the middle position of the am3 codon, the version frequencies were calculated from duplicate transfections. No reversion frequency, and the error rate of each enzyme were described enzyme reversion frequency of9.5 X IO6 was subtracted from the under “Experimental Procedures.” The reversion frequency was cal- values below. culated from duplicate transfections. The reversion frequencies of Units Fraction Reversion Error uncopied @X174am3 DNA (8.8 X lo6 for M e + 50 X ACG and 5.6 Age rate (x IO3) copied frequency (x 106) X 1O“j for Mn2+ + 30 X A) were subtracted from the values below. The error rates were not compensated for pool bias, since we have month not demonstrated that error rateis directly proportional to the ratio 11170 2766.0 0.29 6 of incorrect to correct deoxyribonucleotide triphosphate concentra11220 722 12 1.0 tions for the DNA polymerases used in these studies (20). 11180 19 1.0 911 1/220 0.22 161 28 1.4 Units Fraction Reversion Error Pool frequency 1/150 246 0.12 4.6 ( X lo3) copied ( X 109 rate 1/290 406 5.6 0.73 month

6

M%++50xACG

28

6 28

Mn2++ 30 X A

3.7 7.3 0.87 1.5 3.0

0.22 0.70 0.95 0.24 0.50 0.84

11.7 15.5 49.7 9.5 12.7 22.7

1/3100 1/7200 1/3100 1/4000 1/6300 1/5900

0.49 0.98 1.8 0.22 0.43 0.87

0.032 0.026 0.042 0.019 0.064 0.068

28.4 26.9 31.7 9.9 26.2 25.2

1/180 1/150 1/210 1/310 1/390 1/430

1.8

DNA polymerase as a result of our previous demonstration of a n age-dependent decrease in theproliferative capacity of this organ (9). New modifications of the 6x174 fidelity measurement systemallows us, for the first time, to estimate accuracy of DNA synthesis in aging animals on a natural DNA template. When the accuracy with which both species of DNA a biologically active template polymerase synthesized DNA on was measured by two methods, eachemploying two different reaction conditions, no age-dependent decrease in thefidelity of DNA polymerase-a or-p was detected. Furthermore, DNA polymerase-a for bothages wascharacteristically less faithful

1310

Fidelity of DNA Polymerases in Aging Mice

in reactions activated by Mn2+rather than Mg2+ (11).Likewise, the approximately 20-fold greater accuracy of DNA polymerase compared to DNA polymerase-@observed for both ages when assayed under the same reaction conditions was in accord with the relative fidelity of DNA polymerase-a and -@ from other sources (28). In addition, no age-dependent difference in the chromatographic behavior or thermolability of either DNA polymerase was observed. We conclude that DNA polymerase-a and -p are not detectably altered in the properties examined as a consequence ofage. These findings, particularly the failure to detect an age-related decrease in the accuracy of either DNA polymerase, do not support the tenets of the error catastrophe of aging. Previous studies to detect age-related alterations in catalytic and antigenic properties of various proteins isolated from a variety of sources have yielded conflicting results (7, 22, 23, 29-33). This is true of reports for the fidelity of isolated DNA polymerases. The accuracy of DNA polymerase-a, -@, and -7 isolated from cultured fibroblasts which have gone through a high number of population doublings has been observed consistently to be lower than that of DNA polymerases isolated from fibroblast cultures which have undergone a low number of population doublings (4-6). On the other hand, the fidelity of DNA polymerase-a isolated from phytohemagglutininstimulated human lymphocytes was found to be the same for young and old donors (7). Similarly, no age-dependent difference in fidelity was detected for either partially purified or chomatin-associated DNA polymerase-@from mature liver of the mice M. musculus or Peromyscus leucopus (8). This dichotomy of results based on the source of DNA polymerizing activity may reflect fundamental differences in the mechanism of aging in uiuo and aging in cultured cell systems. Supporting this hypothesis are the observations of Krauss and Linn (6) that thefidelity of DNA polymerases of cultured fibroblasts decreased in a young culture when it became confluent. This suggests that the reported decreases in accuracy of DNA polymerases purified from cells aging in culture may be a function of physiological changes induced by a nonproliferating state rather than any time-dependent change in the function of cellular enzyme. From the evidence obtained with isolated DNA polymerases cited above, it is obviousthat therole of mutagenesis in aging is ambiguous. It is possible that changes in the fidelity of cellular DNA synthesis can occur without alterations in the intrinsic accuracy of DNA polymerases. Of interest in this respect are the changes in age in the fraction of total DNApolymerizing activity due to DNA polymerase-a or DNA [email protected] has been demonstrated previously that DNA polymerase-@is inherently less accurate than DNA polymerase-a (33). Therefore, even if the fidelity of each species of DNA polymerase does not change with age, an increase in the contribution of less faithful forms to totalpolymerizing activity would result in a decrease in the accuracy of DNA synthesis. We have observed a decrease in the ratio of DNA polymerase-a DNA polymerase-@activity in regenerating liver of aging mice (9). It remains to be determined, however, to what extent the changes in the proportions of the various DNA polymerases can be attributed to a decreased fraction of

replicating cells within the regenerating liver of an aged animal. Even though DNA replication in eucaryotes is likely to be mediated by a multienzyme complex, DNA polymerasea from young and old animals indicatesthat nucleotide selection by this enzyme is unaffected by aging. Hence, polymerase-a perse does not contributeto error accumulation during aging. If accessory proteins enhance accuracy, they would have to be selectively altered during aging to affect overall mutation rates. Similarly, the lack of changes in DNA polymerase-@argues against increased error accumulation during DNA repair in aging animals. It should be noted that the difficulty by which eucaryotic DNA polymerases copy singlestranded DNA templates and the presence of deoxyribonucleases in cellular extracts have until now presented serious obstacles to theuse of the 4x174fidelity assay. However, the use of the 15-nucleotide oligomer as a primer on 4x174 am3 DNA templates facilitates studies with crude extract^.^ The close proximity of the 3’-hydroxyl terminus of this primer reduces the time required for DNA synthesis to proceed past the am3 site, thereby decreasing the fraction of 4x174 templates inactivated by deoxyribonucleases. Acknowledgments-Weare indebted to Charles Ogburnfor his indispensable help in animal maintenance and surgery. The help of Marlene Koplitz is gratefully acknowledged. REFERENCES 1. Orgel, L. E. (1973) Nature ( L o n d . ) 243,441-444

2. Holliday, R., and Tarrant, G. M. (1972) Nature (Lond.)238,26-30 3. Fry, M., and Weisman-Shomer, P. (1976) Biochemistry 15,4319-4329 4. Linn, S., Karis, M., and Holliday, R. (1976) Proc. Natl. Acad. Sci. U. S. A. 73,2818-2822 5. Murray, V., and Holiday, R. (1981) J. Mol. Biol. 1 4 6 , 55-76 6. Krauss, S. K., and Linn, S. (1982) Biochemistry 2 1 , 1002-1009 7. Agarwal, S. S., Tuffner, M., and Loeb, L. A. (1978) J. Cell. Physiol. 9 6 , 235-244 8. Fry, M., Loeh, L. A,, and Martin, G. M. (1981) J . Cell. Physiol. 1 0 6 , 435444 9. Frv. M.. Silber. J. R.., Loeb., L. A,., and Martin. G. M. (1984) . , J. Cell. Phvsiol. 118,”225-232 10. Loeb, L. A. (1969) J. Biol. Chern. 244,1672-1681 11. Kunkel, T. A,, andLoeb, L. A. (1979) J. Biol. Chem. 254,5718-5725 12. Jovin, T. M., Englund, P. T., and Bertsch, L. L. (1969) J. Biol. Chem. 2 4 4 , 2996-3008 13. Lynch, W. E., Surrey, S., and Lieberman, I. (1975) J. Bid. Chem. 2 5 0 , 8179-8183 14. Knopf, K. W., and Weissbach, A. (1977) Biochemistry 16,3190-3194 15. Stalker, D. M., Mosbaugh, D. W., and Meyer, R.R. (1976) Biochemistry 15.3114-3121 16. Fry, M . (1983) in Enzymes of Nucleic Acid Synthesis and Modification (Jacob, S. T., ed) Vol. I, pp. 39-92, CRC Press, Boca Raton, FL 17. Battula, N., and Loeb, L. A. (1974) J. Biol. Chem. 249,4086-4093 18. Maniatis, T., Fritsch, E. F., and Sambrook, J. (1982) Molecular Cloning, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 19. Weymouth, L. A., and Loeb, L. A. (1978) Proc. Natl. Acad. Sci. U. S. A. 7 5 , 1924-1928 20. Kunkel, T. A,, and Loeb, L. A. (1980) J. Biol. Chem. 255,9961-9966 21. Goodman, M. F., Keener, S., Guidotti, S., and Branscomb, E. W. (1983) J. Biol. Chem. 258,3469-3475 22. Goldstein, S., and Singal, D. P. (1974) Nature ( L o n d . ) 2 5 1 , 719-721 23. Wolf, J. H., and Cutler, R. G. (1975) Exp. Gerontol. 10, 101-109 24. Korn, D., Fisher, P. A,, and Wang, T.S. F. (1981) Prog. Nucleic Acid Res. Mol. Biol. 2 6 , 63-86 25. Hanawalt. P. C.. Coouer. P. K.. Ganesan. A. K.. and Smith. C. A. (1979) Annu. Reu. Biochem. 48, 783-836 26. Lindahl, T., and Nyburg, B. (1972) Biochemistry 11,3610-3618 27. Schendel, P. F. (1981) CRC Crit. Reu. Toricol. 8 , 311-362 28. Kunkel, T. A,, and Loeb, L. A. (1981) Science (Wash. D.C.) 213,765-767 29. Gershon, H., and Gershon, D. (1970) Nature (Lond.)2 2 5 , 1214-1217 30. Lewis, C. M., and Holliday, R. (1970) Nature ( L o n d . ) 228,877-880 31. Pendergrass, W. R., Martin, G. M., and Bornstein, P. (1976)J. Cell. Physiol. 87,3-14 32. Goldstein, S., and Moerman, E. J. (1975) Nature (Lond.)2 5 5 , 157-159 33. Loeb. L. A,. ar.d Kunkel, T. A. (1982) Annu. Reu. Biochem. 52,429-457 _

I

’

J. Abbotts and J. Silber, unpublished results.