Age-associated changes in the template-reading fidelity of DNA polymerase α from regenerating rat liver

Mechanisms of Ageing and Development 92 (1996) 143 – 157

Age-associated changes in the template-reading fidelity of DNA polymerase a from regenerating rat liver Takahiko Taguchi*, Mochihiko Ohashi Department of Molecular Biology, Tokyo Metropolitan Institute of Gerontology, 35 -2 Sakae-cho, Itabashi-ku, Tokyo 173, Japan Received 16 August 1996; revised 2 November 1996; accepted 8 November 1996

Abstract DNA polymerases (deoxynucleosidetriphosphate: DNA deoxynucleotidyltransferase EC 2.7.7.7.) were extracted from regenerating livers from young and aged rats. DNA polymerase a was separated and partially purified by DEAE-cellulose column chromatography, polyethyleneglycol precipitation, and phosphocellulose column chromatography, and fidelity levels were then monitored with the synthetic template-primer poly (dG-dC). The fidelity level of the DNA polymerase from regenerating liver of a 4-month-old rat was very high, while that of the DNA polymerase from a 24-month-old rat was significantly decreased. To confirm this result, DNA was synthesized on poly (dG-dC) in a reaction mixture containing [32P]dTTP, and the synthetic polynucleotide was purified and digested with HhaI restriction endonuclease. After hydrolysis, the oligonucleotides were developed by two dimensional thin layer chromatography on PEI cellulose plates. Spots containing [32P]dTMP were observed when DNA polymerase from a 24 month-old rat was used, but none was found in polynucleotides synthesized using DNA polymerase from a 4 month-old rat. Nearest neighbor analysis suggested that dG-dT and dC-dT pairs were constructed by mis-incorporation due to DNA polymerase a. © 1996 Elsevier Science Ireland Ltd. Keywords: Rat; Regenerating liver; DNA polymerases; Synthetic template-primer; Fidelity; Aging

* Corresponding author. 0047-6374/96/$15.00 © 1996 Elsevier Science Ireland Ltd. All rights reserved. PII S 0 0 4 7 - 6 3 7 4 ( 9 6 ) 0 1 8 1 6 - 7

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1. Introduction To ensure the accurate transfer of genetic information to progeny cells, DNA must be duplicated in each generation with high fidelity. Actually, measurements of spontaneous mutation frequencies indicate that the average frequency of basepair substitution is in the range of 10 − 8 to 10 − 11 misincorporations per basepair replicated [1,2]. Generally, it is accepted that the replication fidelities of DNA polymerases are very high. However, the accuracies of DNA polymerases are very low, with reported error rates of 10 − 4 to 10 − 6 [3,4]. Additionaly, it is reported that mutagenic DNA polymerase shows decreased fidelity [5]. Furthermore, the presence of damaged bases such as 8-hydroxy-2%-deoxyguanosine in the template DNA also produces mismatched base pairs at high frequency [6]. As safeguards against these replication errors, some functions such as proofreading, mismatch repair, and the excision repair system contribute to the faithful copying of progeny DNA. An accumulation of genetic instability in somatic cells has been proposed as a possible cause of aging processes [7]. In support of this hypothesis, it has been reported that senescent animal cells or late-passage cultured cells contain a diversity of altered genes [8 – 14]. In addition to DNA alterations, it has been reported that senescent animal cells and late-passage cultured cells contain a diversity of altered proteins [15 – 17]. Any of these might be a reflection of genetic instability. On the other hand, several investigators have been unable to provide conclusive evidence of the presence of altered proteins by measuring electrophoretic mobility, immunological titration, heat-stability, or the viral susceptibility of cellular proteins during the aging process in mice [18,19] or human diploid fibroblasts [20 – 22]. These discrepancies may be resolved by a more detailed characterization of protein structures and other parameters in cellular materials of various ages in vitro and in vivo. During the passage of dividing cells in vitro and in vivo, a high level of template-reading accuracy by a DNA polymerase is required to maintain cellular properties. However, the incorporation of non-complementary nucleotides into homopolymer templates is increased when an abnormal template-primer containing bromouracil bases is used instead of thymines [23] or when templates that have been modified by g-ray irradiation are used [24]. The incorporation of non-complementary nucleotides into a synthetic template-primer was also observed using a DNA polymerase derived from the T4 phage mutant [5]. Linn et al. [25] reported that a DNA polymerase obtained from late-passage human fibroblasts, MRC-5, was more error-prone than that from early-passage cells when the fidelity of polymerization was monitored by several synthetic templateprimers. This suggested that senescent cells might produce an altered DNA polymerase with low fidelity. Fry and Weisman-Shomer [26] reported independently that age-related alteration in the properties of the two nuclear DNA polymerases A(a) and A(b) in cultured chick embryonic fibroblasts may reflect structural or conformational changes in these enzymes. Thus, age-related alter-

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ations in the fidelity of DNA polymerases from cultured cells and tissues of animal origin has been discussed. The fidelities of DNA polymerases extracted from in vitro cultured cells decreases with increasing population doubling levels [25–28]. On the other hand, there are conflicting reports that the fidelity of DNA polymerase decreases [29,30] and remains unchanged [31–33] with age in vivo. In the present paper, we wish to describe age-associated changes in the template-reading fidelity of DNA polymerase a from regenerating rat livers, and introduce a new method that clearly indicates the misincorporation.

2. Materials and methods

2.1. Tissues Regenerating livers were obtained from Wistar-strain rats (male), 4-months and 24-months of age, 48 h after partial hepatectomy according to the method of Higgins and Anderson [34].

2.2. Chemical compounds Chemicals were purchased as follows; deoxynucleoside triphosphates from Boehringer Mannheim-Yamanouchi, Tokyo, Japan; [3H]deoxynucleoside triphosphates from Dupont/New England Nuclear, Boston, MA; deoxynucleoside monophosphate, deoxynucleoside diphosphate, (dC-dG)2, (dC-dG)3, (dC-dG)4, (dC-dG)5, and poly (dG-dC) from P-L Biochemicals, Milwaukee, WI.

2.3. DNA polymerases Extraction of a DNA polymerase from tissue was performed by the following procedures. The tissue was homogenized in a Teflon homogenizer with 4 volumes of 0.005 M Tris – HCl buffer, pH 8.0, containing 0.34 M sucrose, 25 mM KCl, and 5 mM MgCl2. The homogenate was centrifuged at 100 000 × g for 120 min and the supernatant was used as the crude DNA polymerase extract. Partial purification by DEAE-cellulose and phosphocellulose column chromatography was carried out by a procedure similar to that described in previous reports [35]. Polyethyleneglycol precipitation was performed before phosphocellulose column chromatography to remove DNA in the enzyme fraction. An equal volume of 0.04 M Tris – HCl buffer, pH 7.4, containing 3.5 M KCl and 2 mM EDTA was added to the DNA polymerase a fraction separated by DEAE-cellulose column chromatography. After 30 min, polyethyleneglycol 6000 was added and the final concentration raised to 10% (w/w). This solution was left to stand at 0°C for 60 min, then the polyethyleneglycol 6000 was removed as a precipitate after centrifugation at 10 000× g for 20 min. The molecular forms of DNA polymerase were determined by the methods described previously [36,37].

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2.4. Standard assay method for DNA polymerase acti6ity

DNA polymerase activity was measured by the same procedure as described by Taguchi, et al. [35]. The standard reaction mixture contained 0.625 mCi per 0.125 nmol of [3H]dTTP, 6.25 nmol each of dATP, dCTP, and dGTP, 0.5 mmol of MgCl2, 0.75 mmol of dithiothreitol, 1.25 mmol of Tris–HCl buffer, pH 8.3, 5 mg of activated calf thymus DNA, and 25 ml of enzyme fraction in a final volume of 62.5 ml. The reaction mixture was incubated for 60 min at 37°C. At the end of the incubation, an aliquot of 50 ml from each reaction mixture was applied to a Whatman 3MM paper disc, 2.4 cm in diameter, which had been previously immersed in 0.1 M Na4P2O7 and dried. The discs were then quickly immersed in a large volume of cold 5% trichloroacetic acid (TCA) solution. After 15 min, the TCA solution was decanted and the disc rinsed three times with TCA solution and then placed in a 95% ethanol solution. The discs were dried and put into counting vials containing 10 ml of toluene scintillator containing 0.5 g of 2,5diphenyloxazole (PPO) and 1,4 bis-2(5-phenyloxazole)-benzene (POPOP) in one l of toluene. The radioactivity was counted by means of an Aloka LSC-650 liquid scintillation spectrometer (Aloka, Tokyo).

2.5. Assay for the fidelity le6el of DNA polymerase

For the assay of fidelity, the amount of the complementary nucleotide incorporated into poly (dG-dC) was calculated from the amount of incorporated [3H]dGMP in a separate assay. The reaction mixture contained 3.125 mCi per 0.112 nmol of [3H]dGTP, 6.25 nmol each of dCTP and dGTP, 0.5 mmol of MgCl2, 0.75 mmol of dithiothreitol, 1.25 mmol of Tris–HCl buffer, pH 8.3, 2.5 mg of poly (dG-dC), and 25 ml of enzyme fraction in a final volume of 62.5 ml. The reaction was initiated by adding enzyme fraction and the reactions were incubated at 37°C for 60 min. At the end of reaction, an aliquot of 50 ml was removed and applied to a paper disc. The rinsing and counting of radioactivity were carried out by the same procedures described for the standard assay method for DNA polymerase activity. To measure infidelity, the amount of non-complementary nucleotide incorporated into poly (dG-dC) was measured based on the amount of [32P]dTMP incorporated in the same reaction mixture described above except using 10 mCi per 0.0031 nmol of [a-32P]dTTP instead of [3H]dGTP. Non-complementary nucleotides were also incorporated by contamination with terminal transferase. Therefore, terminal transferase activity was measured in a reaction mixture without cold complementary deoxynucleoside triphosphates. In addition, incorporation due to endogenous DNA fragments in the enzyme preparation was estimated by removing the template-primer from the reaction mixture.

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2.6. Calculation of the infidelity le6el The exact amount of non-complementary nucleotide incorporation was the amount left after subtracting the nucleotide incorporations due to terminal transferase and endogenous DNA fragments from the total non-complementary nucleotide incorporation. The level of infidelity is expressed as one true noncomplementary nucleotide per total complementary nucleotides.

2.7. Analysis of HhaI endonuclease digestion products To clarify the presence of incorporated dTMP by misreading into poly (dG-dC), the following experiment was carried out. DNA synthesis in vitro was performed in the same reaction mixture to measure the amount of misincorporated non-complimentary nucleotides. After the incubation for in vitro DNA synthesis, an equal volume of saturated phenol was added, and the mixture was shaken vigorously and centrifuged to separate the phases. The copolymer in the upper aqueous layer was then purified by repeated ethanol precipitation. This copolymer was dissolved in 62.5 ml of a solution composed of 0.5 mmol of MgCl2, 0.75 mmol of dithiothreitol, and 1.25 mmol of Tris – HCl buffer, pH 8.3, and the reaction was started by the addition of 100 units of HhaI restriction endonuclease. The solution was then incubated at 37°C for 5 h. The addition of HhaI endonuclease was repeated 5 times at intervals of 1 h. After the incubation, an aliquot of 2 ml was applied directly onto a polyethyleneimine (PEI)–cellulose thin layer plate (Macherey-Nagel, Germany), washed with 10% NaCl solution, water, 2N formic acid adjusted to pH 2.2 with pyridine, and water, and air dried. Twodimensional chromatography on PEI–cellulose thin layer plates was carried out by a procedure similar to that described by Mirzabekov and Griffin [38]. That is, 2 ml of reaction mixture after HhaI digestion was applied to a PEI–cellulose plate and developed in the first dimension for oligonucleotides produced by HhaI digestion with 1.3 M lithium formate–7 M urea (pH 3.5) up to 10 cm above the origin, and then developed a further 8 cm with 1.8 M lithium formate – 7 M urea (pH 3.5). The plate was washed three times with methanol, dried at room temperature, and then developed in the second dimension with 0.8 M lithium chloride–7 M urea–0.02 M Tris–HCl, pH 8.0, up to 18 cm from the origin. The plate was washed three times with methanol, dried at room temperature, and finally autoradiographed. Oligonucleotide sequences were estimated from the positions of oligonucleotide markers and deoxynucleoside mono-, di- and tri-phosphates on PEI–cellulose plates. Oligonucleotide markers labeled with [32P] at the 5%-end with [g-32P]ATP and polynucleotide kinase from T4 phage were developed on PEI–cellulose plates and autoradiographed. Deoxynucleoside mono-, di- and triphosphates on PEI – cellulose plates were developed and visualized by UV shadowing.

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2.8. Nearest neighbor analysis An aliquot of DNA synthesized in vitro on poly (dG-dC) in a reaction mixture containing [32P]dTTP and then purified was hydrolyzed in the same reaction mixture used for HhaI except that 200 units of micrococcal endonuclease, 0.01 units of bovine spleen phosphodiesterase, and 10 mM CaCl2 were used instead of HhaI and MgCl2. The reaction was started by the addition of deoxyribonuclease and continued at 37°C for 2 h. Two ml of reaction mixture were applied to the origin of a PEI – cellulose plate and developed with 1 M LiCl. Finally, the plate was washed and submitted to autoradiography.

3. Results The DNA polymerase used in this experiment was obtained from the regenerating livers of young or old rats, then partially purified by DEAE–cellulose column chromatography, polyethyleneglycol precipitation, and phosphocellulose column chromatography. This DNA polymerase preparation was inhibited by aphidicolin, and sedimented at an S value of 7.4 on a 5–20% linear sucrose density gradient (w/w) containing 0.3 M KCl. For the determination of DNA polymerase a or d, deoxyribonuclease activity was measured in the DNA polymerase fraction by the degradation of [3H]DNA (100 000 cpm/6.7 mg) prepared with in vitro DNA synthesis on activated calf thymus DNA. As assayed by this method, we found no deoxyribonuclease activity (data not shown). These are characteristics of DNA polymerase a itself. The fidelity of DNA polymerase a preparations prepared separately from the regenerating livers of three 4-month-old and three 24-month-old rats were measured (Table 1). The age-associated changes in the fidelity of DNA polymerase a was found by means of DNA synthesis using poly (dG-dC). The fidelity levels of three DNA polymerase a preparations from young rats were very high with no noncomplementary nucleotides incorporated (Table 1). On the other hand, the fidelity levels of DNA polymerase a from aged rats were very low, within a range of about 1/10 000 to 1/13 000 (Table 1). These experiments were carried out carefully in order to avoid the effects of terminal transferase and endogenous DNA contamination in the enzyme preparations. We were afraid that these molecules would specifically contaminate DNA polymerase a preparations from aged rats. To examine for contamination by terminal transferase, the amount of [32P]dTMP incorporated was measured in reaction mixtures without dCTP and dGTP using DNA polymerase a preparations from young and old rats. The results showed no terminal transferase activity in any of the DNA polymerase a preparations (results not shown). However, slight [32P]dTMP incorporation was measured with some enzyme preparations using reaction mixtures without template-primer. Although the amount of this incorporation corresponded to about one-twentieth of the non-complementary nucleotide incorporation observed in the complete reaction, it was near the background level

24 24 24

DNP-a DNP-a DNP-a * DNP-a, DNA polymerase a.

4 4 4

Age (months)

DNP-a* DNP-a DNP-a

DNA polymerase

1 2 3

1 2 3

Exp no.

poly (dG-dC) poly (dG-dC) poly (dG-dC)

poly (dG-dC) poly (dG-dC) poly (dG-dC)

Templateprimer

280.548 295.645 338.079

307.779 376.331 318.510

Correct nucleotide incorporated (pmol) [3H]dGMP

0.054 0.047 0.053

0.000 0.000 0.000

Incorrect nucleotide incorporated (pmol) [a-32P]dTMP

1/10 000 1/12 580 1/12 760

B1/600 000 B1/750 000 B1/630 000

Level of fidelity

Table 1 Fidelity levels of DNA polymerase a from regenerating livers of 4- and 24-month-old rats. The experimental procedures are those described in Section 2

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( B 0.002 pmol). This amount could be neglected even if DNA fragments contaminated the fraction. DNA polymerase fidelity is very difficult to determine on extremely small amounts of materials. We observed the presence of nucleotides mis-incorporated by DNA polymerase in the newly synthesized DNA product. All of the sequence of poly (dG-dC) forms a site for restriction endonuclease HhaI which recognizes 5%-GCGC, cutting to GCG and C as indicated in Fig. 1A. It is estimated that the complete digestion products of poly (dG-dC) by HhaI are CG and CGCG. If dTMP is incorporated into poly (dG-dC), then the oligomers CTCGCG, CGTGCG, CGCTCGCG, and CGCGTGCG, in addition to CG and CGCG, will be produced by HhaI digestion (Fig. 1B and C). These oligonucleotides can easily be separated on PEI – cellulose plates by thin layer chromatography. On the other hand, to determine the sequence of oligonucleotides containing [32P]dTMP, similar oligonucleotides with known sequences and mononucleotides as markers are mapped on plates as shown in Fig. 2. DNA synthesis was performed in a reaction mixture containing [32P]dTMP and poly (dG-dC) by DNA polymerase a (the same enzyme used in experiment 1 in Table 1) from an aged rat. The DNA was extracted and purified by phenol

Fig. 1. HhaI digestion and digestion products. (A) is HhaI digests of poly (dG-dC). (B) and (C) are digests of poly (dG-dC) containing dTMP.

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Fig. 2. Identification of spots corresponding to deoxynucleoside mono-, di-, and tri-phosphates and oligo (dG-dC)n on a PEI–cellulose plate. Detection of each spot is described in Section 2. The three crosses indicate the origins. Two are the origins for the external marker d(pG)2 in the first and second dimension.

extraction and ethanol precipitation; finally, the DNA was thoroughly digested by HhaI. A 2 ml sample was applied to the origin of a PEI–cellulose plate, developed in two dimensions, and autoradiographed. Four faint spots were observed on the autoradiogram (Fig. 3). It is estimated that these spots represent CTCGCG, CGTGCG, CGCTCGCG, and CGCGTGCG based on the results shown in Fig. 2. However, none of these spots was observed in a sample prepared by the same methods after DNA synthesis by DNA polymerase a from a young rat. Based on the observation of spots containing dTMP, it is suggested that the presence of G-T and C-T mispairs occurs during in vitro DNA synthesis on poly (dG-dC) by DNA polymerase a from aged rats. The amounts of G-T and C-T mispairs were similar. These results were supported by nearest neighbor analysis (Fig. 4). [32P] from dTMP was mis-incorporated to deoxyguanosine and deoxycytidine in similar amounts by this analysis. This shows that error-prone DNA polymerase a mis-incorporates equally at the both dGMP and dCMP in the template.

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4. Discussion The partially purified DNA polymerase used in the above experiments was inhibited by aphidicolin. This shows that the DNA polymerase was a or d type [35,39]. DNA polymerase d was found by Byrnes et al., but is not separable from the 3%“ 5% exonuclease activity [40]. Thus, it was shown that the active site of this exonuclease is present in the DNA polymerase molecule itself [41] and this enzyme performs proofreading during DNA synthesis [42]. In our experiment, however, there was no measurable nuclease activity in the final DNA polymerase preparation. Therefore, the DNA polymerase used in this experiment was a type. In these experiments, the presence of altered DNA polymerase a characterized by low fidelity was observed in regenerating liver from 24-month-old rats. On the other

Fig. 3. Analysis of GC oligomers containing dTMP. DNA synthesis was performed using DNA polymerase a from a 24-month-old rat on a GC copolymer in a reaction mixture containing [a32 P]dTMP. The DNA in the reaction mixture was purified by phenol extraction and ethanol precipitation, then hydrolyzed by HhaI restriction endonuclease. The digested products were applied to a PEI–cellulose plate and developed in two dimensions for analysis of the oligomers. Details are described in Section 2.

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Fig. 4. DNA synthesis and purification were the same as for Fig. 3. DNA digestion was performed by micrococcal endonuclease and bovine spleen phosphodiesterase. The labeled mononucleotide products and 4 known deoxynucleoside monophosphates were analyzed by PEI – cellulose thin layer chromatography and spots were detected by autoradiography or UV shadowing. Details are described in Section 2.

hand, error-prone DNA polymerase a was not found in regenerating liver from 4-month-old rats (Table 1). These experiments were carefully carried out in order to avoid the effects of terminal transferase and endogenous DNA contamination in the enzyme preparations. No non-complementary nucleotide incorporation due to terminal transferase was measured in any DNA polymerase preparation, but very small amounts of non-complementary nucleotide incorporation (about 0.002 pmol) were observed using reaction mixtures without poly (dG-dC) for some DNA polymerase preparations despite the removal of DNA by polyethyleneglycol precipitation. However, the values were very small and negligible. These results suggest that because of error-prone DNA polymerase in aged animals, incorrect nucleotides are incorporated into synthesizing DNA strands during the elongation process. Decreased fidelities of DNA polymerase have been reported in tissue samples from aged animals [29,30,43,44] and senescent cultured cells [25–28]. However,

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there are some reports that no age-dependent differences are found in the fidelity of DNA polymerase [31 – 33,45]. In these reports, the fidelity levels of DNA polymerases from young as well as aged animals were rather low, and this might account for the discrepancies. In all cases, the measurement of fidelity is very difficult. We examined the concentrated oligonucleotide products containing mis-incorporated nucleotides and compared the products originating from polynucleotides synthesized by DNA polymerase from young and old rats. When poly (dG-dC) is completely digested by HhaI [46], the digestion products are CG and CGCG because all of poly (dG-dC) is a cleavage site for HhaI restriction endonuclease (Fig. 1A). If dTMP is mis-incorporated into poly (dG-dC), then the HhaI digestion products expected are CTCGCG, CGTGCG, CGCTCGCG, and CGCGTGCG in addition to CG and CGCG (Fig. 1B and C). These oligomers containing dTMP can be concentrated and separated on thin layer plates. Using [32P] labeled dTTP, these spots can be observed by autoradiograpy. The results of the experiment performed under this scheme are shown in Fig. 3. Very faint spots were found when DNA synthesis was carried out using DNA polmerase from the regenerating liver of old rats, and their sequences were determined based on the results of related known oligomers as shown in Fig. 2. The sequence of each spot is indicated in Fig. 3 and were found to be the expected sequences. The rather dark spot at the origin is due to incompletely digested long oligomers. None of these faint or dark spots were observed in the case of enzyme from young rats (data not shown). This suggests that the fidelity of DNA polymerase from young rats is very high. We were concerned about the problem of whether the mis-incorporation depends upon the base in the template-primer. Nearest neighbor analysis showed no difference between guanine and cytosine in the template strand in terms of incorrect nucleotide incorporation by the enzyme from aged rats (Fig. 4). Furthermore, our preliminary data showed that DNA polymerase from aged rats mis-incorporates dAMP at approximately equal levels on poly (dG-dC), and that incorrect nucleotide incorporations are also found on poly (dA-dT), poly dA-dT10, and poly dC-dG10 (data not shown). These findings indicate that the incorporation of non-complementary nucleotides into newly synthesized polynucleotide strands occurs randomly and is not restricted to a specific purine or pyrimidine base; furthermore, mutations of the transition and transversion types are introduced by altered DNA polymerases in regenerating liver cells of aged rats. We have no evidence related to the occurrence of altered DNA polymerase during the aging process. One proposed possibility is that the mutation in the DNA polymerase gene may be caused by DNA damage such as oxidation and alkylation. In fact, DNA polymerase mutants have been found in phages [5,47–49], viruses [50], bacteria [51 – 53], and mammals [54]. If non-complementary nucleotides are inserted in the newly synthesized DNA molecules and are not detected by the mismatch repair system, then these regions may act as sites of gene mutation in daughter cells. These mutant cells containing incorrect nucleotide sequences in their cistrons may produce altered proteins. However, we believe that the sites of mutation by error-prone DNA polymerases

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are random and the characteristics of the mutant cells differ from one another. If our supposition is correct, the amount of each altered protein will be very small compared with the corresponding normal protein. Therefore, it may be difficult to detect each altered protein as previously described ([18–21]). On the other hand, the expression of the DNA polymerase a gene is greatly amplified in proliferating cells such as regenerating liver cells. Therefore, the altered DNA polymerase a, which is characterized by its low fidelity, is easy to detect even if the ratio of normal to altered DNA polymerase a molecules is not change. These random mutations may lead to decreases in some cellular functions or to cell death by alterations of essential proteins. Large decreases in cell numbers in tissues means that the tissues can not maintain their primary functions. Decreases in cell number have been observed in most tissues in aged animals. If an important characteristic of the aging phenomenon is a decline in various functions, then mutations caused by mis-reading by DNA polymerase may be an essential factor in the aging process.

Acknowledgements The authors would like to express their gratitude to Dr Tetsuo Ono and Dr Masami Muramatsu for valuable suggestions and discussion and Dr Margaret Dooley-Ohto for her assistance with the manuscript. This work was supported in part by a Grant-in-Aid for Cancer Research from the Ministry of Education, Science and Culture, Japan, and in part by a project grant (for research on Parameters of Biomedical Aging) from the Institute of Physical and Chemical Research, Japan.

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