Reduction of DNA synthesis in aging but still proliferating cells

It is well known that cell proliferation (and hence, DNA synthesis) declines in human diploid fibroblast-like cells with increasing passage number. It is not clear whether DNA synthesis declines in the remaining cells that are still actively proliferating. Estimations of cell kinetic parameters permitted extrapolations to be made that reflected the declining numbers of cells still capable of DNA replication. DNA synthesis declined with culture age in intact cells, permeabilized cells, and in the isolated nuclear matrix even when corrected for declining numbers of proliferating cells. With age, DNA polymerase alpha and beta activity in cell lysates declined, but when corrected for the remaining proliferating cells, only polymerase alpha activity declined; DNA polymerase alpha and beta activity bound to the nuclear matrix declined, but when corrected for declining proliferation, no decline was apparent for either enzyme. There was an increase in the number of S1-nuclease sensitive sites and breaks in the parental DNA of the dividing cells in older cultures. It is suggested that in aging cultures, not only does overall DNA synthesis decline owing to decreasing cell proliferation, but also that DNA synthesis declines in the remaining proliferating cells, that this decline is not due to decreasing amounts of DNA polymerase bound to the nuclear matrix, and that alterations in DNA structure occur.     

Repair of benzo[a]pyrene-initiated DNA damage in human cells requires activation of DNA polymerase alpha

Normal human fibroblasts treated with r-7,t-8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) yielded DNA polymerase alpha with elevated levels of activity, incorporated [3H]thymidine as a function of unscheduled DNA synthesis, and exhibited restoration of normal DNA-strand length as a function of unscheduled DNA synthesis. Lipoprotein-deficient fibroblasts treated with BPDE did not show elevated levels of DNA polymerase alpha activity, exhibited minimal [3H]thymidine incorporation, and had fragmented DNA after 24 h of repair in the absence of lipoprotein or phosphatidylinositol supplementation. When DNA polymerase beta activity was inhibited, cells with normal lipoprotein uptake exhibited [3H]thymidine incorporation into BPDE-damaged DNA but did not show an increase in DNA-strand length. DNA polymerase alpha activity and [3H]thymidine incorporation in lipoprotein-deficient fibroblasts increased to normal levels when the cells were permeabilized and low-density lipoproteins or phosphatidylinositol were introduced into the cells. DNA polymerase alpha isolated from normal human fibroblasts, but not from lipoprotein-deficient fibroblasts, showed increased specific activity after the cells were treated with BPDE. When BPDE-treated lipoprotein-deficient fibroblasts were permeabilized and 32P-ATP was introduced into the cells along with lipoproteins, 32P-labeled DNA polymerase alpha with significantly increased specific activity was isolated from the cells. These data suggest that treatment of human fibroblasts with BPDE initiates unscheduled DNA synthesis, as a function of DNA excision repair, which is correlated with increased activity of DNA polymerase alpha, and that increased DNA polymerase alpha activity may be correlated with phosphorylation of the enzyme in a reaction that is stimulated by low-density lipoprotein or by the lipoprotein component, phosphatidylinositol.     

The roles of DNA polymerases alpha, beta, and gamma in DNA repair synthesis induced in hamster and human cells by different DNA damaging agents

The involvement of DNA polymerases alpha, beta, and gamma in DNA repair synthesis was investigated in subcellular preparations of cultured hamster and human cells. A variety of DNA damaging agents, including bleomycin, neocarzinostatin, UV irradiation, and alkylating agents, were utilized to induce DNA repair. The sensitivity of repair synthesis, as well as replicative synthesis and purified DNA polymerase beta activity, to inhibition by the DNA polymerase inhibitors dideoxythymidine triphosphate, aphidicolin, cytosine arabinoside triphosphate, and N-ethylmaleimide was determined. No evidence was obtained for a major role of polymerase gamma in any type of repair synthesis. In both hamster and human cells, the sensitivity of bleomycin- and neocarzinostatin-induced repair synthesis to ddTTP inhibition was essentially identical with that observed for purified polymerase beta, indicating these repair processes proceeded through a mechanism utilizing polymerase beta. Repair synthesis induced by UV irradiation and alkylating agents was not sensitive to ddTTP, indicating repair of these lesions occurred through a pathway primarily utilizing a different DNA polymerase; presumably polymerase alpha. However, replicative synthesis was much more sensitive to polymerase alpha inhibitors than was repair synthesis induced by UV irradiation or alkylating agents. Neither the amount of DNA damage nor the amount of induced repair synthesis influenced the degree to which the different DNA polymerases were involved in repair synthesis. The possibility that "patch size" or the actual type of DNA damage determines the extent to which different polymerases participate in DNA repair synthesis is discussed.     

Induction of alpha type DNA polymerases in human cytomegalovirus-infected WI-38 cells.

Productive infection of WI-38 cells with human cytomegalovirus (HCMV) induced the increase in the activity of DNA polymerases as well as the synthesis of viral and cellular DNA. Sedimentation analyses in sucrose gradients of high ionic strength showed that the HCMV infection caused marked increase in the activity of alpha-type polymerases (resolved into alpha1, 8 S, and alpha 2, 6 S, in the present experiments), while the infection little affected the level of beta-type polymerase (about 3.5 S) activity in both the nuclei and cytoplasm. Such increase in alpha-type polymerases was also observed when DNA synthesis in WI-38 cells was enhanced by SV40 infection or by an increased concentration of serum in medium. Phosphonacetate, which selectively blocked the synthesis of HCMV DNA, did not significantly affect the HCMV-mediated induction of DNA polymerases. However, phosphonoacetate added in the reaction mixture for DNA polymerase assay inhibited the activity of the HCMV-induced polyperase alpha, but not of the polymerases alpha2 and beta. These results support the idea that alpha-type polymerases are involved in the replicative synthesis of cellular and viral DNA.     

Effect of the antitumor antibiotic bleomycin on DNA polymerase activity

Treatment with bleomycin activates considerably a repair synthesis of DNA in rat liver chromatin in vitro and can cause loosening of the nucleoprotein complex, which facilitates the accessibility or repair enzymes for lesions in chromatin DNA. The bleomycin action on DNA-template increases severalfold the rate of synthesis catalyzed by DNA polymerase beta inhibits the activity of DNA polymerase I from Escherichia coli and suppresses severalfold the activity of DNA polymerase alpha and DNA polymerase of bacteriophage T4. The effect of bleomycin consists in a prevailing increase of nicks and minimal gaps in DNA as compared to the rise of moderate gaps, thus suggesting that bleomycin is a gamma-mimetic.     

Microtubule-associated protein-2 stimulates DNA synthesis catalyzed by the nuclear matrix

Microtubule-associated protein-2 (MAP-2) isolated from porcine brains stimulated DNA synthesis catalyzed by the nuclear matrix isolated from Physarum polycephalum in the presence of activated DNA as exogenous templates. The degree of the stimulation depended on the amount of the nuclear matrix, but not on that of the template. MAP-2 also stimulated DNA polymerase alpha activity solubilized from nuclei, but not DNA polymerase beta activity. These results suggest that MAP-2 stimulates DNA synthesis by interacting with the putative DNA replication machinery including DNA polymerase alpha bound to the matrix. Similar stimulation occurred in the nuclear matrix isolated from HeLa and rat ascites hepatoma cells, which strongly suggests that MAP-2 is involved in the control of DNA replication in eukaryotic cells.     

The interrelation between DNA synthesis rates and DNA polymerases bound to the nuclear matrix in synchronized HeLa cells

Quantitative rates of DNA synthesis can be determined by DNA:propidium fluorescence measurements of synchronized cells progressing through S-phase. We have previously reported that HeLa cells have discontinuous rates with values of about 2.9, 1.6, and 4.4 pg of DNA/h for early, middle, and late S-phase, respectively. In attempts to understand why two peaks of DNA synthesis rates are observed, we have examined the nuclear DNA polymerases alpha and beta over the S-phase. Nuclear matrices isolated from HeLa cells contained 2% of the alpha polymerase and 12% of the beta polymerase that was present in cell lysates, and about 2% of the original DNA. The amounts of endogenous DNA synthesis in isolated nuclear matrices were comparable to the amounts observed when exogenous DNA was added. DNase treatment abolished the endogenous DNA synthesis but not the exogenous DNA synthesis, suggesting that polymerase alpha binding does not depend on matrix-bound DNA. As synchronized cells progressed through the S-phase, there appeared two peaks of enzymatic activity of alpha polymerase bound to the nuclear matrix which correlated with in vitro DNA synthesis in these nuclear matrices and with the two peaks of quantitative DNA synthesis rates. Two peaks of alpha polymerase activity were also observed with isolated nuclei, but not with cell lysates or cytosol. Our results suggest that, over the S-phase, the differential binding of polymerase alpha to the nuclear matrix determines the differential rates of DNA synthesis.     

Exogenous 8-oxo-dG is not utilized for nucleotide synthesis but enhances the accumulation of 8-oxo-Gua in DNA through error-prone DNA synthesis

7,8-Dihydro-8-oxoguanine (8-oxo-Gua) and its nucleoside in cytosol are derived from the repair of oxidative DNA and the cleanup of oxidatively damaged DNA precursors, respectively. While the harmful effects of 8-oxo-Gua present in DNA have been studied extensively, few have reported its effects on cytosolic function. Our previous study showed that the addition of 8-oxo-dG to culture media caused an accumulation of 8-oxo-Gua in nuclear DNA in several leukemic cells including KG-1, which lack 8-oxoguanine glycosylase 1 (OGG1) activity due to mutational loss. However, the mechanism underlying 8-oxo-Gua level increases in DNA has not been addressed. In this study, we elucidated the metabolic fate of 8-oxo-Gua-containing nucleotide and the effect of exogenous 8-oxo-dG on DNA synthesis in KG-1 cells. We found that 8-oxo-dGMP was rapidly dephosphorylated to 8-oxo-dG rather than phosphorylated to 8-oxo-dGDP, thus indicating that 8-oxo-Gua-containing molecule is not used as a substrate for DNA synthesis in KG-1 cells. In fact, radiolabeled 8-oxo-dG was incubated but radioactivity was not detected in nuclear DNA of KG-1 cells, showing that 8-oxo-dG is not directly incorporated into DNA. Interestingly, the activity of DNA polymerase beta, which synthesize DNA with low fidelity increased in KG-1 cells treated with 8-oxo-dG, whereas the expression of DNA polymerase alpha decreased. In addition, the accumulation of 8-oxo-Gua in KG-1 DNA was completely inhibited by a specific inhibitor of DNA polymerase beta. Thus, our findings address that the insertion of 8-oxo-dG into KG-1 DNA is not due to the direct incorporation of exogenous 8-oxo-dG, but rather to the inaccurate incorporation of endogenous 8-oxo-dGTP by DNA polymerase beta. It further suggests that 8-oxo-dG in the cytosol may function as an active molecule itself and perturb the well-defined DNA synthesis.     

Rat DNA polymerase beta gene can join in excision repair of Escherichia coli.

Though DNA polymerase I (poll) of Escherichia (E.) coli is understood to play a role in repair synthesis of excision repair, it is still obscure whether DNA polymerase beta (pol beta) plays a similar role in eukaryotic cells. To estimate the role of pol beta in excision repair processes, we inserted the rat pol beta gene into several mutant E. coli defective in a diverse set of enzymatic activities of poll. UV resistance was seen only when the 5'----3' exonuclease (exo) activity of poll molecules remained. Therefore it is suggested that 5'----3' exo activity as well as pol beta activity are essential for repair synthesis of excision repair in eukaryotic cells.     

Evidence that DNA polymerases alpha and beta participate differentially in DNA repair synthesis induced by different agents

The roles of DNA polymerases alpha and beta in DNA replication and repair synthesis were studied in permeable animal cells, using different agents to induce repair synthesis. DNA polymerase inhibitors were used to investigate which polymerases were involved in repair synthesis and in replication. Polymerase alpha was responsible for replication. On the other hand, both polymerases alpha and beta were involved in DNA repair synthesis; the extent to which each polymerase participated depended primarily on the agent used to damage DNA. Polymerase beta was primarily responsible for repair synthesis induced by bleomycin or neocarzinostatin, whereas polymerase alpha played a more prominent role in repair synthesis indiced by N-methyl-N'-nitro-N-nitrosoguanidine or N-nitrosomethyl urea. More DNA damage was induced by the alkylating agents than by bleomycin or neocarzinostatin, suggesting that the extent of involvement of polymerase alpha or beta in DNA repair synthesis is related to the amount or type of DNA damage. In addition, salt concentration was found to have little or no effect on the results obtained with the DNA polymerase inhibitors. Our findings provide an explanation for conflicting reports in the literature concerning the roles of DNA polymerases alpha and beta in DNA repair.     

Modulation of the 3'-->5'-exonuclease activity of human apurinic endonuclease (Ape1) by its 5'-incised Abasic DNA product

The major abasic endonuclease of human cells, Ape1 protein, is a multifunctional enzyme with critical roles in base excision repair (BER) of DNA. In addition to its primary activity as an apurinic/apyrimidinic endonuclease in BER, Ape1 also possesses 3'-phosphodiesterase, 3'-phosphatase, and 3'-->5'-exonuclease functions specific for the 3' termini of internal nicks and gaps in DNA. The exonuclease activity is enhanced at 3' mismatches, which suggests a possible role in BER for Ape1 as a proofreading activity for the relatively inaccurate DNA polymerase beta. To elucidate this role more precisely, we investigated the ability of Ape1 to degrade DNA substrates that mimic BER intermediates. We found that the Ape1 exonuclease is active at both mismatched and correctly matched 3' termini, with preference for mismatches. In our hands, the exonuclease activity of Ape1 was more active at one-nucleotide gaps than at nicks in DNA, even though the latter should represent the product of repair synthesis by polymerase beta. However, the exonuclease activity was inhibited by the presence of nearby 5'-incised abasic residues, which result from the apurinic/apyrimidinic endonuclease activity of Ape1. The same was true for the recently described exonuclease activity of Escherichia coli endonuclease IV. Exonuclease III, the E. coli homolog of Ape1, did not discriminate among the different substrates. Removal of the 5' abasic residue by polymerase beta alleviated the inhibition of the Ape1 exonuclease activity. These results suggest roles for the Ape1 exonuclease during BER after both DNA repair synthesis and excision of the abasic deoxyribose-5-phosphate by polymerase beta.     

2',3'-Dideoxythymidine 5'-triphosphate inhibition of DNA replication and ultraviolet-induced DNA repair synthesis in human cells: evidence for involvement of DNA polymerase delta

It is well established that DNA replication and ultraviolet-induced DNA repair synthesis in mammalian cells are aphidicolin-sensitive and thus are mediated by one or both of the aphidicolin-sensitive DNA polymerases, alpha and/or delta. Recently, it has been shown that DNA polymerase delta is much more sensitive to inhibition by the nucleotide analogue 2',3'-dideoxythymidine 5'-triphosphate (ddTTP) than DNA polymerase alpha but is less sensitive than DNA polymerase beta [Wahl, A. F., Crute, J. J., Sabatino, R. D., Bodner, J. B., Marraccino, R. L., Harwell, L. W., Lord, E. M., & Bambara, R. A. (1986) Biochemistry 25, 7821-7827]. We find that DNA replication and ultraviolet-induced DNA repair synthesis in permeable human fibroblasts are also more sensitive to inhibition by ddTTP than polymerase alpha and less sensitive than polymerase beta. The Ki for ddTTP of replication is about 40 microM and that of repair synthesis is about 25 microM. These are both much less than the Ki of polymerase alpha (which is greater than 200 microM) but greater than the Ki of polymerase beta (which is less than 2 microM). These data suggest that DNA polymerase delta participates in DNA replication and ultraviolet-induced DNA repair synthesis in human cells.     

Involvement of deoxyribonucleic acid polymerase beta in nuclear deoxyribonucleic acid synthesis.

The effects on DNA synthesis in vitro in mouse L929-cell nuclei of differential extraction of DNA polymerases alpha and beta were studied. Removal of all measurable DNA polymerase alpha and 20% of DNA polymerase beta leads to a 40% fall in the replicative DNA synthesis. Removal of 70% of DNA polymerase beta inhibits replicative synthesis by 80%. In all cases the nuclear DNA synthesis is sensitive to N-ethylmaleimide and aCTP (arabinosylcytosine triphosphate), though less so than DNA polymerase alpha. Addition of deoxyribonuclease I to the nuclear incubation leads to synthesis of high-molecular-weight DNA in a repair reaction. This occurs equally in nuclei from non-growing or S-phase cells. The former nuclei lack DNA polymerase alpha and the reaction reflects the sensitivity of DNA polymerase beta to inhibiton by N-ethylmaleimide and aCTP.     

Levels of cellular DNA polymerases in synchronized bovine paravovirus-infected cells.

Infection of synchronized bovine fetal spleen cells with bovine parvovirus results in changes in the levels and patterns of DNA polymerases alpha and gamma during the cell cycle. The pattern of DNA polymerase alpha activity closely paralled viral DNA synthesis and the production of progeny virus, and levels, of this enzyme were threefold greater than in mock-infected cells during the period of maximal viral DNA synthesis. DNA polymerase gamma activity remained slightly elevated during viral DNA replication. Levels and patterns of DNA polymerase beta were similar in mock- and virus-infected cells.     

Comparative effects of the 5'-triphosphates of 9-beta-(2'-azido-2'-deoxy-D-arabinofuranosyl)adenine and 9-beta-D-arabinofuranosyladenine on DNA polymerases from L1210 leukemia cells.

9-beta-(2'-Azido-2'-deoxy-D-arabiofuranosyl)adenine (arazide) causes greater and significantly more persistent inhibition of [3H]-thymidine incorporation into the DNA of neoplastic cells than the related agent 9-beta-D-arabinofuranosyladenine (araA). To elucidate the mechanism(s) responsible, we compared the effects of arazide and araA 5'-triphosphates on DNA polymerases alpha and beta of L1210 leukemia cells. Both nucleoside triphosphate analogs inhibited DNA polymerase alpha activity by competing with dATP; only araATP was inhibitory to DNA polymerase beta. Arazide triphosphate was at least four times more active than araATP as an inhibitor of DNA polymerase alpha. Preincubation of DNA polymerase alpha with either agent did not result in enzyme inactivation. The results suggest that interference with DNA polymerase alpha activity by arazide triphosphate may be in part responsible for the inhibition of DNA synthesis produced by arazide in neoplastic cells.     

Analysis of the ATPase subassembly which initiates processive DNA synthesis by DNA polymerase III holoenzyme.

The gamma complex (gamma delta delta' chi psi) subassembly of DNA polymerase III holoenzyme transfers the beta subunit onto primed DNA in a reaction which requires ATP hydrolysis. Once on DNA, beta is a "sliding clamp" which tethers the polymerase to DNA for highly processive synthesis. We have examined beta and the gamma complex to identify which subunit(s) hydrolyzes ATP. We find the gamma complex is a DNA dependent ATPase. The beta subunit, which lacks ATPase activity, enhances the gamma complex ATPase when primed DNA is used as an effector. Hence, the gamma complex recognizes DNA and couples ATP hydrolysis to clamp beta onto primed DNA. Study of gamma complex subunits showed no single subunit contained significant ATPase activity. However, the heterodimers, gamma delta and gamma delta', were both DNA-dependent ATPases. Only the gamma delta ATPase was stimulated by beta and was functional in transferring the beta from solution to primed DNA. Similarity in ATPase activity of DNA polymerase III holoenzyme accessory proteins to accessory proteins of phage T4 DNA polymerase and mammalian DNA polymerase delta suggests the basic strategy of chromosome duplication has been conserved throughout evolution.     

Nucleotide excision repair DNA synthesis by excess DNA polymerase beta: a potential source of genetic instability in cancer cells.

The nucleotide excision repair pathway contributes to genetic stability by removing a wide range of DNA damage through an error-free reaction. When the lesion is located, the altered strand is incised on both sides of the lesion and a damaged oligonucleotide excised. A repair patch is then synthesized and the repaired strand is ligated. It is assumed that only DNA polymerases delta and/or epsilon participate to the repair DNA synthesis step. Using UV and cisplatin-modified DNA templates, we measured in vitro that extracts from cells overexpressing the error-prone DNA polymerase beta exhibited a five- to sixfold increase of the ultimate DNA synthesis activity compared with control extracts and demonstrated the specific involvement of Pol beta in this step. By using a 28 nt gapped, double-stranded DNA substrate mimicking the product of the incision step, we showed that Pol beta is able to catalyze strand displacement downstream of the gap. We discuss these data within the scope of a hypothesis previously presented proposing that excess error-prone Pol beta in cancer cells could perturb the well-defined specific functions of DNA polymerases during error-free DNA transactions.     

DNA repair synthesis in mouse spermatogenesis involves DNA polymerase beta activity

The role of DNA polymerase alpha-DNA primase complex and DNA polymerase beta in DNA replication and ultraviolet-induced DNA repair synthesis has been analyzed in mouse spermatogenesis. Autoradiographic experiments with germ cells in culture, indicating an involvement of DNA polymerase alpha and/or delta in DNA replication, and of DNA polymerase beta in DNA repair synthesis, have been confirmed by studying partially purified enzymes. These findings support the idea that, different from other biological systems, in meiotic and post meiotic male mouse germ cells DNA polymerase beta is the main DNA polymerase form needed for DNA repair.     

DNA synthesis on discontinuous templates by human DNA polymerases: implications for non-homologous DNA recombination.

DNA polymerases catalyze the synthesis of DNA using a continuous uninterrupted template strand. However, it has been shown that a 3'-->5' exonuclease-deficient form of the Klenow fragment of Escherichia coli DNA polymerase I as well as DNA polymerase of Thermus aquaticus can synthesize DNA across two unlinked DNA templates. In this study, we used an oligonucleotide-based assay to show that discontinuous DNA synthesis was present in HeLa cell extracts. DNA synthesis inhibitor studies as well as fractionation of the extracts revealed that most of the discontinuous DNA synthesis was attributable to DNA polymerase alpha. Additionally, discontinuous DNA synthesis could be eliminated by incubation with an antibody that specifically neutralized DNA polymerase alpha activity. To test the relative efficiency of each nuclear DNA polymerase for discontinuous synthesis, equal amounts (as measured by DNA polymerase activity) of DNA polymerases alpha, beta, delta (+/- PCNA) and straightepsilon (+/- PCNA) were used in the discontinuous DNA synthesis assay. DNA polymerase alpha showed the most discontinuous DNA synthesis activity, although small but detectable levels were seen for DNA polymerases delta (+PCNA) and straightepsilon (- PCNA). Klenow fragment and DNA polymerase beta showed no discontinuous DNA synthesis, although at much higher amounts of each enzyme, discontinuous synthesis was seen for both. Discontinuous DNA synthesis by DNA polymerase alpha was seen with substrates containing 3 and 4 bp single-strand stretches of complementarity; however, little synthesis was seen with blunt substrates or with 1 bp stretches. The products formed from these experiments are structurally similar to that seen in vivo for non-homologous end joining in eukaryotic cells. These data suggest that DNA polymerase alpha may be able to rejoin double-strand breaks in vivo during replication.