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.     

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.     

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.     

Effect of DNA polymerase inhibitors on DNA repair in intact and permeable human fibroblasts: evidence that DNA polymerases delta and beta are involved in DNA repair synthesis induced by N-methyl-N'-nitro-N-nitrosoguanidine

The involvement of DNA polymerases alpha, beta, and delta in DNA repair synthesis induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) was investigated in human fibroblasts (HF). The effects of anti-(DNA polymerase alpha) monoclonal antibody, (p-n-butylphenyl)deoxyguanosine triphosphate (BuPdGTP), dideoxythymidine triphosphate (ddTTP), and aphidicolin on MNNG-induced DNA repair synthesis were investigated to dissect the roles of the different DNA polymerases. A subcellular system (permeable cells), in which DNA repair synthesis and DNA replication were differentiated by CsCl gradient centrifugation of BrdUMP density-labeled DNA, was used to examine the effects of the polymerase inhibitors. Another approach investigated the effects of several of these inhibitors on MNNG-induced DNA repair synthesis in intact cells by measuring the amount of [3H]thymidine incorporated into repaired DNA as determined by autoradiography and quantitation with an automated video image analysis system. In permeable cells, MNNG-induced DNA repair synthesis was inhibited 56% by 50 micrograms of aphidicolin/mL, 6% by 10 microM BuPdGTP, 13% by anti-(DNA polymerase alpha) monoclonal antibodies, and 29% by ddTTP. In intact cells, MNNG-induced DNA repair synthesis was inhibited 57% by 50 micrograms of aphidicolin/mL and was not significantly inhibited by microinjecting anti-(DNA polymerase alpha) antibodies into HF nuclei. These results indicate that both DNA polymerases delta and beta are involved in repairing DNA damage caused by MNNG.     

The overexpression of specialized DNA polymerases in cancer

Specialized DNA polymerases are required to bypass DNA damage lesions that would otherwise cause replication arrest and cell death. When operating on non-canonical templates, such as undamaged DNA or on non-cognate lesions, these polymerases exhibit considerably reduced fidelity, resulting in the generation of mutations. Ectopic overexpression of these polymerases can also lead to an increased mutation rate and an enhanced capability of DNA repair, suggesting that they could potentially act as oncogenes if they were overexpressed in cancers. Here, we examine expression patterns of DNA polymerases in matched normal and tumor samples from a diverse range of tissues. As well as investigating the specialized polymerases beta, lambda, iota and kappa, we also investigate the expression of the replicative polymerases alpha, delta and epsilon. The data presented provide evidence for the overexpression of specialized polymerases in tumors, with more than 45% of the 68 tumor samples studied demonstrating greater than two-fold enhanced expression of at least one specialized polymerase. Of particular note, DNA polymerase beta (pol beta) was found to be overexpressed at both the mRNA and protein level in approximately one third of all tumor types studied, with overexpression being particularly frequent in uterus, ovary, prostate and stomach samples. Pols lambda, and iota were also found to be overexpressed to a significant extent in a range of tumor types, albeit less frequently than pol beta. In contrast, pol kappa was rarely found to be overexpressed in tumors but was found to be commonly underexpressed in many samples. Downregulation of pol beta expression by siRNA resulted in an increased sensitivity to the chemotherapeutic agent cisplatin, suggesting a role for this polymerase in providing tolerance to cisplatin-induced damage. These observations suggest that specialised DNA polymerases, and particularly pol beta, could be considered both as caretaker genes altered during tumorigenesis, and as potential drug targets to sensitise tumors to chemotherapy.     

Defective repair of DNA single- and double-strand breaks in the bleomycin- and X-ray-sensitive Chinese hamster ovary cell mutant, BLM-2

Using filter elution techniques, we have measured the level of induced single- and double-strand DNA breaks and the rate of strand break rejoining following exposure of two Chinese hamster ovary (CHO) cell mutants to bleomycin or neocarzinostatin. These mutants, designated BLM-1 and BLM-2, were isolated on the basis of hypersensitivity to bleomycin and are cross-sensitive to a range of other free radical-generating agents, but exhibit enhanced resistance to neocarzinostatin. A 1-h exposure to equimolar doses of bleomycin induces a similar level of DNA strand breaks in parental CHO-K1 and mutant BLM-1 cells, but a consistently higher level is accumulated by BLM-2 cells. The rate of rejoining of bleomycin-induced single- and double-strand DNA breaks is slower in BLM-2 cells than in CHO-K1 cells. BLM-1 cells show normal strand break repair kinetics. The level of single- and double-strand breaks induced by neocarzinostatin is lower in both BLM-1 and BLM-2 cells than in CHO-K1 cells. The rate of repair of neocarzinostatin-induced strand breaks is normal in BLM-1 cells but retarded somewhat in BLM-2 cells. Thus, there is a correlation between the level of drug-induced DNA damage in BLM-2 cells and the bleomycin-sensitive, neocarzinostatin resistant phenotype of this mutant. Strand breaks induced by both of these agents are also repaired with reduced efficiency by BLM-2 cells. The neocarzinostatin resistance of BLM-1 cells appears to be a consequence of a reduced accumulation of DNA damage. However, the bleomycin-sensitive phenotype of BLM-1 cells does not apparently correlate with any alteration in DNA strand break induction or repair, as analysed by filter elution techniques, suggesting an alternative mechanism of cell killing.     

Bleomycin-induced DNA repair synthesis in permeable human fibroblasts: mediation of long-patch and short-patch repair by distinct DNA polymerases

Treatment of permeable human fibroblasts with bleomycin elicits DNA repair synthesis that is only partially sensitive to aphidicolin, an inhibitor of mammalian DNA polymerases alpha and delta. Inhibition of long-patch repair synthesis by omission of the three unlabeled deoxyribonucleoside triphosphates (dNTPs) selectively eliminates the aphidicolin-sensitive component. The majority of this residual aphidicolin-resistant repair synthesis is contained in ligated patches as revealed by resistance to exonuclease III. Determination of repair patch length by bromodeoxyuridine-induced density shift under conditions where essentially all of the repair synthesis is sensitive or resistant to aphidicolin yielded values of approximately 20 and 4 nucleotides per patch, respectively. On the basis of these data and the relative sensitivity of bleomycin-induced repair synthesis to N2-(p-n-butylphenyl)-2'-deoxyguanosine 5'-triphosphate (BuPdGTP), 2',3'-dideoxythymidine 5'-triphosphate (ddTTP), and N-ethylmaleimide (NEM), long-patch repair is attributed to DNA polymerase delta and short-patch repair to DNA 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.     

DNA polymerase beta is the major dRP lyase involved in repair of oxidative base lesions in DNA by mammalian cell extracts.

The repair of oxidative base lesions in DNA is a coordinated chain of reactions that includes removal of the damaged base, incision of the phosphodiester backbone at the abasic sugar residue, incorporation of an undamaged nucleotide and sealing of the DNA strand break. Although removal of a damaged base in mammalian cells is initiated primarily by a damage-specific DNA glycosylase, several lyases and DNA polymerases may contribute to the later stages of repair. DNA polymerase beta (Pol beta) was implicated recently as the major polymerase involved in repair of oxidative base lesions; however, the identity of the lyase participating in the repair of oxidative lesions is unclear. We studied the mechanism by which mammalian cell extracts process DNA substrates containing a single 8-oxoguanine or 5,6-dihydrouracil at a defined position. We find that, when repair synthesis proceeds through a Pol beta-dependent single nucleotide replacement mechanism, the 5'-deoxyribosephosphate lyase activity of Pol beta is essential for repair of both lesions.     

Involvement of DNA polymerase alpha in host cell reactivation of UV-irradiated herpes simplex virus.

Aphidicolin is a potent inhibitor of both host cell DNA polymerase alpha and herpes simplex virus (HSV)-induced DNA polymerase but has no effect on DNA polymerases beta and gamma of host cells. By using an aphidicolin-resistant mutant (Aphr) of HSV, a possible involvement of DNA polymerase alpha in host cell reactivation of UV-damaged HSV was studied. Plaque formation by UV-irradiated Aphr was markedly inhibited by 1 microgram of aphidicolin per ml, which did not affect the plating efficiency of nonirradiated Aphr. Aphidicolin added before 12 h postinfection inhibited plaque formation by irradiated Aphr, which became aphidicolin insensitive after 36 h postinfection. The results strongly suggest that host cell DNA polymerase alpha is involved in the repair of UV-irradiated HSV DNA.     

Involvement of DNA polymerase delta in DNA repair synthesis in human fibroblasts at late times after ultraviolet irradiation

DNA repair synthesis following UV irradiation of confluent human fibroblasts has a biphasic time course with an early phase of rapid nucleotide incorporation and a late phase of much slower nucleotide incorporation. The biphasic nature of this curve suggests that two distinct DNA repair systems may be operative. Previous studies have specifically implicated DNA polymerase delta as the enzyme involved in DNA repair synthesis occurring immediately after UV damage. In this paper, we describe studies of DNA polymerase involvement in DNA repair synthesis in confluent human fibroblasts at late times after UV irradiation. Late UV-induced DNA repair synthesis in both intact and permeable cells was found to be inhibited by aphidicolin, indicating the involvement of one of the aphidicolin-sensitive DNA polymerases, alpha or delta. In permeable cells, the process was further analyzed by using the nucleotide analogue (butylphenyl)-2'-deoxyguanosine 5'-triphosphate, which inhibits DNA polymerase alpha several hundred times more strongly than it inhibits DNA polymerase delta. The (butylphenyl)-2'-deoxyguanosine 5'-triphosphate inhibition curve for late UV-induced repair synthesis was very similar to that for polymerase delta. It appears that repair synthesis at late times after UV irradiation, like repair synthesis at early times, is mediated by DNA polymerase delta.     

The response of normal and ataxia-telangiectasia cells to bleomycin: relationships between chromosome damage, cell cycle delay and cell killing

In agreement with our earlier observation (Scott and Zampetti-Bosseler, 1982) on X-irradiated normal and ataxia-telangiectasia (A-T) fibroblasts, we now report that after bleomycin or neocarzinostatin treatment also, A-T cells exhibit less G2 delay than normal cells. We confirm that A-T cells sustain more chromosome damage and lethality than normal cells after bleomycin. These observations support the hypothesis (Painter and Young, 1980) that A-T cells are defective in the recognition of certain lesions which normally lead to delays in progression through the cell cycle, during which they are repaired, and which, if unrepaired, lead to cell-lethal chromosome damage. However, we find that after bleomycin, as opposed to X-rays, the contribution of this type of lesion to cell death is minimal. The predominant lesions leading to cell death after bleomycin are not manifested at chromosome aberrations and do not lead to G2 delay or DNA-synthesis inhibition. A-T cells are defective in the recognition and/or repair of both types of lesion.     

DNA Polymerases β and λ Mediate Overlapping and Independent Roles in Base Excision Repair in Mouse Embryonic Fibroblasts

Base excision repair (BER) is a DNA repair pathway designed to correct small base lesions in genomic DNA. While DNA polymerase beta (pol β) is known to be the main polymerase in the BER pathway, various studies have implicated other DNA polymerases in back-up roles. One such polymerase, DNA polymerase lambda (pol λ), was shown to be important in BER of oxidative DNA damage. To further explore roles of the X-family DNA polymerases λ and β in BER, we prepared a mouse embryonic fibroblast cell line with deletions in the genes for both pol β and pol λ. Neutral red viability assays demonstrated that pol λ and pol β double null cells were hypersensitive to alkylating and oxidizing DNA damaging agents. In vitro BER assays revealed a modest contribution of pol λ to single-nucleotide BER of base lesions. Additionally, using co-immunoprecipitation experiments with purified enzymes and whole cell extracts, we found that both pol λ and pol β interact with the upstream DNA glycosylases for repair of alkylated and oxidized DNA bases. Such interactions could be important in coordinating roles of these polymerases during BER.     

DNA polymerases required for repair of UV-induced damage in Saccharomyces cerevisiae.

The ability of yeast DNA polymerase mutant strains to carry out repair synthesis after UV irradiation was studied by analysis of postirradiation molecular weight changes in cellular DNA. Neither DNA polymerase alpha, delta, epsilon, nor Rev3 single mutants evidenced a defect in repair. A mutant defective in all four of these DNA polymerases, however, showed accumulation of single-strand breaks, indicating defective repair. Pairwise combination of polymerase mutations revealed a repair defect only in DNA polymerase delta and epsilon double mutants. The extent of repair in the double mutant was no greater than that in the quadruple mutant, suggesting that DNA polymerases alpha and Rev3p play very minor, if any, roles. Taken together, the data suggest that DNA polymerases delta and epsilon are both potentially able to perform repair synthesis and that in the absence of one, the other can efficiently substitute. Thus, two of the DNA polymerases involved in DNA replication are also involved in DNA repair, adding to the accumulating evidence that the two processes are coupled.     

Interplay between DNA polymerases beta and lambda in repair of oxidation DNA damage in chicken DT40 cells

DNA polymerase lambda (Pol lambda) is a DNA polymerase beta (Pol beta)-like enzyme with both DNA synthetic and 5'-deoxyribose-5'-phosphate lyase domains. Recent biochemical studies implicated Pol lambda as a backup enzyme to Pol beta in the mammalian base excision repair (BER) pathway. To examine the interrelationship between Pol lambda and Pol beta in BER of DNA damage in living cells, we disrupted the genes for both enzymes either singly or in combination in the chicken DT40 cell line and then characterized BER phenotypes. Disruption of the genes for both polymerases caused hypersensitivity to H(2)O(2)-induced cytotoxicity, whereas the effect of disruption of either polymerase alone was only modest. Similarly, BER capacity in cells after H(2)O(2) exposure was lower in Pol beta(-/-)/Pol lambda(-/-) cells than in Pol beta(-/-), wild-type, and Pol lambda(-/-) cells, which were equivalent. These results suggest that these polymerases can complement for one another in counteracting oxidative DNA damage. Similar results were obtained in assays for in vitro BER capacity using cell extracts. With MMS-induced cytotoxicity, there was no significant effect on either survival or BER capacity from Pol lambda gene disruption. A strong hypersensitivity and reduction in BER capacity was observed for Pol beta(-/-)/Pol lambda(-/-) and Pol beta(-/-) cells, suggesting that Pol beta had a dominant role in counteracting alkylation DNA damage in this cell system.     

The repair of bleomycin-induced DNA damage and its relationship to chromosome aberration repair.

Previous studies using the technique of premature chromosome condensation indicated that nearly one-half of the bleomycin-induced chromatid breaks and gaps in CHO cells could be repaired within 1 h (repair starting at 30 min) after treatment. Cycloheximide and streptovitacin A (but not hydroxyurea or hycanthone) inhibited chromosome repair. The purpose of this study was to measure the kinetics of DNA repair after bleomycin treatment using the alkaline elution technique and to determine whether various inhibitors could block this repair. After bleomycin treatment, the major proportion of the repair of DNA damage occurred within 15 min, with significant repair evident by 2 min. This fast repair component was inhibited by 0.2% EDTA. A slower repair component was observed to occur up to 60 min after bleomycin treatment. None of the inhibitors tested were found to have a significant effect on the repair of bleomycin damage at the DNA level. Since chromosome breaks were observed not to begin repair until after 30 min while over 50% of the DNA was repaired by 15 min, these results suggest that the DNA lesions that are repaired quickly are not important in the formation of chromosome aberrations. Further, since cycloheximide and streptovitacin A blocked chromosome repair but had little measurable effect on DNA repair, these results suggest that the DNA lesions responsible for chromosome damage represent only a small proportion of the total DNA lesions produced by bleomycin.     

Structure of repaired sites in human DNA synthesized in the presence of inhibitors of DNA polymerases alpha and beta in human fibroblasts

Excision repair of ultraviolet damage in human fibroblasts was partially inhibited by drugs that block DNA polymerases alpha or beta (cytosine arabinoside, aphidicolin and dideoxythymidine) causing a reduction in unscheduled synthesis and an accumulation of single-strand breaks. The strand breaks accumulated in the presence of aphidicolin could be resealed within 30 min after removal of the drug, but those accumulated by cytosine arabinoside took many hours. Digestion of repaired DNA with exonuclease III or S1 nuclease revealed that even the highest concentration of polymerase inhibitors, singly or in combination, that produced maximal accumulation of single-strand breaks only blocked 37-86% of repair sites. Use of single-strand break frequencies to measure the number of repair events can therefore be in error by as much as a factor of 3. The blocked patches with free 3'OH termini were, on average, 22% of normal length, corresponding to between 6 and 17 bases (assuming a normal patch of 25-75 bases in length). Patches that remained unsealed in vivo were also resistant to sealing by T4 ligase in vitro. The data are more consistent with a mechanism of repair in which long single-strand gaps are first made by excision enzymes and subsequently filled in by DNA polymerase alpha. Strand displacement or nick translation mechanisms seem unlikely.     

Altered spectra of hypermutation in DNA repair-deficient mice

Affinity maturation of the humoral immune response is based on the ability of immunoglobulin variable genes to undergo a process of rapid and extensive somatic mutation followed by antigenic selection for antibodies with higher affinity. While the behaviour of this somatic hypermutation phenomenon has been well characterized over the last 20 years, the molecular mechanism responsible for inserting mutations has remained shrouded. To better understand this mechanism, we studied the interplay between hypermutation and other DNA associated activities such as DNA repair. There was no effect on the frequency and pattern of hypermutation in mice deficient for nucleotide excision repair, base excision repair and ataxia-telangiectasia mutated gene repair of double strand breaks. However, variable genes from mice lacking some components of mismatch repair had an increased frequency of tandem mutations and had more mutations of G and C nucleotides. These results suggest that the DNA polymerase(s) involved in the hypermutation pathway produces a unique spectra of mutations, which is then altered by mismatch repair and antigenic selection. We, also describe the differential pattern of expression of some nuclear DNA polymerases in hypermutating versus non-hypermutating B lymphocytes. The rapidly dividing germinal centre B cells expressed DNA polymerases alpha, beta, delta, epsilon and zeta, whereas the resting non-germinal centre cells did not express polymerases alpha or epsilon at detectable levels, although they did express polymerases beta, delta and zeta. The lack of expression of polymerase epsilon in the non-germinal centre cells suggests that this enzyme has a critical role in chromosomal replication but does not participate in DNA repair in these cells.