Protection against methylation-induced cytotoxicity by DNA polymerase beta-dependent long patch base excision repair

Using a plasmid-based uracil-containing DNA substrate, we found that the long patch base excision repair (BER) activity of a wild-type mouse fibroblast extract was partially inhibited by an antibody to DNA polymerase beta (beta-pol). This suggests that beta-pol participates in long patch BER, in addition to single-nucleotide BER. In single-nucleotide BER, the deoxyribose phosphate (dRP) in the abasic site is removed by the lyase activity of beta-pol. Methoxyamine (MX) can react with the aldehyde of an abasic site, making it refractory to the beta-elimination step of the dRP lyase mechanism, thus blocking single-nucleotide BER. MX exposure sensitizes wild-type, but not beta-pol null mouse embryonic fibroblasts, to the cytotoxic effects of methyl methanesulfonate (MMS) and methylnitrosourea. Expression of beta-pol in the null cells restores the ability of MX to modulate sensitivity to MMS. The beta-pol null cells are known to be hypersensitive to MMS and methylnitrosourea, and in the presence of MX (i.e. under conditions where single-nucleotide BER is blocked) the null cells are still considerably more sensitive than wild-type. The data are consistent with a role of beta-pol in long patch BER, which helps protect cells against methylation damage-induced cytotoxicity.     

Involvement of DNA polymerase beta in protection against the cytotoxicity of oxidative DNA damage

We had shown previously that DNA polymerase beta (beta-pol) null mouse fibroblasts, deficient in base excision repair (BER), are hypersensitive to monofunctional methylating agents but not to hydrogen peroxide (H2O2). This is surprising because beta-pol is thought to be involved in BER of oxidative as well as methylated DNA damage. We confirm these findings here in early-passage cells. However, with time in culture, beta-pol null cells become hypersensitive to H2O2 and other reactive oxygen species-generating agents. Analysis of in vitro BER reveals a strong deficiency in single-nucleotide BER of 8-oxoguanine (8-oxoG) by both early- and late-passage beta-pol null cell extracts. Therefore, in early-passage wild-type and beta-pol null cells, the capacity for single-nucleotide BER of 8-oxoG does not correlate with cellular sensitivity to H2O2. Expression of beta-pol protein in the late-passage null cells almost completely reverses the H2O2-hypersensitivity phenotype. Methoxyamine (MX) treatment sensitizes late-passage wild-type cells to H2O2 as expected for beta-pol-mediated single-nucleotide BER; however in beta-pol null cells, MX has no effect. The data indicate a role(s) of beta-pol-dependent repair in protection against the cytotoxicity of oxidative DNA damage in wild-type cells.     

Hypersensitivity of DNA polymerase beta null mouse fibroblasts reflects accumulation of cytotoxic repair intermediates from site-specific alkyl DNA lesions

Monofunctional alkylating agents react with DNA by S(N)1 or S(N)2 mechanisms resulting in formation of a wide spectrum of cytotoxic base adducts. DNA polymerase beta (beta-pol) is required for efficient base excision repair of N-alkyl adducts, and we make use of the hypersensitivity of beta-pol null mouse fibroblasts to investigate such alkylating agents with a view towards understanding the DNA lesions responsible for the cellular phenotype. The inability of O(6)-benzylguanine to sensitize wild-type or beta-pol null cells to S(N)1-type methylating agents indicates that the observed hypersensitivity is not due to differential repair of cytotoxic O-alkyl adducts. Using a 3-methyladenine-specific agent and an inhibitor of such methylation, we find that inefficient repair of 3-methyladenine is not the reason for the hypersensitivity of beta-pol null cells to methylating agents, and further that 3-methyladenine is not the adduct primarily responsible for methyl methanesulfonate (MMS)- and methyl nitrosourea-induced cytotoxicity in wild-type cells. Relating the expected spectrum of DNA adducts and the relative sensitivity of cells to monofunctional alkylating agents, we propose that the hypersensitivity of beta-pol null cells reflects accumulation of cytotoxic repair intermediates, such as the 5'-deoxyribose phosphate group, following removal of 7-alkylguanine from DNA. In support of this conclusion, beta-pol null cells are also hypersensitive to the thymidine analog 5-hydroxymethyl-2'-deoxyuridine (hmdUrd). This agent is incorporated into cellular DNA and elicits cytotoxicity only when removed by glycosylase-initiated base excision repair. Consistent with the hypothesis that there is a common repair intermediate resulting in cytotoxicity following treatment with both types of agents, both MMS and hmdUrd-initiated cell death are preceded by a similar rapid concentration-dependent suppression of DNA synthesis and a later cell cycle arrest in G(0)/G(1) and G(2)M phases.     

Strategic down-regulation of DNA polymerase beta by antisense RNA sensitizes mammalian cells to specific DNA damaging agents.

Previously, mouse NIH 3T3 cells were stably transfected with human DNA polymerase beta (beta-pol) cDNA in the antisense orientation and under the control of a metallothionein promoter [Zmudzka, B.Z. and Wilson, S.H. (1990) Som. Cell Mol. Gen., 16, 311-320]. To assess the feasibility of enhancing the efficacy of chemotherapy by an antisense approach and to confirm a role for beta-pol in cellular DNA repair, we looked for increased sensitivity to DNA damaging agents under conditions where beta-pol is down-regulated in the antisense cell line. Such a sensitization is anticipated only where beta-pol is rate-limiting in a DNA repair pathway. A number of agents were tested: cis-diamminedichloroplatinum II (cisplatin); 1,3-bis(2-chloroethyl)-1- nitrosourea (BCNU); ionizing radiation and the radio-mimetic drug bleomycin; the bifunctional alkylating agents nitrogen mustard and L-phenylalanine mustard (melphalan); the monofunctional alkylating agent methyl methane sulfonate (MMS) and ultraviolet (UV) radiation. In the cases of cisplatin and UV radiation, a significant enhancement of cytotoxicity was observed. Damage as a result of both of these agents is thought to be repaired by the nucleotide excision repair (NER) pathway. The results suggest that, in this cell line, beta-pol is involved in and is rate-limiting in NER. We propose that down-regulation of beta-pol by antisense approaches might be used to enhance the cytotoxic effects of cisplatin and other DNA damaging chemotherapeutic agents.     

Base excision repair intermediates induce p53-independent cytotoxic and genotoxic responses

DNA alkylation damage is primarily repaired by the base excision repair (BER) machinery in mammalian cells. In repair of the N-alkylated purine base lesion, for example, alkyl adenine DNA glycosylase (Aag) recognizes and removes the base, and DNA polymerase beta (beta-pol) contributes the gap tailoring and DNA synthesis steps. It is the loss of beta-pol-mediated 5'-deoxyribose phosphate removal that renders mouse fibroblasts alkylation-hypersensitive. Here we report that the hypersensitivity of beta-pol-deficient cells after methyl methanesulfonate-induced alkylation damage is wholly dependent upon glycosylase-mediated initiation of repair, indicating that alkylated base lesions themselves are tolerated in these cells and demonstrate that beta-pol protects against accumulation of toxic BER intermediates. Further, we find that these intermediates are initially tolerated in vivo by a second repair pathway, homologous recombination, inducing an increase in sister chromatid exchange events. If left unresolved, these BER intermediates trigger a rapid block in DNA synthesis and cytotoxicity. Surprisingly, both the cytotoxic and genotoxic signals are independent of both the p53 response and mismatch DNA repair pathways, demonstrating that p53 is not required for a functional BER pathway, that the observed damage response is not part of the p53 response network, and that the BER intermediate-induced cytotoxic and genotoxic effects are distinct from the mechanism engaged in response to mismatch repair signaling. These studies demonstrate that, although base damage is repaired by the BER pathway, incomplete BER intermediates are shuttled into the homologous recombination pathway, suggesting possible coordination between BER and the recombination machinery.     

The accumulation of MMS-induced single strand breaks in G1 phase is recombinogenic in DNA polymerase beta defective mammalian cells

DNA polymerase (Pol) β null mouse embryonic fibroblasts provide a useful cell system to investigate the effects of alterations in base excision repair (BER) on genome stability. These cells are characterized by hypersensitivity to the cytotoxic effects of methyl methanesulfonate (MMS) and by decreased repair of the MMS-induced DNA single strand breaks (SSB). Here, we show that, in the absence of Pol β, SSB accumulate in G₁ phase cells, accompanied by the formation of proliferating cell nuclear antigen foci in the nuclei. When replicating Pol β null cells are treated with MMS, a rapid phosphorylation of histone H2AX is detected in the nuclei of S phase cells, indicating that double strand breaks (DSB) are formed in response to unrepaired SSB. This is followed by relocalization within the nuclei of Rad51 protein, which is essential for homologous recombination (HR). These findings are compatible with a model where, in mammalian cells, unrepaired SSB produced during BER are substrates for the HR pathway via DSB formation. This is an example of a coordinated effort of two different repair pathways, BER and HR, to protect mammalian cells from alkylation-induced cytotoxicity.     

Regulated over-expression of DNA polymerase beta mediates early onset cataract in mice

Base excision repair (BER) is a tightly coordinated mechanism for repair of DNA base damage (via alkylation and oxidation) and base loss. From E. coli to yeast to human cells, subtle alterations in expression of BER proteins lead to mutagenic or genome instability phenotypes. DNA polymerase beta (beta-pol), the major BER polymerase, has been found to be over-expressed in human tumor tissues and more recently it has been shown that over-expression of beta-pol results in a mutator and genome instability phenotype. These previous reports imply that beta-pol over-expression is deleterious and suggests that such an imbalance may cause an overall functional deficiency in the BER pathway. In the present study, we have developed a bicistronic tetracycline-responsive transgenic system to over-express beta-pol in mice. We find that over-expression of beta-pol in the lens epithelium results in the early onset of severe cortical cataract, with cataractogenesis beginning within 4 days after birth. In utero and post-natal suppression of transgenic Flag-beta-pol expression by doxycycline administration completely prevents cataract formation through adulthood, yet cataract is subsequently observed following removal of doxycycline and re-expression of the transgene. Cataract development accompanies increased expression of cyclooxygenase-2 in the lenticular fibers of the lens, implicating oxidative stress in the development of this cataractous phenotype. Although the mechanism for the transgene mediated cataractogenesis is not clear at this time, it is nevertheless intriguing that increased expression of beta-pol leads to such a phenotype. These results suggest that either a beta-pol expression imbalance negatively affects overall fidelity and/or BER capacity or that beta-pol has a role in lens epithelial cell differentiation.     

FEN1 stimulation of DNA polymerase beta mediates an excision step in mammalian long patch base excision repair

In mammalian cells, single-base lesions, such as uracil and abasic sites, appear to be repaired by at least two base excision repair (BER) subpathways: "single-nucleotide BER" requiring DNA synthesis of just one nucleotide and "long patch BER" requiring multi-nucleotide DNA synthesis. In single-nucleotide BER, DNA polymerase beta (beta-pol) accounts for both gap filling DNA synthesis and removal of the 5'-deoxyribose phosphate (dRP) of the abasic site, whereas the involvement of various DNA polymerases in long patch BER is less well understood. Recently, we found that beta-pol plays a role in mammalian cell extract-mediated long patch BER, in that formation of a key excision product, 5'-dRP-trinucleotide (5'-dRP-N(3)), is dependent upon beta-pol (Dianov, G. L., Prasad, R., Wilson, S. H., and Bohr, V.A. (1999) J. Biol. Chem. 274, 13741-13743). The structure-specific endonuclease flap endonuclease 1 (FEN1) has also been suggested to be involved in long patch BER excision. Here, we demonstrate by immunodepletion experiments that 5'-dRP-N(3) excision in long patch BER of uracil-DNA in a human lymphoid cell extract is, indeed, dependent upon FEN1. Next, we reconstituted the excision step of long patch BER using purified human proteins and an oligonucleotide substrate with 5'-dRP at the margin of a one-nucleotide gap. Formation of the excision product 5'-dRP-N(3) was dependent upon both strand displacement DNA synthesis by beta-pol and FEN1 excision. FEN1 stimulated strand displacement DNA synthesis of beta-pol. FEN1 acting either alone, or without DNA synthesis by beta-pol, produced a two-nucleotide excision product, 5'-dRP-N(1), but not 5'-dRP-N(3). These results demonstrate that human FEN1 and beta-pol can cooperate in long patch BER excision and specify the predominant excision product seen with a cell extract.     

Identification of small molecule synthetic inhibitors of DNA polymerase beta by NMR chemical shift mapping

DNA polymerase beta (beta-pol) plays a central role in repair of damaged DNA bases by base excision repair (BER) pathways. A predominant phenotype of beta-pol null mouse fibroblasts is hypersensitivity to the DNA-methylating agent methyl methanesulfonate. Residues in the 8-kDa domain of beta-pol that seem to interact with a known natural product beta-pol inhibitor, koetjapic acid, were identified by NMR chemical shift mapping. The data implicate the binding pocket as the hydrophobic cleft between helix-2 and helix-4, which provides the DNA binding and deoxyribose phosphate lyase activities of the enzyme. Nine structurally related synthetic compounds, containing aromatic or other hydrophobic groups in combination with two carboxylate groups, were then tested. They were found to bind to the same or a very similar region on the surface of the enzyme. The ability of these compounds to potentiate methyl methanesulfonate cytotoxicity, an indicator of cellular BER capacity, in wild-type and beta-pol null mouse fibroblasts, was next ascertained. The most active and beta-pol-specific of these agents, pamoic acid, was further characterized and found to be an inhibitor of the deoxyribose phosphate lyase and DNA polymerase activities of purified beta-pol on a BER substrate. Our results illustrate that NMR-based mapping techniques can be used in the design of small molecule enzyme inhibitors including those with potential use in a clinical setting.     

Hypersensitivity phenotypes associated with genetic and synthetic inhibitor-induced base excision repair deficiency

Single-base lesions in DNA are repaired predominantly by base excision repair (BER). DNA polymerase beta (pol beta) is the polymerase of choice in the preferred single-nucleotide BER pathway. The characteristic phenotype of mouse fibroblasts with a deletion of the pol beta gene is moderate hypersensitivity to monofunctional alkylating agents, e.g., methyl methanesulfonate (MMS). Increased sensitivity to MMS is also seen in the absence of pol beta partner proteins XRCC1 and PARP-1, and under conditions where BER efficiency is reduced by synthetic inhibitors. PARP activity plays a major role in protection against MMS-induced cytotoxicity, and cells treated with a combination of non-toxic concentrations of MMS and a PARP inhibitor undergo cell cycle arrest and die by a Chk1-dependent apoptotic pathway. Since BER-deficient cells and tumors are similarly hypersensitive to the clinically used chemotherapeutic methylating agent temozolomide, modulation of DNA damage-induced cell signaling pathways, as well as BER, are attractive targets for potentiating chemotherapy.     

HMGB1 is a cofactor in mammalian base excision repair

Deoxyribose phosphate (dRP) removal by DNA polymerase beta (Pol beta) is a pivotal step in base excision repair (BER). To identify BER cofactors, especially those with dRP lyase activity, we used a Pol beta null cell extract and BER intermediate as bait for sodium borohydride crosslinking. Mass spectrometry identified the high-mobility group box 1 protein (HMGB1) as specifically interacting with the BER intermediate. Purified HMGB1 was found to have weak dRP lyase activity and to stimulate AP endonuclease and FEN1 activities on BER substrates. Coimmunoprecipitation experiments revealed interactions of HMGB1 with known BER enzymes, and GFP-tagged HMGB1 was found to accumulate at sites of oxidative DNA damage in living cells. HMGB1(-/-) mouse cells were slightly more resistant to MMS than wild-type cells, probably due to the production of fewer strand-break BER intermediates. The results suggest HMGB1 is a BER cofactor capable of modulating BER capacity in cells.     

Homologous recombination protects mammalian cells from replication-associated DNA double-strand breaks arising in response to methyl methanesulfonate

DNA-methylating agents of the S_N2 type target DNA mostly at ring nitrogens, producing predominantly N-methylated purines. These adducts are repaired by base excision repair (BER). Since defects in BER cause accumulation of DNA single-strand breaks (SSBs) and sensitize cells to the agents, it has been suggested that some of the lesions on their own or BER intermediates (e.g. apurinic sites) are cytotoxic, blocking DNA replication and inducing replication-mediated DNA double-strand breaks (DSBs). Here, we addressed the question of whether homologous recombination (HR) or non-homologous end-joining (NHEJ) or both are involved in the repair of DSBs formed following treatment of cells with methyl methanesulfonate (MMS). We show that HR defective cells (BRCA2, Rad51D and XRCC3 mutants) are dramatically more sensitive to MMS-induced DNA damage as measured by colony formation, apoptosis and chromosomal aberrations, while NHEJ defective cells (Ku80 and DNA-PK_CS mutants) are only mildly sensitive to the killing, apoptosis-inducing and clastogenic effects of MMS. On the other hand, the HR mutants were almost completely refractory to the formation of sister chromatid exchanges (SCEs) following MMS treatment. Since DSBs are expected to be formed specifically in the S-phase, we assessed the formation and kinetics of repair of DSBs by γH2AX quantification in a cell cycle specific manner. In the cytotoxic dose range of MMS a significant amount of γH2AX foci was induced in S, but not G1- and G2-phase cells. A major fraction of γH2AX foci colocalized with 53BP1 and phosphorylated ATM, indicating they are representative of DSBs. DSB formation following MMS treatment was also demonstrated by the neutral comet assay. Repair kinetics revealed that HR mutants exhibit a significant delay in DSB repair, while NHEJ mutants completed S-phase specific DSB repair with a kinetic similar to the wildtype. Moreover, DNA-PKcs inhibition in HR mutants did not affect the repair kinetics after MMS treatment. Overall, the data indicate that agents producing N-alkylpurines in the DNA induce replication-dependent DSBs. Further, they show that HR is the major pathway of protection of cells against DSB formation, killing and genotoxicity following S_N2-alkylating agents.     

Caloric restriction promotes genomic stability by induction of base excision repair and reversal of its age-related decline

Caloric restriction is a potent experimental manipulation that extends mean and maximum life span and delays the onset and progression of tumors in laboratory rodents. While caloric restriction (CR) clearly protects the genome from deleterious damage, the mechanism by which genomic stability is achieved remains unclear. We provide evidence that CR promotes genomic stability by increasing DNA repair capacity, specifically base excision repair (BER). CR completely reverses the age-related decline in BER capacity (P<0.01) in all tissues tested (brain, liver, spleen and testes) providing aged, CR animals with the BER phenotype of young, ad libitum-fed animals. This CR-induced reversal of the aged BER phenotype is accompanied by a reversal in the age-related decline in DNA polymerase beta (beta-pol), a rate-limiting enzyme in the BER pathway. CR significantly reversed the age-related loss of beta-pol protein levels (P<0.01), mRNA levels (P<0.01) and enzyme activity (P<0.01) in all tissues tested. Additionally, in young (4-6-month-old) CR animals a significant up-regulation in BER capacity, beta-pol protein and beta-pol mRNA is observed (P<0.01), demonstrating an early effect of CR that may provide insight in distinguishing the anti-tumor from the anti-aging effects of CR. This up-regulation in BER by caloric restriction in young animals corresponds to increased protection from carcinogen exposure, as mutation frequency is significantly reduced in CR animals exposed to either DMS or 2-nitropropane (2-NP) (P<0.01). Overall the data suggest an important biological consequence of moderate BER up-regulation and provides support for the hormesis theory of caloric restriction.     

Preventing oxidation of cellular XRCC1 affects PARP-mediated DNA damage responses

Poly(ADP-ribose) polymerase-1 (PARP-1) binds intermediates of base excision repair (BER) and becomes activated for poly(ADP-ribose) (PAR) synthesis. PAR mediates recruitment and functions of the key BER factors XRCC1 and DNA polymerase β (pol β) that in turn regulate PAR. Yet, the molecular mechanism and implications of coordination between XRCC1 and pol β in regulating the level of PAR are poorly understood. A complex of PARP-1, XRCC1 and pol β is found in vivo, and it is known that pol β and XRCC1 interact through a redox-sensitive binding interface in the N-terminal domain of XRCC1. We confirmed here that both oxidized and reduced forms of XRCC1 are present in mouse fibroblasts. To further understand the importance of the C12–C20 oxidized form of XRCC1 and the interaction with pol β, we characterized cell lines representing stable transfectants in Xrcc1^?/? mouse fibroblasts of wild-type XRCC1 and two mutants of XRCC1, a novel reduced form with the C12–C20 disulfide bond blocked (C12A) and a reference mutant that is unable to bind pol β (V88R). XRCC1-deficient mouse fibroblasts are extremely hypersensitive to methyl methanesulfonate (MMS), and transfected wild-type and C12A mutant XRCC1 proteins similarly reversed MMS hypersensitivity. However, after MMS exposure the cellular PAR level was found to increase to a much greater extent in cells expressing the C12A mutant than in cells expressing wild-type XRCC1. PARP inhibition resulted in very strong MMS sensitization in cells expressing wild-type XRCC1, but this sensitization was much less in cells expressing the C12A mutant. The results suggest a role for the oxidized form of XRCC1 in the interaction with pol β in (1) controlling the PAR level after MMS exposure and (2) enabling the extreme cytotoxicity of PARP inhibition during the MMS DNA damage response.     

Relationships between yeast Rad27 and Apn1 in response to apurinic/apyrimidinic (AP) sites in DNA.

Yeast Rad27 is a 5'-->3' exonuclease and a flap endo-nuclease. Apn1 is the major apurinic/apyrimidinic (AP) endonuclease in yeast. The rad27 deletion mutants are highly sensitive to methylmethane sulfonate (MMS). By examining the role of Rad27 in different modes of DNA excision repair, we wish to understand why the cytotoxic effect of MMS is dramatically enhanced in the absence of Rad27. Base excision repair (BER) of uracil-containing DNA was deficient in rad27 mutant extracts in that (i) the Apn1 activity was reduced, and (ii) after DNA incision by Apn1, hydrolysis of 1-5 nucleotides 3' to the baseless sugar phosphate was deficient. Thus, some AP sites may lead to unprocessed DNA strand breaks in rad27 mutant cells. The severe MMS sensitivity of rad27 mutants is not caused by a reduction of the Apn1 activity. Surprisingly, we found that Apn1 endonuclease sensitizes rad27 mutant cells to MMS. Deleting the APN1 gene largely restored the resistance of rad27 mutants to MMS. These results suggest that unprocessed DNA strand breaks at AP sites are mainly responsible for the MMS sensitivity of rad27 mutants. In contrast, nucleotide excision repair and BER of oxidative damage were not affected in rad27 mutant extracts, indicating that Rad27 is specifically required for BER of AP sites in DNA.     

Homologous recombination prevents methylation-induced toxicity in Escherichia coli

Methylating agents such as N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) and methyl methane sulfonate (MMS) produce a wide variety of N- and O-methylated bases in DNA, some of which can block replication fork progression. Homologous recombination is a mechanism by which chromosome replication can proceed despite the presence of lesions. The two major recombination pathways, RecBCD and RecFOR, which repair double-strand breaks (DSBs) and single-strand gaps respectively, are needed to protect against toxicity with the RecBCD system being more important. We find that recombination-deficient cell lines, such as recBCD recF, and ruvC recG, are as sensitive to the cytotoxic effects of MMS and MNNG as the most base excision repair (BER)-deficient (alkA tag) isogenic mutant strain. Recombination and BER-deficient double mutants (alkA tag recBCD) were more sensitive to MNNG and MMS than the single mutants suggesting that homologous recombination and BER play essential independent roles. Cells deleted for the polA (DNA polymerase I) or priA (primosome) genes are as sensitive to MMS and MNNG as alkA tag bacteria. Our results suggest that the mechanism of cytotoxicity by alkylating agents includes the necessity for homologous recombination to repair DSBs and single-strand gaps produced by DNA replication at blocking lesions or single-strand nicks resulting from AP-endonuclease action.     

Poly(ADP-ribose) polymerase activity prevents signaling pathways for cell cycle arrest after DNA methylating agent exposure

Mouse fibroblasts, deficient in DNA polymerase beta, are hypersensitive to monofunctional DNA methylating agents such as methyl methanesulfonate (MMS). Both wild-type and, in particular, repair-deficient DNA polymerase beta null cells are highly sensitized to the cytotoxic effects of MMS by 4-amino-1,8-naphthalimide (4-AN), an inhibitor of poly(ADP-ribose) polymerase (PARP) activity. Experiments with synchronized cells suggest that exposure during S-phase of the cell cycle is required for the 4-AN effect. 4-AN elicits a similar extreme sensitization to the thymidine analog, 5-hydroxymethyl-2'-deoxyuridine, implicating the requirement for an intermediate of DNA repair. In PARP-1-expressing fibroblasts treated with a combination of MMS and 4-AN, a complete inhibition of DNA synthesis is apparent after 4 h, and by 24 h, all cells are arrested in S-phase of the cell cycle. Continuous incubation with 4-AN is required to maintain the cell cycle arrest. Caffeine, an inhibitor of the upstream checkpoint kinases ATM (ataxia telangiectasia-mutated) and ATR (ATM and Rad3-related), has no effect on the early inhibition of DNA synthesis, but cells are no longer able to maintain the block after 8 h. Instead, the addition of caffeine leads to arrest of cells in G(2)/M rather than S-phase after 24 h. Analysis of signaling pathways in cell extracts reveals an activation of Chk1 after treatment with MMS and 4-AN, which can be suppressed by caffeine. Our results suggest that inhibition of PARP activity results in sensitization to MMS through maintenance of an ATR and Chk1-dependent S-phase checkpoint.     

Changes of 8-OH-dG levels in DNA and its base excision repair activity in rat lungs after inhalation exposure to hexavalent chromium

The co-genotoxic effects of cadmium are well recognized and it is assumed that most of these effects are due to the inhibition of DNA repair. We used the comet assay to analyze the effect of low, non-toxic concentrations of CdCl2 on DNA damage and repair-induced in Chinese hamster ovary (CHO) cells by UV-radiation, by methyl methanesulfonate (MMS) and by N-methyl-N-nitrosourea (MNU). The UV-induced DNA lesions revealed by the comet assay are single-strand breaks which are the intermediates formed during nucleotide excision repair (NER). In cells exposed to UV-irradiation alone the formation of DNA strand breaks was rapid, followed by a fast rejoining phase during the first 60 min after irradiation. In UV-irradiated cells pre-exposed to CdCl2, the formation of DNA strand breaks was significantly slower, indicating that cadmium inhibited DNA damage recognition and/or excision. Methyl methanesulfonate and N-methyl-N-nitrosourea directly alkylate nitrogen and oxygen atoms of DNA bases. The lesions revealed by the comet assay are mainly breaks at apurinic/apyrimidinic (AP) sites and breaks formed as intermediates during base excision repair (BER). In MMS treated cells the initial level of DNA strand breaks did not change during the first hour of recovery; thereafter repair was detected. In cells pre-exposed to CdCl2 the MMS-induced DNA strand breaks accumulated during the first 2h of recovery, indicating that AP sites and/or DNA strand breaks were formed but that further steps of BER were blocked. In MNU treated cells the maximal level of DNA strand breaks was detected immediately after the treatment and the breaks were repaired rapidly. In CdCl2 pre-treated cells the formation of MNU-induced DNA single-strand breaks was not affected, while the repair was slower, indicating inhibition of polymerization and/or the ligation step of BER. Cadmium thus affects the repair of UV-, MMS- and MNU-induced DNA damage, providing further evidence, that inhibition of DNA repair is an important mechanism of cadmium induced mutagenicity and carcinogenicity.     

DNA polymerase β-dependent cell survival independent of XRCC1 expression

Base excision repair (BER) is a primary mechanism for repair of base lesions in DNA such as those formed by exposure to the DNA methylating agent methyl methanesulfonate (MMS). Both DNA polymerase β (pol β)- and XRCC1-deficient mouse fibroblasts are hypersensitive to MMS. This is linked to a repair deficiency as measured by accumulation of strand breaks and poly(ADP-ribose) (PAR). The interaction between pol β and XRCC1 is important for recruitment of pol β to sites of DNA damage. Endogenous DNA damage can substitute for MMS-induced damage such that BER deficiency as a result of either pol β- or XRCC1-deletion is associated with sensitivity to PARP inhibitors. Pol β shRNA was used to knock down pol β in Xrcc1^+/+ and Xrcc1^−/− mouse fibroblasts. We determined whether pol β-mediated cellular resistance to MMS and PARP inhibitors resulted entirely from coordination with XRCC1 within the same BER sub-pathway. We find evidence for pol β-dependent cell survival independent of XRCC1 expression for both types of agents. The results suggest a role for pol β-dependent, XRCC1-independent repair. PAR immunofluorescence data are consistent with the hypothesis of a decrease in repair in both pol β knock down cell variants.