Julie K. Horton

The lyase activity of the DNA repair protein β-polymerase protects from DNA-damage-induced cytotoxicity

Small DNA lesions such as oxidized or alkylated bases are repaired by the base excision repair (BER) pathway. BER includes removal of the damaged base by a lesion-specific DNA glycosylase, strand scission by apurinic/apyrimidinic endonuclease, DNA resynthesis and ligation. BER may be further subdivided into DNA β-polymerase (β-pol)-dependent single-nucleotide repair and β-pol-dependent or -independent long patch repair subpathways. Two important enzymatic steps in mammalian single-nucleotide BER are contributed by β-pol: DNA resynthesis of the repair patch and lyase removal of 5′-deoxyribose phosphate (dRP). Fibroblasts from β-pol null mice are hypersensitive to monofunctional DNA-methylating agents, resulting in increases in chromosomal damage, apoptosis and necrotic cell death. Here we show that only the dRP lyase activity of β-pol is required to reverse methylating agent hypersensitivity in β-pol null cells. These results indicate that removal of the dRP group is a pivotal step in BER in vivo. Persistence of the dRP moiety in DNA results in the hypersensitivity phenotype of β-pol null cells and may signal downstream events such as apoptosis and necrotic cell death.

XRCC1 and DNA polymerase β in cellular protection against cytotoxic DNA single-strand breaks

Single-strand breaks (SSBs) can occur in cells either directly, or indirectly following initiation of base excision repair (BER). SSBs generally have blocked termini lacking the conventional 5′-phosphate and 3′-hydroxyl groups and require further processing prior to DNA synthesis and ligation. XRCC1 is devoid of any known enzymatic activity, but it can physically interact with other proteins involved in all stages of the overlapping SSB repair and BER pathways, including those that conduct the rate-limiting end-tailoring, and in many cases can stimulate their enzymatic activities. XRCC1−/− mouse fibroblasts are most hypersensitive to agents that produce DNA lesions repaired by monofunctional glycosylase-initiated BER and that result in formation of indirect SSBs. A requirement for the deoxyribose phosphate lyase activity of DNA polymerase β (pol β) is specific to this pathway, whereas pol β is implicated in gap-filling during repair of many types of SSBs. Elevated levels of strand breaks, and diminished repair, have been demonstrated in MMS-treated XRCC1−/−, and to a lesser extent in pol β−/− cell lines, compared with wild-type cells. Thus a strong correlation is observed between cellular sensitivity to MMS and the ability of cells to repair MMS-induced damage. Exposure of wild-type and pol β−/− cells to an inhibitor of PARP activity dramatically potentiates MMS-induced cytotoxicity. XRCC1−/− cells are also sensitized by PARP inhibition demonstrating that PARP-mediated poly(ADP-ribosyl)ation plays a role in modulation of cytotoxicity beyond recruitment of XRCC1 to sites of DNA damage.

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 β (β-pol) contributes the gap tailoring and DNA synthesis steps. It is the loss of β-pol-mediated 5′-deoxyribose phosphate removal that renders mouse fibroblasts alkylation-hypersensitive. Here we report that the hypersensitivity of β-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 β-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.

Involvement of DNA polymerase β 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.

Mutations associated with base excision repair deficiency and methylation-induced genotoxic stress

The long-term effect of exposure to DNA alkylating agents is entwined with the cells genetic capacity for DNA repair and appropriate DNA damage responses. A unique combination of environmental exposure and deficiency in these responses can lead to genomic instability; this “gene–environment interaction” paradigm is a theme for research on chronic disease etiology. In the present study, we used mouse embryonic fibroblasts with a gene deletion in the base excision repair (BER) enzymes DNA β-polymerase (β-pol) and alkyladenine DNA glycosylase (AAG), along with exposure to methyl methanesulfonate (MMS) to study mutagenesis as a function of a particular gene–environment interaction. The β-pol null cells, defective in BER, exhibit a modest increase in spontaneous mutagenesis compared with wild-type cells. MMS exposure increases mutant frequency in β-pol null cells, but not in isogenic wild-type cells; UV light exposure or N-methyl-N′-nitro-N-nitrosoguanidine exposure increases mutant frequency similarly in both cell lines. The MMS-induced increase in mutant frequency in β-pol null cells appears to be caused by DNA lesions that are AAG substrates, because overexpression of AAG in β-pol null cells eliminates the effect. In contrast, β-pol/AAG double null cells are slightly more mutable than the β-pol null cells after MMS exposure. These results illustrate that BER plays a role in protecting mouse embryonic fibroblast cells against methylation-induced mutations and characterize the effect of a particular combination of BER gene defect and environmental exposure.

Characterization of a chlorambucil-resistant human ovarian carcinoma cell line overexpressing glutathione S-transferase μ

Ovarian carcinoma cells 10-fold resistant to the alkylating agent chlorambucil (CBL) were isolated after repeated exposure of the parent cells to gradually escalating concentrations of the drug. The resistant variant, A2780(100), was highly cross-resistant (9-fold) to melphalan and showed lower-level resistance to other cross-linking agents. The resistant A2780(100) cells had almost 5-fold higher glutathione S-transferase (GST) activity than the parental A2780 cells with 1-chloro-2,4-dinitrobenzene (CDNB) as substrate. The pi-class GST(s) was the major isoform(s) in both cell lines. However, the resistant A2780(100) cells had at least 11-fold higher GST mu as compared with the parental cells, in which this isoform was barely detectable. A significant induction of GST mu was observed in A2780 cells, but not in the resistant cells, 18 hr after a single exposure to 100 microM CBL. The induction of GST mu by CBL was both time- and concentration-dependent. Assays of the conjugation of CBL with GSH showed that the human mu-class GST had 3.6- and 5.2-fold higher catalytic efficiency relative to the pi- and alpha-class GSTs, respectively. This difference was reflected in the relatively higher (about 6-fold) efficiency of CBL conjugation in A2780(100) cells as compared with the parental cells. These results have demonstrated for the first time a near-linear correlation between CBL resistance and overexpression of mu-class GSTs and suggest that this overexpression maybe responsible, at least in part, for the acquired resistance of ovarian carcinoma cells to CBL, and possibly the other bifunctional alkylating agents. Consistent with this hypothesis, we found evidence for decreased formation of DNA lesions in A2780(100) compared with the drug-sensitive A2780 cells after exposure to CBL.

Alkylation DNA damage in combination with PARP inhibition results in formation of S-phase-dependent double-strand breaks.

The combination of poly(ADP-ribose)polymerase (PARP) inhibitors and alkylating agents is currently being investigated in cancer therapy clinical trials. However, the DNA lesions producing the synergistic cell killing effect in tumors are not fully understood. Treatment of human and mouse fibroblasts with the monofunctional DNA methylating agent methyl methanesulfonate (MMS) in the presence of a PARP inhibitor has been shown to trigger a cell cycle checkpoint response. Among other changes, this DNA damage response to combination treatment includes activation of ATM/Chk2 and phosphorylation of histone H2A.X. These changes are consistent with DNA double-strand break (DSB) formation during the response, but the measurement of DSBs has not been addressed. Such DSB evaluation is important in understanding this DNA damage response because events other than DSB formation are known to lead to ATM/Chk2 activation and H2A.X phosphorylation. Here, we examined the structural integrity of genomic DNA after the combined treatment of cells with MMS and a PARP inhibitor, i.e., exposure to a sub-lethal dose of MMS in the presence of the PARP inhibitor 4-amino-1,8-napthalimide (4-AN). We used pulsed field gel electrophoresis (PFGE) for measurement of DSBs in both human and mouse embryonic fibroblasts, and flow cytometry to follow the phosphorylated form of H2A.X (gamma-H2A.X). The results indicate that DSBs are formed with the combination treatment, but not following treatment with either agent alone. Our data also show that formation of gamma-H2A.X correlates with PARP-1-expressing cells in S-phase of the cell cycle. The observations support the model that persistence of PARP-1 at base excision repair intermediates, as cells move into S-phase, leads to DSBs and the attendant checkpoint responses.

Predicting Enhanced Cell Killing through PARP Inhibition

PARP inhibitors show promise as combination and single agents in cancer chemotherapy. Here, we evaluate results obtained with mouse fibroblasts and the common laboratory PARP inhibitor 4-amino-1,8-naphthalimide (4-AN) and analyze the potential for enhanced cytotoxicity following the combination of a DNA-damaging agent and a PARP inhibitor. Methylated DNA bases are repaired by the monofunctional glycosylase-initiated single-nucleotide base excision repair (BER) pathway. An intermediate of this process has a single-nucleotide gap in double-stranded DNA containing the 5′-deoxyribose phosphate (dRP) group at one margin. This 5′-dRP group is removed by the lyase activity of pol β prior to gap filling; then completion of repair is by DNA ligation. PARP-1 binds to and is activated by the 5′-dRP group–containing intermediate, and poly(ADP-ribos)ylation is important for efficient repair. 4-AN–mediated sensitization to the methylating chemotherapeutic agent temozolomide is extreme, producing a level of cytotoxicity not seen with either agent alone. In contrast, with agents producing oxidative DNA damage repaired by bifunctional glycosylase-initiated BER, there is only weak sensitization by cotreatment with PARP inhibitor. Other clinically used DNA-damaging agents repaired by different DNA repair pathways also reveal minimal 4-AN–mediated sensitization. This information has potentially important implications for strategic use of PARP inhibitors in chemotherapy. Mol Cancer Res; 11(1); 13–18. ©2012 AACR.

Increased PARP-1 association with DNA in alkylation damaged, PARP-inhibited mouse fibroblasts

Treatment of base excision repair–proficient mouse fibroblasts with the DNA alkylating agent methyl methanesulfonate (MMS) and a small molecule inhibitor of PARP-1 results in a striking cell killing phenotype, as previously reported. Earlier studies showed that the mechanism of cell death is apoptosis and requires DNA replication, expression of PARP-1, and an intact S-phase checkpoint cell signaling system. It is proposed that activity-inhibited PARP-1 becomes immobilized at DNA repair intermediates, and that this blocks DNA repair and interferes with DNA replication, eventually promoting an S-phase checkpoint and G2-M block. Here we report studies designed to evaluate the prediction that inhibited PARP-1 remains DNA associated in cells undergoing repair of alkylation-induced damage. Using chromatin immunoprecipitation with anti–PARP-1 antibody and qPCR for DNA quantification, a higher level of DNA was found associated with PARP-1 in cells treated with MMS plus PARP inhibitor than in cells without inhibitor treatment. These results have implications for explaining the extreme hypersensitivity phenotype after combination treatment with MMS and a PARP inhibitor. Mol Cancer Res; 10(3); 360–8. ©2012 AACR.

Base excision repair and design of small molecule inhibitors of human DNA polymerase β.

Base excision repair (BER) can protect a cell after endogenous or exogenous genotoxic stress, and a deficiency in BER can render a cell hypersensitive to stress-induced apoptotic and necrotic cell death, mutagenesis, and chromosomal rearrangements. However, understanding of the mammalian BER system is not yet complete as it is extraordinarily complex and has many back-up processes that complement a deficiency in any one step. Due of this lack of information, we are unable to make accurate predictions on therapeutic approaches targeting BER. A deeper understanding of BER will eventually allow us to conduct more meaningful clinical interventions. In this review, we will cover historical and recent information on mammalian BER and DNA polymerase β and discuss approaches toward development and use of small molecule inhibitors to manipulate BER. With apologies to others, we will emphasize results obtained in our laboratory and those of our collaborators.