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.

Oxidative stress in cells can lead to accumulation of reactive oxygen species and oxidation of DNA precursors. Oxidized purine nucleotides can be inserted into DNA during replication and repair. The main pathway for correcting oxidized bases in DNA is base excision repair (BER), and in vertebrates DNA polymerase β (pol β) provides gap filling and tailoring functions. Here we report that the DNA ligation step of BER is compromised after pol β insertion of oxidized purine nucleotides into the BER intermediate in vitro. These results suggest the possibility that BER mediated toxic strand breaks are produced in cells under oxidative stress conditions. We observe enhanced cytotoxicity in oxidizing-agent treated pol β expressing mouse fibroblasts, suggesting formation of DNA strand breaks under these treatment conditions. Increased cytotoxicity following MTH1 knockout or treatment with MTH1 inhibitor suggests the oxidation of precursor nucleotides.

Mouse fibroblasts, deficient in DNA polymerase ␤, are hypersensitive to monofunctional DNA methylating agents such as methyl methanesulfonate (MMS). Both wild-type and, in particular, repair-deficient DNA polymerase ␤ 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.Removal of unnatural bases or single base lesions from DNA is predominantly by a glycosylase-initiated base excision repair (BER) 1 pathway. Methylated bases are excised by N-methylpurine-DNA glycosylase, a monofunctional glycosylase. In the preferred "single-nucleotide" BER pathway, this is followed by strand cleavage on the 5Ј side of the sugar by apurinic/apyrimidinic endonuclease, gap filling and cleavage on the 3Ј side by the DNA synthesis and deoxyribose phosphate (dRP) lyase activities, respectively, of DNA polymerase ␤ (␤-pol), and finally sealing of the nick by a DNA ligase (1). The essential role of ␤-pol in this pathway has been established using extracts from wild-type and ␤-pol null mouse fibroblasts (2). The requirement for ␤-pol, more specifically the dRP lyase activity of ␤-pol, in protection of cells against the cytotoxicity of methyl DNA lesions, has been demonstrated by the hypersensitivity of ␤-pol null cells to the monofunctional methylating agent methyl methanesulfonate (MMS) (2, 3). It is thought that the MMS hypersensitivity phenotype of ␤-pol null cells reflects accumulation of cytotoxic repair intermediates, such as the 5Ј dRP group after removal of methylated bases from DNA (4).Repair of oxidative DNA damage occurs by an alternate sub-pathway of single-nucleotide BER utilizing bifunctional glycosylases that have an associated lyase activity that nicks the DNA strand 3Ј to the abasic site after base removal. Singlen...