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...
DNA polymerase  (-pol) plays a central role in repair of damaged DNA bases by base excision repair (BER) pathways. A predominant phenotype of -pol null mouse fibroblasts is hypersensitivity to the DNA-methylating agent methyl methanesulfonate. Residues in the 8-kDa domain of -pol that seem to interact with a known natural product -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 -pol null mouse fibroblasts, was next ascertained. The most active and -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 -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.
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