Poly(ADP-ribose) Polymerase Activity Prevents Signaling Pathways for Cell Cycle Arrest after DNA Methylating Agent Exposure

Abstract

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...

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

“…It is interesting to note that mouse fibroblasts with a double knockout of PARP-1 and pol β are reported to be no more hypersensitive to MNU than cells with a single gene deletion [32]. The observed hypersensitivity of PARP-1 −/− cells to hmdUrd [66] is consistent with the idea that PARP-1 is activated in response to DNA strand breaks and with the proposal that sensitivity to hmdUrd results from accumulation of repair intermediates in cells with a deficiency in BER.…”

“…The results demonstrate that PARP activity is critical in protection of cells against MMS-induced DNA adducts both in the presence or absence of pol β-dependent repair pathways. Cells exposed to potent PARP inhibitors are more sensitive to MMS than are PARP-1 −/− cells [66]. In the absence of PARP-1, BER may be less efficient, hence the moderate MMS hypersensitivity.…”

“…Both wild-type and pol β null cells are extremely sensitized to MMS by co-incubation with 4-AN [71] and the extent of sensitization is both time-and 4-AN dose-dependent [66]. Survival following exposure to MMS and utilizing the optimal 4-AN schedule (10 μM for 24 h) is shown in Figure 4.…”

“…2) and MNU, as well as to hydrogen peroxide and ionizing radiation [32,[63][64][65][66], indicating the importance of PARP-1 protein in cellular resistance to these agents. In the case of MMS, the sensitivity ratio between PARP-1 +/+ and −/− cells is similar to that seen in the pol β cells (Fig.…”

Measurement of the Incorporation and Repair of Exogenous 5-Hydroxymethyl-2′-deoxyuridine in Human Cells in Culture Using Gas Chromatography-Negative Chemical Ionization-Mass Spectrometry

“…The ability to experimentally introduce a single type of oxidative DNA damage and titrate its levels into cells has led to considerable interest in this compound as a model for studying uncharacterized DNA repair proteins and pathways (20)(21)(22). The toxicity of HmdU to cultured cells has also led to interest in the compound as a potential chemotherapy agent (23)(24)(25)(26).…”