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.