Mismatch Repair-dependent Iterative Excision at Irreparable O6-Methylguanine Lesions in Human Nuclear Extracts

Abstract

The response of mammalian cells to S n 1 DNA methylators depends on functional MutS␣ and MutL␣. Cells deficient in either of these activities are resistant to the cytotoxic effects of this class of chemotherapeutic drug. Because killing by S n 1 methylators has been attributed to O 6 -methylguanine (MeG), we have constructed nicked circular heteroduplexes that contain a single MeG-T mispair, and we have examined processing of these molecules by mismatch repair in nuclear extracts of human cells. Excision provoked by MeG-T is restricted to the incised heteroduplex strand, leading to removal of the MeG when it resides on this strand. However, when the MeG is located on the continuous strand, the heteroduplex is irreparable. MeG-T-dependent repair DNA synthesis is observed on both reparable and irreparable 3 and 5 heteroduplexes as judged by [ P]dAMP incorporation. Labeling with [␣-32 P]dATP followed by a cold dATP chase has demonstrated that newly synthesized DNA on irreparable molecules is subject to re-excision in a reaction that is MutL␣-dependent, an effect attributable to the presence of MeG on the template strand. Processing of the irreparable 3 heteroduplex is also associated with incision of the discontinuous strand of a few percent of molecules near the thymidylate of the MeG-T base pair. These results provide the first direct evidence for mismatch repairmediated iterative processing of DNA methylator damage, an effect that may be relevant to damage signaling events triggered by this class of chemotherapeutic agent.

DNA mismatch repair: Molecular mechanism, cancer, and ageing

“…Repeated rounds of excision by MMR or "futile cycles" eventually result in lethal DSBs. In vitro studies of MMR using mammalian proteins are consistent with iterative rounds of excision repair providing support for this scheme (York and Modrich, 2006). In E. coli, homologous recombination is required for resistance to methylating agents that give rise to MMR-dependent DSBs.…”

“…Inhibition of TS without FU treatment elicits a G2 arrest regardless of MMR status suggesting that the contribution of MMR to DNA damage signalling is via incorporation of FPs into DNA as opposed to the inhibition of TS and the resulting imbalance in nucleotide pools. MutSα recognizes FdU mispairs in vitro and is a substrate for in vitro MMR assays (Fischer et al, 2007;York and Modrich, 2006). In the clinic, resistance to FU treatment occurs frequently.…”

DNA mismatch repair-induced double-strand breaks

“…Futile cycling by MMR at O 6 -meG base pairs was also proposed as a model [40] and recently data were obtained in vitro that supports this concept [42]. It is not inconceivable that events similar to those in E. coli dam mutants could occur in mammalian cells by the generation of a stable MMR-induced single-strand gap as the first step and replication fork collapse as the second followed by signaling of the apoptotic response.…”

Mammalian mismatch repair: error-free or error-prone?

“…We also identified a direct interaction between CAF-1 and MSH6, but its significance is yet to be elucidated [65]. The role of MMR proteins in DNA damage signaling [66] and in the processing of modified bases [67,68] has not been discussed here, but this topic remains enigmatic and much remains to be discovered. As the MMR field is attracting much attention at the present time, it is likely that further important discoveries are just around the corner.…”