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

Sally J York,Paul Modrich

doi:10.1074/jbc.m603667200

Abstract

The response of mammalian cells to Sn1 DNA methylators depends on functional MutSalpha and MutLalpha. Cells deficient in either of these activities are resistant to the cytotoxic effects of this class of chemotherapeutic drug. Because killing by Sn1 methylators has been attributed to O6-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 [32P]dAMP incorporation. Labeling with [alpha-32P]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 MutLalpha-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 repair-mediated iterative processing of DNA methylator damage, an effect that may be relevant to damage signaling events triggered by this class of chemotherapeutic agent.

Highlights

Subject to DNA alkylation damage by Sn1 DNA methylators, cells deficient in MutS␣ or MutL␣ are resistant to the cytotoxic effects of such agents, including the clinically useful compounds temozolomide and procarbazine
Alkylation tolerance attributable to mismatch repair deficiency has been widely documented in cultured cell systems (2, 4 – 6), but it is important to note that development of procarbazine resistance associated with MSH2 deficiency has been demonstrated for a malignant glioma propagated as a xenograft in athymic mice [40]
To address involvement of the mismatch repair system in the processing of Sn1 methylator lesions, we have examined the fate of heteroduplexes containing a single MeG-T mispair in nuclear extracts

Summary

Introduction

2 An Investigator of the Howard Hughes Medical Institute. To whom correspondence should be addressed: Howard Hughes Medical Institute and Dept. The mismatch repair-dependent damage response has been most thoroughly studied for lesions produced by Sn1 DNA methylators. Two models, which are not mutually exclusive, have been proposed to explain the MutS␣- and MutL␣-dependent damage response to DNA methylators. The futile cycling model posits that replication bypass of a template strand MeG produces a base pair anomaly that activates the mismatch repair system. Recruitment of a damage recognition complex consisting of MutS␣, MutL␣, and perhaps other activities to a MeG lesion in the absence of an excision-repair response is sufficient to trigger kinase activation [22]. Several DNA polymerases have been shown to display a significant preference for incorporation of thymidylate opposite a template MeG (24 –26)

Methods

Results

Conclusion