Mismatch repair-dependent iterative excision at irreparable O6-methylguanine lesions in human nuclear extracts.
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.
York
Sally J
SJ
Howard Hughes Medical Institute and Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710, USA.
Modrich
Paul
P
D000255:Adenosine Triphosphate / Q000737:chemistry; D020137:Base Pair Mismatch; D001483:Base Sequence; D002467:Cell Nucleus / Q000378:metabolism; D019175:DNA Methylation; D004260:DNA Repair; D045643:DNA Repair Enzymes; D006147:Guanine / Q000031:analogs & derivatives / Q000737:chemistry; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D000070956:MutL Proteins; D051719:MutS DNA Mismatch-Binding Protein / Q000378:metabolism; D009363:Neoplasm Proteins / Q000378:metabolism; D009692:Nucleic Acid Heteroduplexes / Q000737:chemistry
C500929:MutLalpha protein, human; D009363:Neoplasm Proteins; D009692:Nucleic Acid Heteroduplexes; D006147:Guanine; D000255:Adenosine Triphosphate; C008449:O-(6)-methylguanine; D000070956:MutL Proteins; D051719:MutS DNA Mismatch-Binding Protein; D045643:DNA Repair Enzymes