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J. Biol. Chem., Vol. 281, Issue 32, 22674-22683, August 11, 2006

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

Sally J. York¹ and Paul Modrich²

From the Howard Hughes Medical Institute and Departments of Biochemistry and Medicine, Duke University Medical Center, Durham, North Carolina 27710

The response of mammalian cells to S_n1 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_n1 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 [-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 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 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.

Received for publication, April 17, 2006

^* This work was supported in part by National Institutes of Health Grants R01 GM45190, P01 CA92584 (to P. M.), and 1P50 CA108786 (to S. J. Y.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. 1–5.

¹ Supported in part by a Physician Postdoctoral Fellowship from the Howard Hughes Medical Institute.

² An Investigator of the Howard Hughes Medical Institute. To whom correspondence should be addressed: Howard Hughes Medical Institute and Dept. of Biochemistry, Box 3711, Duke University Medical Center, Durham, NC 27710. Tel.: 919-684-2775; Fax: 919-681-7874; E-mail: modrich{at}biochem.duke.edu.

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