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J. Biol. Chem., Vol. 282, Issue 51, 37181-37190, December 21, 2007

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Saccharomyces cerevisiae MutL Is a Mismatch Repair Endonuclease^*

Farid A. Kadyrov, Shannon F. Holmes^¶1, Mercedes E. Arana^¶1, Olga A. Lukianova, Mike O'Donnell^||**2, Thomas A. Kunkel^¶, and Paul Modrich³

From the Department of Biochemistry and Howard Hughes Medical Institute, Duke University Medical Center, Durham, North Carolina 27710, the Laboratory of Structural Biology and ^¶Laboratory of Molecular Genetics, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709, and the ^||Laboratory of DNA Replication and ^**Howard Hughes Medical Institute, Rockefeller University, New York 10021-6399

MutL homologs are crucial for mismatch repair and genetic stability, but their function is not well understood. Human MutL (MLH1-PMS2 heterodimer) harbors a latent endonuclease that is dependent on the integrity of a PMS2 DQHA(X)₂E(X)₄E motif (Kadyrov, F. A., Dzantiev, L., Constantin, N., and Modrich, P. (2006) Cell 126, 297-308). This sequence element is conserved in many MutL homologs, including the PMS1 subunit of Saccharomyces cerevisiae MutL, but is absent in MutL proteins from bacteria like Escherichia coli that rely on d(GATC) methylation for strand directionality. We show that yeast MutL is a strand-directed endonuclease that incises DNA in a reaction that depends on a mismatch, yMutS, yRFC, yPCNA, ATP, and a pre-existing strand break, whereas E. coli MutL is not. Amino acid substitution within the PMS1 DQHA(X)₂E(X)₄E motif abolishes yMutL endonuclease activity in vitro and confers strong genetic instability in vivo, but does not affect yMutL ATPase activity or the ability of the protein to support assembly of the yMutL·yMutS·heteroduplex ternary complex. The loaded form of yPCNA may play an important effector role in directing yMutL incision to the discontinuous strand of a nicked heteroduplex.

Received for publication, September 11, 2007 , and in revised form, October 18, 2007.

^* This research was supported in part by National Institutes of Health Grants GM45190 (to P. M.) and GM 38839 (to M. O.), and by the Intramural Research Program of the NIEHS, National Institutes of Health (to T. A. K.). 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. S1-S4.

¹ Both authors contributed equally to this work.

² Investigator of the Howard Hughes Medical Institute.

³ Investigator of the Howard Hughes Medical Institute. To whom correspondence should be addressed. Tel.: 919-684-2775; E-mail: modrich{at}biochem.duke.edu.

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