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J. Biol. Chem., Vol. 281, Issue 13, 8399-8408, March 31, 2006

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The DNA Binding Activity of MutL Is Required for Methyl-directed Mismatch Repair in Escherichia coli^*

Adam Robertson, Steven R. Pattishall, and Steven W. Matson^¶1

From the Department of Biology, Curriculum in Genetics and Molecular Biology, and ^¶Program in Molecular and Cellular Biophysics, University of North Carolina, Chapel Hill, North Carolina 27599

The DNA binding properties of the mismatch repair protein MutL and their importance in the repair process have been controversial for nearly two decades. We have addressed this issue using a point mutant of MutL (MutL-R266E). The biochemical and genetic data suggest that DNA binding by MutL is required for dam methylation-directed mismatch repair. We demonstrate that purified MutL-R266E retains wild-type biochemical properties that do not depend on DNA binding, such as basal ATP hydrolysis in the absence of DNA and the ability to interact with other mismatch repair proteins. However, purified MutL-R266E binds DNA poorly in vitro as compared with MutL, and consistent with this observation, its DNA-dependent biochemical activities, like DNA-stimulated ATP hydrolysis and helicase II stimulation, are severely compromised. In addition, there is a modest effect on stimulation of MutH-catalyzed nicking. Finally, genetic assays show that MutL-R266E has a strong mutator phenotype, demonstrating that the mutant is unable to function in dam methylation-directed mismatch repair in vivo.

Received for publication, August 19, 2005 , and in revised form, January 25, 2006.

^* This work was supported by a grant from the National Institutes of Health (to S. W. M.). 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.

¹ To whom correspondence should be addressed: Dept. of Biology CB #3280, Coker Hall, University of North Carolina, Chapel Hill, NC 27599-3280. Tel.: 919-962-0005; Fax: 919-962-1625; E-mail: smatson{at}bio.unc.edu.

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