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J. Biol. Chem., Vol. 278, Issue 49, 49505-49511, December 5, 2003

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Hydrolytically Deficient MutS E694A Is Defective in the MutL-dependent Activation of MutH and in the Mismatch-dependent Assembly of the MutS · MutL · Heteroduplex Complex^*

Celia Baitinger, Vickers Burdett, and Paul Modrich, Investigator of the Howard Hughes Medical Institute.¶

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

The roles of ATP binding and hydrolysis by MutS in mismatch repair are poorly understood. MutS E694A, in which Glu-694 of the Walker B motif is substituted with alanine, is defective in hydrolysis of bound ATP and has been reported to support MutL-dependent activation of the MutH d(GATC) endonuclease in a trans DNA activation assay (Junop, M. S., Obmolova, G., Rausch, K., Hsieh, P., and Yang, W. (2001) Mol. Cell 7, 1–12). Because the MutH trans activation assay used in these previous studies was characterized by high background and low efficiency, we have re-evaluated the activities of MutS E694A. In contrast to native MutS, which can be isolated in a nucleotide-free form, purified MutS E694A contains 1.0 mol of bound ATP per dimer equivalent, and substoichiometric levels of bound ADP (0.08–0.58 mol/dimer), consistent with the suggestion that the ADP·MutS·ATP complex comprises a significant fraction of the protein in solution (Bjornson, K. P. and Modrich, P. (2003) J. Biol. Chem. 278, 18557–18562). In the presence of Mg²⁺, endogenous ATP is hydrolyzed with a rate constant of 0.12 min^-1 at 30 °C, and hydrolysis yields a protein that displays increased specificity for heteroduplex DNA. As observed with wild type MutS, ATP can promote release of MutS E694A from a mismatch. However, the mutant protein is defective in the methyl-directed, mismatch- and MutL-dependent cis activation of MutH endonuclease on a 6.4-kilobase pair heteroduplex, displaying only 1 to 2% of the activity of wild type MutS. The mutant protein also fails to support normal assembly of the MutS·MutL·DNA ternary complex. Although a putative ternary complex can be observed in the presence of MutS E694A, assembly of this structure displays little if any dependence on a mismatched base pair.

Received for publication, August 7, 2003 , and in revised form, September 15, 2003.

^* This work was supported in part by NIGMS National Institutes of Health Grant GM23719. 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.

^¶ To whom correspondence should be addressed. Tel.: 919-684-2775; Fax: 919-681-7874; E-mail: modrich{at}biochem.duke.edu.

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