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

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 degreesC, 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.