Mismatch repair plays a major role in genome stabilization, and available information indicates that the substrate specificity and mechanism of the repair pathway have been highly conserved during evolution. The Escherichia coli methyl-directed pathway, which is the best understood of the known mismatch repair systems, provides a paradigm for study of the mechanism of this complex pathway. The applicant and his colleagues have isolated ten activities that are sufficient for methyl-directed mismatch repair in vitro, and have identified two partial reactions that embody its most interesting features: (i) The mismatch-provoked, MutS-, MutL-, and ATP-dependent activation of MutH endonuclease, which is the initiation step of methyl-directed repair; and (ii) The MutS- and MutL-dependent loading of DNA helicase II at the strand break introduced by MutH, a partial reaction that corresponds to the key step in mismatch-provoked excision. To further clarify the mechanism of mismatch repair, they will attempt to identify protein-protein and protein-DNA interactions that are of importance in these partial reactions, and to establish the role of the MutS associated ATPase in these systems. In collaboration with a crystallographer, Dr. Loreena Beese, they will pursue structural work on MutS and MutS.heteroduplex complexes with the aim of establishing the molecular basis of mismatch recognition by the protein. Lastly, the applicant will explore """"""""reagent applications"""""""" of mismatch repair activities for mutation detection and for fractionation of a population of DNA molecules, a subset of which may contain one or more mismatched base pairs.
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