application abstract) Mismatch repair plays a major role in genome stabilization, and available information indicates that the substrate specificity and mechanism of the reaction 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 reaction. Dr. Paul Modrich and his colleagues have identified eleven activities that are involved in mismatch repair, including MutS, MutL, MutH, DNA helicase II, and DNA polymerase III holoenzyme. This application addresses several features of the mechanism of this interesting reaction: (i) MutS and its eukaryotic homologs are responsible for mismatch recognition, but also possess a slow ATPase that is required for MutS function in mismatch repair. It is now clear that these proteins leave the mismatch in an ATP-dependent reaction to move along the helix contour, an effect that is believed to play an important role in the coupling of mismatch recognition to the recognition of a strand signal elsewhere on the helix that confers strand specificity on the reaction. However, the mechanism of this movement is controversial. They plan experiments that they hope will resolve this question with respect to the bacterial protein. (ii) In collaboration with the laboratory of Lorena Beese, they are pursuing structural analysis of bacterial MutS in order to clarify the basis of its ability to recognize mismatched base pairs. (iii) They showed that MutS and MutL load DNA helicase II at the strand break introduced by MutH, the key step in initiation of mismatch-provoked excision. They have also shown that MutL functions as an activator of the helicase. They hope to clarify the molecular basis of this activation. (iv) Several protein-DNA assemblies have been documented during the course of the methyl-directed reaction, although their nature has only been addressed in qualitative terms. They hope to establish the molecular composition of these complexes. This phase of the work will also address the basis of the specific requirement for DNA polymerase III holoenzyme in the repair synthesis step of the reaction.
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