Molecular Studies Of Protein-DNA Interactions

Diabetes, Digestive, Kidney Diseases

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Abstract

We seek to understand at a molecular level the various ways by which an organism maintains the integrity of its genome while accommodating the need for genetic diversity. Our research efforts currently focus on a highly conserved DNA repair pathway, DNA mismatch repair. Mismatch repair, exemplified by the E. coli methyl-directed mismatch repair pathway, targets base pair mismatches that arise through DNA replication errors, homologous recombination and spontaneous DNA damage. Inactivation of mismatch repair results in a large increase in the rate of spontaneous mutation and is associated with both sporadic and hereditary cancers. Previously, we determined the crystal structure of a key mismatch repair complex consisting of MutS bound to a mismatch DNA. Both structural and biochemical studies provide evidence for a key intermediate in mismatch repair involving a complex of MutS and MutL bound to a DNA mismatch. We have investigated the role of nucleotide binding and hydrolysis in the formation of this complex that signals downstream excision events in repair. The structural data also identified a Phe-X-Glu motif involved in mismatch recognition. Using nonpolar base shape analogs of A and T, we have assessed the role of the conserved Glu residue and find that it plays an important role in mismatch discrimination. We are currently using atomic force microscopy, AFM, to better understand how mismatch repair proteins function in the context of large, multiprotein complexes. Some mismatch repair proteins play important roles in modulating homologous recombination and chromosome pairing during meiosis, though the molecular mechanisms underlying these functions are not known. We are attempting to identify intermediates of recombination that are targets for mismatch repair proteins.