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. Studies of the E. coli mismatch repair proteins, MutS and MutL reveal the formation of an ATP-dependent ternary complex involving MutS, MutL and the mismatched DNA that we propose is the intermediate that signals downstream repair events. Studies of these ternary complexes reveal that MutL stabilizes the interaction of MutS with mismatched DNA in the presence of ATP. Atomic force microscopy analysis of these repair complexes reveals the presence of both kinked and unkinked DNAs bound to the mismatch binding site of MutS and provide novel insights into mismatch recognition by MutS proteins. These studies also reveal how key residues in the mismatch binding site of MutS confer mismatch specificity on excision repair. We are currently investigating molecular mechanisms of mismatch recognition by mammalian MutS homologs, MSH2-MSH3 and MSH2-MSH6. DNA binding by these two heterodimeric proteins is modulated by ATP binding and hydrolysis. We are using a variety of approaches to determine how these proteins target mismatches and DNA damage leading to the activation of cell cycle checkpoints and apoptotic pathways.