MOLECULAR STUDIES OF PROTEIN-DNA INTERACTIONS

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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 diversity. Our research efforts currently focus on two related processes, homologous recombination and DNA mismatch repair. We are interested in studies of proteins that target a key DNA intermediate in homologous recombination known as the Holliday junction. This junction marks the point of exchange of two homologous DNAs that are undergoing recombination. Using artificially constructed Holliday junctions, we have determined that a histone octamer is a barrier to migration of the junction. However, motor proteins like E. coli RuvAB and presumptive eukaryotic counterparts can promote movement of the junction through a nucleosome. Mismatch repair, exemplified by the E. coli methyl-directed mismatch repair pathway, plays critical roles in maintaining the integrity of a genome. Mismatches can arise through DNA replication errors, homologous recombination and spontaneous DNA damage. Components of the bacterial mismatch repair system encoded by the mutS and mutL genes in E. coli, are highly conserved in both prokayotes and eukaryotes with defects in human genes encoding mismatch repair enzymes being implicated in hereditary colon cancer. We are interested in understanding the molecular mechanism involved in mismatch recognition by the MutS protein. Through photocross-linking and site-directed mutagenesis, we have identified a critical phenylalanine residue involved in heteroduplex DNA binding in both Taq and E. coli MutS proteins.