Cooperative Interactions in DNA Repair

Our goal is to elucidate mechanisms by which DNA-repair proteins perform their essential biological functions. As immediate objectives, we will investigate the interactions of human 06 -alkylguanine-DNA alkyltransferase (AGT) with 06-alkylguanine (lesion)-containing and lesion-free DNAs. AGT repairs promutagenic 06 -alkylguanine residues in DNA. It binds DNA with substantial cooperativity but little sequence- or base composition dependence. These results argue against mechanisms of target recognition that depend strongly on sequence. An alternate possibility, which comprises the central hypothesis of this application, is that cooperative DNA binding and access to DNA modulate the binding distributions of AGT and its rate of DNA-repair. To test this hypothesis we will pursue three specific aims. These are: 1. To determine how binding cooperativity, supercoiling and the presence of nucleosomes affect the equilibrium distribution of AGT among available DNA sites and between 06 -alkyl guanine-containing and lesion-free sequences. 2. To identify amino acids that are present at the protein-protein interface in the cooperative AGT-DNA complex. To test the consequences of mutation of these residues on DNA binding in vitro and on DNA repair, in vitro and in vivo. 3. To identify the roles played by cooperative binding, supercoiling and nucleosomes in the kinetic mechanisms of lesion-search by AGT and on its rate of DNA repair. At the conclusion of this research, we will have identified the role played by cooperative binding in lesion-search, -binding and -repair, and we will have tested the notion that the rate of lesion-search depends on the structure of the DNA template. Together, these results will test the hypothesis that differences in DNA structure and accessibility determine the mechanism(s) by which AGT scans the genome for lesions and repairs them.