Y-Family DNA polymerases (DNAPs) are usually required to do translesion synthesis (TLS) of DNA damaged by chemicals and radiation. Opposite [+ta]-B[a]P-N2-dG (the major adduct of the environmental mutagen/carcinogen benzo[a]pyrene), E. coli DNAP IV correctly inserts dCTP, while DNAP V inserts dATP (the first step in the G->T mutational pathway). We are investigating the structural differences in DNAP IV vs. DNAP V that dictate this dNTP insertional difference opposite [+ta]-B[a]P-N2-dG. (DNAPs IV and V are the functional orthologs of human DNAPs kappa and eta, respectively, suggesting that studies in E. coli potentially may be revealing vis-a-vis human cells.) No X-ray structures exist for DNAPs IV, V, kappa or eta, so we built models taking a homology modeling approach. The models suggest the hypothesis that the architecture in two regions of Y-Family DNAPs--the roof and the chimney--are likely to have an influence on dNTP insertional pattern. The roof lies above the base of the dNTP, while the chimney is an opening (hole) in the protein surface on the minor groove side of the roof. DNAPs IV/kappa have a non-bulky roof and a large chimney opening, which accommodates the pyrene moiety of [+ta]-B[a]P-N2-dG and allows adduct-dG:dCTP pairing. In contrast, DNAPs V/eta have a bulky roof and a small chimney opening, which enforces a different kind of adduct-dG:dNTP structure. Molecular modeling studies in support of this hypothesis will be presented, along with experimental studies on mutant DNAPs IV and V with amino acid changes in the roof and the chimney.

98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA