Y-Family DNA polymerases may use two different dNTP shapes for insertion: A hypothesis and its implications

Chemicals and radiation can damage DNA leading to the formation of adducts/lesions, which – if not removed by DNA repair pathways – usually block replicative DNA polymerases (DNAPs). To overcome such potentially lethal blockage, cells have lesion bypass DNAPs, which are often in the Y-Family and include several classes. One class includes human DNAP κ and E. coli DNAP IV, and they insert dCTP in the non-mutagenic pathway opposite [+ta]-B[a]P-N2-dG, which is the major adduct formed by the environmental carcinogen benzo[a]pyrene. Another class includes hDNAP η and ecDNAP V, and they insert dATP opposite [+ta]-B[a]P-N2-dG in the dominant G → T mutagenic pathway. Herein we develop a hypothesis for why the IV/κ-class preferentially does cellular dCTP insertion. On the minor groove side of the active site, Y-Family DNAPs have a cleft/hole that can be analyzed based on an analogy to a “chimney.” Our models of DNAP IV show a large chimney opening from which the pyrene of [+ta]-B[a]P-N2-dG can protrude, which allows canonical adduct-dG:dCTP pairing. In contrast, our models of DNAP V have small chimney openings that forces adduct-dG downward in the active site such that canonical adduct-dG:dCTP pairing is not possible. Based on X-ray structures, sequence alignment and our modeled structures of Y-Family DNAPs, chimney opening size seems primarily controlled by one amino acid (“flue-handle”), which dictates whether nearby amino acids (“flue”) plug the chimney or not. Based on this analysis, a correlation is apparent: the flue is closed in V/η-class DNAPs giving small chimney openings, while the flue is open for the IV/κ-class giving large chimney openings. Secondarily, a hypothesis is developed for why the V/η-class might preferentially do cellular dATP insertion opposite [+ta]-B[a]P-N2-dG: the small chimney forces adduct-dG lower in the active site, possibly leading to catalysis using a non-canonical dNTP shape that permits syn-adenine:adduct-dG base pairing. In summary, a hypothesize is developed that the pyrene moiety of [+ta]-B[a]P-N2-dG protrudes from the large chimney opening of DNAP IV, thus permitting canonical dCTP:adduct-dG pairing, while the small chimney opening of DNAP V forces [+ta]-B[a]P-N2-dG lower down in the active site, in which syn-adenine can pair with adduct-dG via a non-canonical dNTP shape.