Y-Family DNA Polymerases Have Three Structural Elements That Promote Accurate dCTP Insertion And Minimize dATP/dGTP/dTTP Misinsertion Opposite a N2-dG Adduct of Benzo[a]pyrene

Abstract

ABSTRACTTo bypass DNA damage, cells have Y-Family DNA polymerases (DNAPs). One Y-Family-class includes DNAP κ and DNAP IV, which accurately insert dCTP opposite N2-dG adducts, including from the carcinogen benzo[a]pyrene (BP). Another class includes DNAP η and DNAP V, which insert accurately opposite UV-damage, but inaccurately opposite BP-N2-dG. To investigate structural differences between Y-Family-classes, Dpo4 (a canonical η/V-class-member) is modified to make it more κ/IV-like, as evaluated via primer-extension studies with a BP-N2-dG-containing template. Three protein structural elements are identified that promote fidelity. (1) Watson-Crick-like [dCTP:BP-N2-dG] pairing requires the BP-moiety to be in the minor groove. Thus, as expected, dCTP insertion is facilitated by having large openings in the protein surface that can accommodate BP-bulk in the minor groove. The BP-moiety is also in the minor groove during dATP and dTTP misinsertion, though evidence suggests that each of these three minor groove BP-conformations differ. (2) Plugging an opening on the major groove side of the protein suppresses dGTP misinsertion, implying BP-N2-dG bulk is in the major groove during Hoogsteen syn-adduct-dG:dGTP pairing. (3) Y-Family DNAPs have non-covalent bridges (NCBs) holding their little finger-domain in contact with their catalytic core-domain; dATP/dGTP/dTTP misinsertions are suppressed by the quantity and quality of NCBs, including one near and another distal to the active site on the minor groove side. In conclusion, three protein structural elements enhance dCTP and/or suppress dATP/dGTP/dTTP insertion; four different BP-adduct conformations are responsible for the four different dNTP insertional pathways opposite BP-N2-dG; generalizations about Y-Family structure are also considered.