Mechanism of error-free and semitargeted mutagenic bypass of an aromatic amine lesion by Y-family polymerase Dpo4

Abstract

The aromatic amine carcinogen 2-aminofluorene (AF) forms covalent adducts with DNA, predominantly with guanine at the C8 position. Such lesions are bypassed by Y-family polymerases such as Dpo4 via error-free and error-prone mechanisms. We show that Dpo4 catalyzes elongation from a correct 3′-terminal C opposite [AF]G in a nonrepetitive template sequence with low efficiency. This extension leads to cognate full-length product, as well as mis-elongated products containing base mutations and deletions. Crystal structures of the Dpo4 ternary complex with the 3′-terminal primer C base opposite [AF]G in the anti conformation and with the AF-moiety positioned in the major groove, revealed both accurate and misalignment-mediated mutagenic extension pathways. The mutagenic template/primer-dNTP arrangement is promoted by interactions between the polymerase and the bulky lesion, rather than by a base pairstabilized misaligment. Further extension leads to semi-targeted mutations via this proposed polymerase-guided mechanism.