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 cytosine 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 cytosine base opposite [AF]G in the anti conformation and with the AF moiety positioned in the major groove, reveal 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 pair-stabilized misaligment. Further extension leads to semitargeted mutations via this proposed polymerase-guided mechanism.
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We thank Y.Cheng for help with expression and purification of Dpo4, A. Serganov for data collection from the [AF]G•C-1 and [AF]G•A-2 crystals and L. Wang (New York Univ.) for providing the coordinates of the [AF]G-modified DNA duplexes. The research was supported by US National Institutes of Health grants CA46533 to D.J.P., CA75449 to S.B. and CA99194 to N.E.G. Partial support for computational infrastructure and computer systems management was also provided to S.B. by CA28038. We would like to thank the staff at the Northeastern Collaborative Access Team beamlines of the Advanced Photon Source (APS), Argonne National Laboratory, supported by award RR-15301 from the National Center for Research Resources at the National Institute of Health, for assistance with data collection. Use of the Advanced Photon Source is supported by the U.S. Department of Energy, Office of Basic Energy Sciences, under Contract No. DE-AC02-06CH11357.