O6-methylguanine (O6MeG) is a minor, yet highly genotoxic lesion that promotes misinsertion of T opposite O6MeG by various DNA polymerases in vitro. Human DNA polymerase � (pol �) has shown to insert T opposite O6MeG ~30-fold than C, albeit with a ~1000-fold reduced insertion efficiency relative to the C insertion opposite G. Currently, structural basis for this slow, yet promutagenic insertion opposite O6MeG by pol � is unknown. To gain insights into this, we have solved five X-ray structures of pol � bound to O6MeG-containing DNA: a binary structure with a gap opposite O6MeG, two ternary pre-catalytic structures with an incoming dC/T nucleotide and templating O6MeG, and two binary post-insertion structures. All the previously reported pol � ternary structures with a nascent base pair mismatch possessed either an alternate metal ion (e.g. Mn2+) or an active-site residue mutation, preventing it from occurring a thorough understanding of the misinsertion mechanism of the enzyme. Our O6MeG�C/T ternary complex structures, the first structures of wild-type pol � with a nascent base pair mismatch and Mg2+ ion, suggest that pol � slows the nucleotide insertion opposite O6MeG by forming an open protein conformation, a staggered nascent base pairing, and one-metal-binding state, rather than forming a closed protein conformation, a co-planar base paring, and two-metal-binding state typically observed for correct insertion. These ternary complex structures also suggest that a difference between the water-mediated H-bond networks found in the insertion sites of the O6MeG�C/T ternary complexes may contribute to the preferential T insertion opposite O6MeG.

Structural basis for slow yet preferential misincorporation of dTTP opposite O6-methylguanine by human DNA polymerase �

247th National Meeting (2014)

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