Structural basis for highly promutagenic replication across O6‐methylguanine by human DNA polymerase β (551.3)

Abstract

O6‐methylguanine (O6MeG) is a minor, yet highly genotoxic lesion that promotes misincorporation of T opposite O6MeG by various DNA polymerases in vitro. Human DNA polymerase β (polβ) has been shown to insert T opposite O6MeG ~30‐fold more frequently than C, albeit with a ~1000‐fold reduced insertion efficiency relative to the insertion of C opposite G. Currently, the structural basis for this slow, yet promutagenic phenomenon is unknown. To gain insight into this process, we solved seven X‐ray structures of polβ bound to O6MeG‐containing DNA: a binary structure with a gap opposite O6MeG, two ground state and two pre‐catalytic ternary structures with an incoming dC/T nucleotide paired with O6MeG, and two binary post‐insertion structures. The two ground‐state ternary structures with active site Mg2+ showed an open protein conformation and staggered base pair conformation, rather than the closed protein conformation and co‐planar base pair conformation typically observed for correct insertion. This structural difference suggests that polβ slows nucleotide incorporation opposite O6MeG by inducing an alternate conformation that is suboptimal for nucleotidyl transfer. The two pre‐catalytic ternary structures with active site Mn2+ showed that, remarkably, the T:O6MeG‐Mn2+ structure adopts closed protein conformation and Watson‐Crick‐type base pairing, whereas the C:O6MeG‐Mn2+ structure adopts open protein conformation and staggered base pairing. These striking structural differences between the T:O6MeG‐Mn2+ and the C:O6MeG‐Mn2+ complexes clearly explain preferential T misincorporation over C opposite O6MeG during replication by the enzyme. Our polβ structures featuring metal ion‐dependent conformational reorganization reveal structural basis for the slow, yet highly promutagenic replication across O6MeG by polβ.Grant Funding Source: Supported by the NIH ES23101