Genetic evidence for reconfiguration of DNA polymerase θ active site for error-free translesion synthesis in human cells

The action mechanisms revealed by the biochemical and structural analyses of replicative and translesion synthesis (TLS) DNA polymerases (Pols) are retained in their cellular roles. In this regard, DNA polymerase ? differs from other Pols in that whereas purified Pol? misincorporates an A opposite 1,N-6-ethenodeoxyadenosine (?dA) using an abasic-like mode, Pol? performs predominantly error-free TLS in human cells. To test the hypothesis that Pol? adopts a different mechanism for replicating through ?dA in human cells than in the purified Pol, here we analyze the effects of mutations in the two highly conserved tyrosine residues, Tyr-2387 and Tyr-2391, in the Pol? active site. Our findings that these residues are indispensable for TLS by the purified Pol but are not required in human cells, as well as other findings, provide strong evidence that the Pol? active site is reconfigured in human cells to stabilize ?dA in the syn conformation for Hoogsteen base pairing with the correct nucleotide. The evidence that a DNA polymerase can configure its active site entirely differently in human cells than in the purified Pol establishes a new paradigm for DNA polymerase function.