Time-Dependent X-ray Crystallography Reveals Intriguing Insights on DNA Polymerase-Catalyzed DNA Synthesis and Lesion Bypass
DNA polymerases are responsible for replicating DNA in an efficient and faithful manner. Over the years the steps of the catalytic mechanism leading up to nucleotide incorporation have been well tested and described. However, the post-chemistry events are difficult to investigate and are less understood. In addition, the perturbation of the catalytic mechanism by DNA damage has been of high interest to the field. To answer these difficult mechanistic questions, our lab has investigated human DNA polymerase β (hPolβ), a key enzyme involved in human base excision repair (BER) pathway. Recently, we utilized nucleotide analogs to structurally investigate the post-catalytic events following hPolβ-catalyzed nucleotidyltransfer and employed time-dependent X-ray crystallography to follow the bypass and extension from a common oxidative DNA lesion, 8-oxoG, often repaired through BER. Contrary to the hypothesis that pyrophosphate (PPi) product release would trigger or occur concurrently with the reverse conformational change of the hPolβ thumb domain after phosphodiester bond formation, we found PPi remain bound following the reverse protein conformational change thereby defining the precise order of post-chemistry events. In addition, we show that the newly identified third divalent metal ion does not appear to facilitate the product release as previously proposed. During the bypass and extension from 8-oxoG, we observe a third divalent metal ion, and identify the structural bases for the relatively unfaithful bypass and subsequent inefficient extension of the mismatched bypass products. Together, these studies help answer key mechanistic questions left unanswered by kinetic and traditional structural approaches as well as shed light on the involvement of hPolβ in DNA repair and human diseases.