In vivo evidence for translesion synthesis by the replicative DNA polymerase delta

摘要

The intolerance of DNA polymerase delta (Pol delta) to incorrect base pairing contributes to its extremely high accuracy during replication, but is believed to inhibit translesion synthesis (TLS). However, chicken DT40 cells lacking the POLD3 subunit of Pol delta are deficient in TLS. Previous genetic and biochemical analysis showed that POLD3 may promote lesion bypass by Pol delta itself independently of the translesion polymerase Pol delta of which POLD3 is also a subunit. To test this hypothesis, we have inactivated Pol delta proofreading inpold3 cells. This significantly restored TLS in pold3 mutants, enhancing dA incorporation opposite abasic sites. Purified proofreading-deficient human Pol delta holoenzyme performs TLS of abasic sites in vitro much more efficiently than the wild type enzyme, with over 90% of TLS events resulting in dA incorporation. Furthermore, proofreading deficiency enhances the capability of Pol delta to continue DNA synthesis over UV lesions both in vivo and in vitro. These data support Pol delta contributing to TLS in vivo and suggest that the mutagenesis resulting from loss of Pol delta proofreading activity may in part be explained by enhanced lesion