High resolution studies of DNA lesion bypass by human DNA polymerase δ holoenzymes

ABSTRACT During DNA replication, DNA lesions present in lagging strand templates are initially encountered by DNA polymerase δ (pol δ). The historical view for what transpires from these encounters is that replication of the afflicted lagging strand template abruptly stops, activating DNA damage tolerance (DDT) pathways that replicate the offending lesion and adjacent DNA sequence, allowing pol δ to resume downstream. However, qualitative studies observed that human pol δ is capable of replicating various DNA lesions, albeit to unknown extents, which raises issues regarding the roles of pol δ and DDT in the replication of DNA lesions. To address these issues, we re-constituted human lagging strand replication to quantitatively characterize initial encounters of pol δ holoenzymes with DNA lesions. The results indicate that pol δ holoenzymes support stable dNTP incorporation opposite and beyond multiple lesions and the extent of these activities depends on the lesion and pol δ proofreading. Furthermore, after encountering a given DNA lesion, subsequent dissociation of pol δ is distributed around the lesion and a portion of pol δ does not dissociate at all. The distributions of these events are dependent on the lesion and pol δ proofreading. These results challenge our understanding of DNA lesion replication and DDT.