Tracking of progressing human DNA polymerase δ holoenzymes reveals distributions of DNA lesion bypass activities

During DNA replication, DNA lesions in lagging strand templates are initially encountered by DNA polymerase δ (pol δ) holoenzymes comprised of pol δ and the PCNA processivity sliding clamp. These encounters are thought to stall replication of an afflicted template before the lesion, activating DNA damage tolerance (DDT) pathways that replicate the lesion and adjacent DNA sequence, allowing pol δ to resume. However, qualitative studies observed that human pol δ can replicate various DNA lesions, albeit with unknown proficiencies, which raises issues regarding the role of DDT in replicating 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 pol δ holoenzymes support 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 does not dissociate. The distributions of these events are dependent on the lesion and pol δ proofreading. Collectively, these results reveal complexity and heterogeneity in the replication of lagging strand DNA lesions, significantly advancing our understanding of human DDT.