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

摘要

During DNA replication, DNA lesions in lagging strand templates are initially encountered by DNA polymerase delta (pol delta) holoenzymes comprised of pol delta 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 delta to resume. However, qualitative studies observed that human pol delta 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 delta holoenzymes with DNA lesions. The results indicate pol delta holoenzymes support dNTP incorporation opposite and beyond multiple lesions and the extent of these activities depends on the lesion and pol delta proofreading. Furthermore, after encountering a given DNA lesion, subsequent dissociation of pol delta is distributed around the lesion and a portion does not dissociate. The distributions of these events are dependent on the lesion and pol delta proofreading. Collectively, these results reveal complexity and heterogeneity in the replication of lagging strand DNA lesions, significantly advancing our understanding of human DDT.