Abstract
Replication stress and DNA damage stall replication forks and impede genome synthesis. During S phase, damage tolerance pathways allow lesion bypass to ensure efficient genome duplication. One such pathway is repriming, mediated by Primase-Polymerase (PrimPol) in human cells. However, the mechanisms by which PrimPol is regulated are poorly understood. Here, we demonstrate that PrimPol is phosphorylated by Polo-like kinase 1 (PLK1) at a conserved residue between PrimPol’s RPA binding motifs. This phosphorylation is differentially modified throughout the cell cycle, which prevents aberrant recruitment of PrimPol to chromatin. Phosphorylation can also be delayed and reversed in response to replication stress. The absence of PLK1-dependent regulation of PrimPol induces phenotypes including chromosome breaks, micronuclei, and decreased survival after treatment with camptothecin, olaparib, and UV-C. Together, these findings establish that deregulated repriming leads to genomic instability, highlighting the importance of regulating this damage tolerance pathway following fork stalling and throughout the cell cycle.
Highlights
The DNA replication machinery regularly encounters obstacles that slow or stall its progression
It enables stalled DNA synthesis to resume by bypassing a diverse range of impediments, without interacting with the obstacle itself, as occurs in translesion synthesis (TLS)
PrimPol-mediated repriming must be tightly regulated, as excessive repriming could lead to increased accumulation of single-stranded DNA (ssDNA) gaps that interfere with other key pathways, such as transcription and replication
Summary
The DNA replication machinery regularly encounters obstacles that slow or stall its progression. Cells have evolved several DNA damage tolerance (DDT) pathways to maintain ongoing replication under perturbed conditions, whose usage is dependent on the environment, type of blockage, and available resources. One such DDT pathway involves repriming DNA synthesis downstream of obstacles to enable stalled replication to resume. PrimPol can reprime DNA synthesis on the leading strand after the fork encounters stalling lesions, such as cyclopyrimidine dimers and structured DNA (e.g., G4 quadruplexes), as well as chain terminators [4,5,6,7]. PrimPol can perform translesion synthesis (TLS) polymerase, replicating across DNA lesions (e.g., 8-oxoG and 6-4 photoproducts) that stall replicative polymerases [6, 7]
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