Temporally distinct post-replicative repair mechanisms fill PRIMPOL-dependent ssDNA gaps in human cells

PRIMPOL repriming allows DNA replication to skip DNA lesions, leading to ssDNA gaps. These gaps must be filled to preserve genome stability. Using a DNA fiber approach to directly monitor gap filling, we studied the post-replicative mechanisms that fill the ssDNA gaps generated in cisplatin-treated cells upon increased PRIMPOL expression or when replication fork reversal is defective because of SMARCAL1 inactivation or PARP inhibition. We found that a mechanism dependent on the E3 ubiquitin ligase RAD18, PCNA monoubiquitination, and the REV1 and POLz translesion synthesis polymerases promotes gap filling in G2. The E2 conjugating enzyme UBC13, the RAD51 recombinase, and REV1-POLz are instead responsible for gap filling in S, suggesting that temporally distinct pathways of gap filling operate throughout the cell cycle. Furthermore, we found that BRCA1 and BRCA2 promote gap filling by limiting MRE11 activity and that simultaneously targeting fork reversal and gap filling enhances chemosensitivity in BRCA-deficient cells.

Automated summary

By studying the mechanisms involved in maintaining genome stability, it was found that different pathways are responsible for filling single-stranded DNA gaps throughout the cell cycle, with different proteins and enzymes acting at different stages. Additionally, BRCA1 and BRCA2 play important roles in limiting MRE11 activity for gap filling, and simultaneously targeting fork reversal and gap filling enhances chemosensitivity in BRCA-deficient cells.