A PCNA-K164R mutation impinges on origin activation and mitotic DNA synthesis

Abstract

Ubiquitination of the replication clamp proliferating cell nuclear antigen (PCNA) at the conserved residue lysine 164 (K164) occurs during normal S phase progression and increases after DNA damage induced replication stress. This signal is crucial for Okazaki fragment (OF) maturation and for the activation of two DNA damage tolerance pathways; error-prone translesion synthesis and error-free template switching. Recently, we demonstrated that PCNA ubiquitination operates in a fork protection pathway parallel to BRCA-RAD51. However, whether PCNA ubiquitination regulates other genome maintenance mechanisms is unclear. Utilizing PCNAK164R cells generated by CRISPR-Cas9 genome editing, we demonstrate that this mutation impacts origin licensing and causes DNA replication defects. Our data suggest that the accumulation of single-stranded (ss) DNA gaps from the previous replication cycle, interferes with the loading of MCM2-7 double hexamers in the following G1 phase. Insufficient origin licensing leads to under-replicated regions throughout the genome that are not resolved by mitotic DNA synthesis (MiDAS). We uncover a novel role for PCNA-K164 ubiquitination in regulating FANCD2 mono-ubiquitination to initiate MiDAS. Our findings demonstrate that the impact of PCNA-K164 ubiquitination is not limited to S/G2 phases but extends to G1 and mitosis.SUMMARY

BRCA1/BARD1 ubiquitinates PCNA in unperturbed conditions to promote replication fork stability and continuous DNA synthesis

“…While RAD18 would ubiquitinate PCNA when the replication fork encounters a replication fork barrier, BRCA1/BARD1 would perform PCNA ubiquitination at low levels to facilitate continuous DNA synthesis in unperturbed conditions (Figure 2E). PCNA K164 ubiquitination has recently been shown to participate in replication fork protection (Thakar et al, 2020) and in preventing the accumulation of ssDNA gaps during DNA replication in unperturbed conditions (Leung et al, 2020), two functions that have been previously also attributed to BRCA1 (Billing et al, 2018;Cong et al, 2021;Daza-Martin et al, 2019;Panzarino et al, 2021). Different groups have shown that BRCA1-deficient cancer cells accumulate ssDNA gaps and thus can be therapeutically exploited with PARP or REV1 inhibitors (Cong et al, 2021;Taglialatela et al, 2021).…”