Search

All cells rely on genomic stability mechanisms to protect against DNA alterations. PCNA is a master regulator of DNA replication and S-phase-coupled repair. PCNA post-translational modifications by ubiquitination and SUMOylation dictate how cells stabilize and re-start replication forks stalled at sites of damaged DNA. PCNA mono-ubiquitination recruits low fidelity DNA polymerases to promote error-prone replication across DNA lesions. Here, we identify the mono-ADP-ribosyltransferase PARP10/ARTD10 as a novel PCNA binding partner. PARP10 knockdown results in genomic instability and DNA damage hypersensitivity. Importantly, we show that PARP10 binding to PCNA is required for translesion DNA synthesis. Our work identifies a novel PCNA-linked mechanism for genome protection, centered on post-translational modification by mono-ADP-ribosylation.

SummaryDamaged DNA templates provide an obstacle to the replication fork and can cause genome instability. In eukaryotes, tolerance to damaged DNA is mediated largely by the RAD6 pathway involving ubiquitylation of the DNA polymerase processivity factor PCNA. Whereas monoubiquitylation of PCNA mediates error-prone translesion synthesis (TLS), polyubiquitylation triggers an error-free pathway. Both branches of this pathway are believed to occur in S phase in order to ensure replication completion. However, we found that limiting TLS or the error-free pathway to the G2/M phase of the cell-cycle efficiently promote lesion tolerance. Thus, our findings indicate that both branches of the DNA damage tolerance pathway operate effectively after chromosomal replication, outside S phase. We therefore propose that the RAD6 pathway acts on single-stranded gaps left behind newly restarted replication forks.

The RAD6 pathway allows replication across DNA lesions by either an error-prone or error-free mode. Error-prone replication involves translesion polymerases and requires monoubiquitylation at lysine (K) 164 of PCNA by the Rad6 and Rad18 enzymes. By contrast, the error-free bypass is triggered by modification of PCNA by K63-linked polyubiquitin chains, a reaction that requires in addition to Rad6 and Rad18 the enzymes Rad5 and Ubc13–Mms2. Here, we show that the RAD6 pathway is also critical for controlling repair pathways that act on DNA double-strand breaks. By using gapped plasmids as substrates, we found that repair in wild-type cells proceeds almost exclusively by homology-dependent repair (HDR) using chromosomal DNA as a template, whereas non-homologous end-joining (NHEJ) is suppressed. In contrast, in cells deficient in PCNA polyubiquitylation, plasmid repair occurs largely by NHEJ. Mutant cells that are completely deficient in PCNA ubiquitylation, repair plasmids by HDR similar to wild-type cells. These findings are consistent with a model in which unmodified PCNA supports HDR, whereas PCNA monoubiquitylation diverts repair to NHEJ, which is suppressed by PCNA polyubiquitylation. More generally, our data suggest that the balance between HDR and NHEJ pathways is crucially controlled by genes of the RAD6 pathway through modifications of PCNA.

Damaged DNA is an obstacle during DNA replication and a cause of genome instability and cancer. To bypass this problem, eukaryotes activate DNA damage tolerance (DDT) pathways that involve ubiquitylation of the DNA polymerase clamp proliferating cell nuclear antigen (PCNA). Monoubiquitylation of PCNA mediates an error-prone pathway by recruiting translesion polymerases, whereas polyubiquitylation activates an error-free pathway that utilizes undamaged sister chromatids as templates. The error-free pathway involves recombination-related mechanisms; however, the factors that act along with polyubiquitylated PCNA remain largely unknown. Here we report that the PCNA-related 9-1-1 complex, which is typically linked to checkpoint signaling, participates together with Exo1 nuclease in error-free DDT. Notably, 9-1-1 promotes template switching in a manner that is distinct from its canonical checkpoint functions and uncoupled from the replication fork. Our findings thus reveal unexpected cooperation in the error-free pathway between the two related clamps and indicate that 9-1-1 plays a broader role in the DNA damage response than previously assumed.