After exposure to DNA-damaging agents that block the progress of the replication fork, monoubiquitination of proliferating cell nuclear antigen (PCNA) mediates the switch from replicative to translesion synthesis DNA polymerases. We show that in human cells, PCNA is monoubiquitinated in response to methyl methanesulfonate and mitomycin C, as well as UV light, albeit with different kinetics, but not in response to bleomycin or camptothecin. Cyclobutane pyrimidine dimers are responsible for most of the PCNA ubiquitination events after UV-irradiation. Failure to ubiquitinate PCNA results in substantial sensitivity to UV and methyl methanesulfonate, but not to camptothecin or bleomycin. PCNA ubiquitination depends on Replication Protein A (RPA), but is independent of ATR-mediated checkpoint activation. After UV-irradiation, there is a temporal correlation between the disappearance of the deubiquitinating enzyme USP1 and the presence of PCNA ubiquitination, but this correlation was not found after chemical mutagen treatment. By using cells expressing photolyases, we are able to remove the UV lesions, and we show that PCNA ubiquitination persists for many hours after the damage has been removed. We present a model of translesion synthesis behind the replication fork to explain the persistence of ubiquitinated PCNA.

DNA polymerase η (polη) belongs to the Y-family of DNA polymerases and facilitates translesion synthesis past UV damage. We show that, after UV irradiation, polη becomes phosphorylated at Ser601 by the ataxia-telangiectasia mutated and Rad3-related (ATR) kinase. DNA damage–induced phosphorylation of polη depends on its physical interaction with Rad18 but is independent of PCNA monoubiquitination. It requires the ubiquitin-binding domain of polη but not its PCNA-interacting motif. ATR-dependent phosphorylation of polη is necessary to restore normal survival and postreplication repair after ultraviolet irradiation in xeroderma pigmentosum variant fibroblasts, and is involved in the checkpoint response to UV damage. Taken together, our results provide evidence for a link between DNA damage–induced checkpoint activation and translesion synthesis in mammalian cells.

The use of translesion synthesis (TLS) polymerases to bypass DNA lesions during replication constitutes an important mechanism to restart blocked/stalled DNA replication forks. Because TLS polymerases generally have low fidelity on undamaged DNA, the cell must regulate the interaction of TLS polymerases with damaged versus undamaged DNA to maintain genome integrity. The Saccharomyces cerevisiae checkpoint proteins Ddc1, Rad17, and Mec3 form a clamp-like structure (the 9-1-1 clamp) that has physical similarity to the homotrimeric sliding clamp proliferating cell nuclear antigen, which interacts with and promotes the processivity of the replicative DNA polymerases. In this work, we demonstrate both an in vivo and in vitro physical interaction between the Mec3 and Ddc1 subunits of the 9-1-1 clamp and the Rev7 subunit of the Polζ TLS polymerase. In addition, we demonstrate that loss of Mec3, Ddc1, or Rad17 results in a decrease in Polζ-dependent spontaneous mutagenesis. These results suggest that, in addition to its checkpoint signaling role, the 9-1-1 clamp may physically regulate Polζ-dependent mutagenesis by controlling the access of Polζ to damaged DNA.

Andrew Jackson, Grant Stewart, Bernd Wollnik and colleagues identify TRAIP mutations in three patients with primordial dwarfism. They show that TRAIP is involved in DNA damage response during genome replication and is necessary for efficient cell cycle progression. DNA lesions encountered by replicative polymerases threaten genome stability and cell cycle progression. Here we report the identification of mutations in TRAIP, encoding an E3 RING ubiquitin ligase, in patients with microcephalic primordial dwarfism. We establish that TRAIP relocalizes to sites of DNA damage, where it is required for optimal phosphorylation of H2AX and RPA2 during S-phase in response to ultraviolet (UV) irradiation, as well as fork progression through UV-induced DNA lesions. TRAIP is necessary for efficient cell cycle progression and mutations in TRAIP therefore limit cellular proliferation, providing a potential mechanism for microcephaly and dwarfism phenotypes. Human genetics thus identifies TRAIP as a component of the DNA damage response to replication-blocking DNA lesions.