USP1 was identified in a siRNA screen as a DUB responsible for the deubiquitylation of PCNA following DNA damage bypass [61
]. USP1 is also involved in the deubiquitylation of FANCD2, a Fanconi anemia effector protein that functions in the repair of DNA interstrand crosslinks [62
]. Considering the opposing roles of the E1/E2/E3 ubiquitinating enzymes and the DUBs, it is not surprising that exposure to UV light, which promotes PCNA monoubiquitylation, also decreases USP1 protein levels. The mechanism for this decrease involves an autocleavage event followed by proteasomal degredation of the cleaved products. The ubiquitylation of PCNA following replication stress is not always accompanied by the disappearance of USP1, though. For example, treatment with hydroxyurea (HU) results in no detectable change in USP1. To explain this phenomenon, it has been suggested that HU disrupts the interaction between USP1 and its activating partner protein UAF1 (USP1-associated factor 1) [12
The importance of PCNA deubiquitylation is highlighted by the finding that depletion of USP1 increases the level of mutagenesis in the cell [61
]. It is thought that persistent PCNA ubiquitylation results in the overuse and subsequent disregulation of the TLS polymerases and allows the error-prone TLS polymerases to replicate undamaged DNA [61
]. Even Polη, which can faithfully replicate past UV-induced cyclobutane pyrimidine dimers, exhibits much lower fidelity compared to replicative polymerases when copying undamaged DNA [63
], and would thus contribute to increased mutagenesis if used inappropriately. Such mutations could ultimately disrupt basic cellular processes and/or cause uncontrolled proliferation, the latter of which is a hallmark of cancer. In addition to being more error-prone than the replicative DNA polymerases, the TLS polymerases also catalyze DNA synthesis much more slowly and thus could potentially cause replication fork collapse. As discussed above, collapsed replication forks are particularly dangerous to cells, as they often result in genomic instability.
Insights into the possible physiological consequences of defects in PCNA deubiquitylation come from a USP1 transgenic mouse model. Usp1−/−
mice displayed an increase in both FANCD2 and PCNA monoubiquitylation, and exhibited a high rate of perinatal lethality, depletion of male germ cells, infertility, hypersensitivity to the crosslinking agent mitomycin C, and chromosome instability. Usp1−/−
mice were also much smaller in size than their wildtype littermates [64
]. It remains to be determined whether these mice exhibit increased point mutation frequency and increased cancer incidence as would be expected given the in vitro
data described above.