Although many genotoxic treatments upregulate the cyclin kinase inhibitor p21, agents such as UV irradiation trigger p21 degradation. This suggests that p21 blocks a process relevant for the cellular response to UV. Here, we show that forced p21 stabilization after UV strongly impairs damaged-DNA replication, which is associated with permanent deficiencies in the recruitment of DNA polymerases from the Y family involved in translesion DNA synthesis), with the accumulation of DNA damage markers and increased genomic instability. Remarkably, such noxious effects disappear when disrupting the proliferating cell nuclear antigen (PCNA) interacting motif of stable p21, thus suggesting that the release of PCNA from p21 interaction is sufficient to allow the recruitment to PCNA of partners (such as Y polymerases) relevant for the UV response. Expression of degradable p21 only transiently delays early replication events and Y polymerase recruitment after UV irradiation. These temporary defects disappear in a manner that correlates with p21 degradation with no detectable consequences on later replication events or genomic stability. Together, our findings suggest that the biological role of UV-triggered p21 degradation is to prevent replication defects by facilitating the tolerance of UV-induced DNA lesions.

After UV irradiation, DNA polymerases specialized in translesion DNA synthesis (TLS) aid DNA replication. However, it is unclear whether other mechanisms also facilitate the elongation of UVdamaged DNA. We wondered if Rad51 recombinase (Rad51), a factor that escorts replication forks, aids replication across UV lesions. We found that depletion of Rad51 impairs S-phase progression and increases cell death after UV irradiation. Interestingly, Rad51 and the TLS polymerase polη modulate the elongation of nascent DNA in different ways, suggesting that DNA elongation after UV irradiation does not exclusively rely on TLS events. In particular, Rad51 protects the DNA synthesized immediately before UV irradiation from degradation and avoids excessive elongation of nascent DNA after UV irradiation. In Rad51-depleted samples, the degradation of DNA was limited to the first minutes after UV irradiation and required the exonuclease activity of the double strand break repair nuclease (Mre11). The persistent dysregulation of nascent DNA elongation after Rad51 knockdown required Mre11, but not its exonuclease activity, and PrimPol, a DNA polymerase with primase activity. By showing a crucial contribution of Rad51 to the synthesis of nascent DNA, our results reveal an unanticipated complexity in the regulation of DNA elongation across UV-damaged templates.PrimPol | polκ | polη | DNA damage tolerance | DNA replication T he DNA-binding protein Rad51 is a central component of homologous recombination repair (HRR). HRR repairs doublestrand breaks (DSBs) in an error-free way and processes one-ended DSBs to reactivate collapsed replication forks (1). During HRR, DSBs are processed by the 3′-to-5′ exonuclease activity of the double strand break repair nuclease (Mre11) to generate protruding 3′ ssDNA at DSBs. The ssDNA is then coated with Rad51, a factor that catalyzes homology search and strand invasion. The loading and stabilization of Rad51/ssDNA complexes are supported by multiple mediators, such as the tumor suppressor BRCA2 (breast cancer 2) (1). Moreover, Rad51 promotes XPF1-and Exo1-mediated DSB formation after gemcitabine-induced irreversible ribonucleotide reductase inhibition, thus promoting cell death (2). The signals that redirect Rad51 into a DSB formation pathway rather than DSB repair are not yet known.The functions of Rad51 are not limited to the processing/ generation of DSBs. Over the past few years, it has become evident that Rad51 escorts ongoing replication forks regardless of the presence of DSBs (3-5). Specifically, Rad51 protects persistently stalled replication forks from Mre11-mediated nucleolytic degradation and facilitates replication fork restart when the replication-halting agent hydroxyurea (HU) or aphidicolin (APH) is removed (6-19). Such novel functions of Rad51 require many HRR factors, including BCRA2, FANCD2 (Fanconi Anemia Complementation group protein D2), CtIP, BRCA1, and the WRN helicase, but are independent of HRR effectors, such as Rad54 (6, 7). Rad51-dependent fork-restart and fork-protection ...