RFWD3/FANCW orchestrates post-replicative filling of ssDNA gaps to promote UV tolerance

DNA lesions can stall replication, generating ssDNA gaps that require filling to maintain genome stability. The E3 ubiquitin ligase RFWD3/FANCW targets RPA and PCNA to regulate replication dynamics and damage tolerance, but its roles in these processes and influence on cell mutability remain incompletely understood. Here, we show that RFWD3 prevents the accumulation of ssDNA gaps in UV-irradiated cells through its E3 ligase activity and interaction with RPA. Mutational signature analysis implicates RFWD3 in the spontaneous mutability of human cells and reveals its involvement in 6-4 photoproducts tolerance. Accordingly, photorepair of 6-4 photoproducts, but not of cyclobutane pyrimidine dimers, alleviates gap accumulation in RFWD3-depleted cells. RFWD3 KO has limited impact on PCNA ubiquitylation, but its depletion sharply decreases UV-induced RPA ubiquitylation. Moreover, cells expressing an RPA mutant that cannot be ubiquitylated accumulate ssDNA gaps epistatically with RFWD3 loss. We propose that RFWD3-dependent RPA ubiquitylation regulates post-replicative ssDNA gaps to promote helix-distorting lesion tolerance. DNA lesions caused by UV light can block replication and generate single-stranded DNA gaps. Here, the authors reveal that the E3 ubiquitin ligase RFWD3 ubiquitylates RPA to prevent gap accumulation and promote DNA damage tolerance. This process helps maintain genome stability and regulates cell mutability.