biostudies-literatureLin YCHHS | NIH | National Institute of General Medical SciencesNCI NIH HHSNIGMS NIH HHSNational Science Foundation13282-13287https://www.ebi.ac.uk/biostudies/studies/S-EPMC6310862biostudies-literatureUnknown115(52)RING finger and WD repeat domain-containing protein 3 (RFWD3) is an E3 ligase known to facilitate homologous recombination by removing replication protein A (RPA) and RAD51 from DNA damage sites. Further, RPA-mediated recruitment of RFWD3 to stalled replication forks is essential for interstrand cross-link repair. Here, we report that in unperturbed human cells, RFWD3 localizes at replication forks and associates with proliferating cell nuclear antigen (PCNA) via its PCNA-interacting protein (PIP) motif. PCNA association is critical for the stability of RFWD3 and for DNA replication. Cells lacking RFWD3 show slower fork progression, a prolonged S phase, and an increase in the loading of several replication-fork components on the chromatin. These findings all point to increased frequency ofProceedings of the National Academy of Sciences of the United States of AmericaPCNA-mediated stabilization of E3 ligase RFWD3 at the replication fork is essential for DNA replication.PMC6310862F31 CA180616R01 GM099669R01 GM12519617230081243372GM099669GM1251961818286Hsu RLin YCPrasanth KVArif MKGiri SChakraborty APrasanth SGWang YWopat SfalsePCNA-mediated stabilization of E3 ligase RFWD3 at the replication fork is essential for DNA replication.RING finger and WD repeat domain-containing protein 3 (RFWD3) is an E3 ligase known to facilitate homologous recombination by removing replication protein A (RPA) and RAD51 from DNA damage sites. Further, RPA-mediated recruitment of RFWD3 to stalled replication forks is essential for interstrand cross-link repair. Here, we report that in unperturbed human cells, RFWD3 localizes at replication forks and associates with proliferating cell nuclear antigen (PCNA) via its PCNA-interacting protein (PIP) motif. PCNA association is critical for the stability of RFWD3 and for DNA replication. Cells lacking RFWD3 show slower fork progression, a prolonged S phase, and an increase in the loading of several replication-fork components on the chromatin. These findings all point to increased frequency of2018-01-01T00:00:00Z2018 Dec2025-04-21T21:39:07.886Z2019-09-14T07:00:57ZS-EPMC63108623053069410.1073/pnas.1814521115