PCNA-mediated stabilization of E3 ligase RFWD3 at the replication fork is essential for DNA replication

Abstract

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 of stalled forks in the absence of RFWD3. The S-phase defect is rescued by WT RFWD3, but not by the PIP mutant, suggesting that the interaction of RFWD3 with PCNA is critical for DNA replication. Finally, we observe reduced ubiquitination of RPA in cells lacking RFWD3. We conclude that the stabilization of RFWD3 by PCNA at the replication fork enables the polyubiquitination of RPA and its subsequent degradation for proper DNA replication.

TheDrosophila melanogasterOrtholog of RFWD3 Functions Independently of RAD51 During DNA Repair

“…RFWD3 mutant cells exhibit increased foci of RPA and RAD51 when treated with MMC (Feeney et al 2017). Consistent with these observations, RFWD3 localizes to replication forks in a proliferating cell nuclear antigen (PCNA)-dependent manner (Lin et al 2018). In addition, RFWD3 is phosphorylated by the DNA damage response kinase ATR (and possibly ATM) (Fu et al 2010;Feeney et al 2017), and this may be required for its function.…”

Target highlights in CASP13: Experimental target structures through the eyes of their authors

“…Furthermore, elongated strands of repeat five appear to be a feature unique to RFWD3 (Figure B panel iv). Interestingly, residues involved in DDR are not part of these insertions, including Trp‐543 and Ile‐639 for binding RPA32, and the QKMDF 624 consensus motif that mediates the interaction with PCNA (proliferating cell nuclear antigen) during DNA replication …”

“…2,3 Evidence has shown that the WDR domain is primarily responsible for the functional interactions that allow RFWD3 to maintain genomic stability. 4,5 Furthermore, heritable mutations, particularly an Ile639Lys point mutation within the WDR domain, may lead to the rare genomic instability disease known as Fanconi anemia (FA), 6 thereby implicating RFWD3 as a potential FA-associated gene (alias: FANCW). [4][5][6] Until recently, the biochemical characterization of RFWD3 has lacked complementary highresolution structural information.…”

“…4,5 Furthermore, heritable mutations, particularly an Ile639Lys point mutation within the WDR domain, may lead to the rare genomic instability disease known as Fanconi anemia (FA), 6 thereby implicating RFWD3 as a potential FA-associated gene (alias: FANCW). [4][5][6] Until recently, the biochemical characterization of RFWD3 has lacked complementary highresolution structural information. Here, we discuss the 1.8 Å resolution X-ray crystal structure of the C-terminal WDR domain of human RFWD3 ( Table 1, Target: T0954; PDB: 6CVZ).…”

“…ates the last eight helices(3)(4)(5)(6)(7)(8)(9)(10). Connecting the helices are the HVSs that form short β-turn loops at both ends of the domain.…”