RFWD3-Mediated Ubiquitination Promotes Timely Removal of Both RPA and RAD51 from DNA Damage Sites to Facilitate Homologous Recombination

Mol Cell. 2017 Jun 1;66(5):622-634.e8. doi: 10.1016/j.molcel.2017.04.022.


RFWD3 is a recently identified Fanconi anemia protein FANCW whose E3 ligase activity toward RPA is essential in homologous recombination (HR) repair. However, how RPA ubiquitination promotes HR remained unknown. Here, we identified RAD51, the central HR protein, as another target of RFWD3. We show that RFWD3 polyubiquitinates both RPA and RAD51 in vitro and in vivo. Phosphorylation by ATR and ATM kinases is required for this activity in vivo. RFWD3 inhibits persistent mitomycin C (MMC)-induced RAD51 and RPA foci by promoting VCP/p97-mediated protein dynamics and subsequent degradation. Furthermore, MMC-induced chromatin loading of MCM8 and RAD54 is defective in cells with inactivated RFWD3 or expressing a ubiquitination-deficient mutant RAD51. Collectively, our data reveal a mechanism that facilitates timely removal of RPA and RAD51 from DNA damage sites, which is crucial for progression to the late-phase HR and suppression of the FA phenotype.

Keywords: BRCA2; Fanconi anemia; ICL repair; RAD51; RFWD3; RPA; homologous recombination.

MeSH terms

  • Adenosine Triphosphatases / genetics
  • Adenosine Triphosphatases / metabolism
  • Ataxia Telangiectasia Mutated Proteins / metabolism
  • Binding Sites
  • Cell Cycle Proteins / genetics
  • Cell Cycle Proteins / metabolism
  • Cell Line, Tumor
  • Chromatin / drug effects
  • Chromatin / enzymology*
  • Chromatin / genetics
  • Chromatin / radiation effects
  • DNA / genetics
  • DNA / metabolism*
  • DNA Damage*
  • Fanconi Anemia / enzymology*
  • Fanconi Anemia / genetics
  • Humans
  • Minichromosome Maintenance Proteins / metabolism
  • Mitomycin / pharmacology
  • Mutation
  • Phosphorylation
  • Proteasome Endopeptidase Complex / metabolism
  • Protein Binding
  • Proteolysis
  • RNA Interference
  • Rad51 Recombinase / genetics
  • Rad51 Recombinase / metabolism*
  • Recombinational DNA Repair* / drug effects
  • Recombinational DNA Repair* / radiation effects
  • Replication Protein A / genetics
  • Replication Protein A / metabolism*
  • Transfection
  • Ubiquitin-Protein Ligases / genetics
  • Ubiquitin-Protein Ligases / metabolism*
  • Ubiquitination*
  • Valosin Containing Protein


  • Cell Cycle Proteins
  • Chromatin
  • Replication Protein A
  • Mitomycin
  • DNA
  • RFWD3 protein, human
  • Ubiquitin-Protein Ligases
  • ATM protein, human
  • ATR protein, human
  • Ataxia Telangiectasia Mutated Proteins
  • RAD51 protein, human
  • Rad51 Recombinase
  • RPA2 protein, human
  • Proteasome Endopeptidase Complex
  • Adenosine Triphosphatases
  • MCM8 protein, human
  • Minichromosome Maintenance Proteins
  • VCP protein, human
  • Valosin Containing Protein