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

Shojiro Inano; Koichi Sato; Yoko Katsuki; Wataru Kobayashi; Hiroki Tanaka; Kazuhiro Nakajima; Shinichiro Nakada; Hiroyuki Miyoshi; Kerstin Knies; Akifumi Takaori-Kondo; Detlev Schindler; Masamichi Ishiai; Hitoshi Kurumizaka; Minoru Takata

doi:10.1016/j.molcel.2017.04.022

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.

Affiliations

¹ Laboratory of DNA Damage Signaling, Department of Late Effects Studies, Radiation Biology Center, Kyoto University, Kyoto 606-8501, Japan; Department of Hematology and Oncology, Graduate School of Medicine, Kyoto University, Kyoto 606-8507, Japan.
² Laboratory of Structural Biology, Graduate School of Advanced Science and Engineering, Waseda University, Tokyo 169-8050, Japan.
³ Laboratory of DNA Damage Signaling, Department of Late Effects Studies, Radiation Biology Center, Kyoto University, Kyoto 606-8501, Japan.
⁴ Department of Bioregulation and Cellular Response, Graduate School of Medicine, Osaka University, Osaka 565-0871, Japan.
⁵ Department of Bioregulation and Cellular Response, Graduate School of Medicine, Osaka University, Osaka 565-0871, Japan; Institute for Advanced Co-Creation Studies, Osaka University, Osaka 565-0871, Japan.
⁶ Department of Physiology, Keio University School of Medicine, Tokyo 160-8582, Japan.
⁷ Department of Human Genetics, Biozentrum, University of Wurzburg, 97074 Wurzburg, Germany.
⁸ Department of Hematology and Oncology, Graduate School of Medicine, Kyoto University, Kyoto 606-8507, Japan.
⁹ Laboratory of DNA Damage Signaling, Department of Late Effects Studies, Radiation Biology Center, Kyoto University, Kyoto 606-8501, Japan. Electronic address: [email protected].

PMID: 28575658
DOI: 10.1016/j.molcel.2017.04.022

Abstract

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

Substances

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