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Mol Cell. 2017 Jun 1;66(5):622-634.e8. 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.

Author information

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: mtakata@house.rbc.kyoto-u.ac.jp.

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

PMID:
28575658
DOI:
10.1016/j.molcel.2017.04.022
[Indexed for MEDLINE]
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