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

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^*

^*Corresponding author for this work

Research output: Contribution to journal › Article › peer-review

92 Citations (Scopus)

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.

Original language	English
Pages (from-to)	622-634.e8
Journal	Molecular Cell
Volume	66
Issue number	5
DOIs	https://doi.org/10.1016/j.molcel.2017.04.022
Publication status	Published - 2017 Jun 1

Keywords

BRCA2
Fanconi anemia
homologous recombination
ICL repair
RAD51
RFWD3
RPA

ASJC Scopus subject areas

Molecular Biology
Cell Biology

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10.1016/j.molcel.2017.04.022

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Inano, S., Sato, K., Katsuki, Y., Kobayashi, W., Tanaka, H., Nakajima, K., Nakada, S., Miyoshi, H., Knies, K., Takaori-Kondo, A., Schindler, D., Ishiai, M., Kurumizaka, H., & Takata, M. (2017). RFWD3-Mediated Ubiquitination Promotes Timely Removal of Both RPA and RAD51 from DNA Damage Sites to Facilitate Homologous Recombination. Molecular Cell, 66(5), 622-634.e8. https://doi.org/10.1016/j.molcel.2017.04.022

Inano, S, Sato, K, Katsuki, Y, Kobayashi, W, Tanaka, H, Nakajima, K, Nakada, S, Miyoshi, H, Knies, K, Takaori-Kondo, A, Schindler, D, Ishiai, M, Kurumizaka, H & Takata, M 2017, 'RFWD3-Mediated Ubiquitination Promotes Timely Removal of Both RPA and RAD51 from DNA Damage Sites to Facilitate Homologous Recombination', Molecular Cell, vol. 66, no. 5, pp. 622-634.e8. https://doi.org/10.1016/j.molcel.2017.04.022

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

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.",

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

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AU - Inano, Shojiro

AU - Sato, Koichi

AU - Katsuki, Yoko

AU - Kobayashi, Wataru

AU - Tanaka, Hiroki

AU - Nakajima, Kazuhiro

AU - Nakada, Shinichiro

AU - Miyoshi, Hiroyuki

AU - Knies, Kerstin

AU - Takaori-Kondo, Akifumi

AU - Schindler, Detlev

AU - Ishiai, Masamichi

AU - Kurumizaka, Hitoshi

AU - Takata, Minoru

PY - 2017/6/1

Y1 - 2017/6/1

N2 - 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.

AB - 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.

KW - BRCA2

KW - Fanconi anemia

KW - homologous recombination

KW - ICL repair

KW - RAD51

KW - RFWD3

KW - RPA

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

Abstract

Keywords

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