Functional analysis of Rad51 activation by Rad55-Rad57 and Swi5-Sfr1

2019 Fiscal Year Annual Research Report

• Project/Area Number: 17K15061
• Research Institution: Tokyo Institute of Technology
• Principal Investigator: Argunhan Bilge 東京工業大学, 科学技術創成研究院, 特任助教 (30792759)
• Project Period (FY): 2017-04-01 – 2020-03-31
• Keywords: DNA repair / Homologous recombination / Rad51 / Swi5-Sfr1 / Rad55-Rad57 / fission yeast

Outline of Annual Research Achievements

Homologous recombination (HR) is a major pathway for the repair of DNA double-strand breaks (DSBs), a severe form of DNA damage. During HR, genetic information from an intact region of the genome is used as a template to recover lost genetic information, leading to the precise repair of a DSB. The Rad51 protein is essential for HR, but several auxiliary factors promote the activity of Rad51, including the Swi5-Sfr1 and Rad55-Rad57 complexes. We employed an interdisciplinary approach to characterize the interactions between these proteins.
Through a combination of NMR-based protein-protein interaction analysis and in vivo proximity crosslinking, I identified two sites within the N-terminal half of Sfr1 that physically interact with Rad51. Mutations in each site reduced the interaction with Rad51, whereas mutations in both sites completely ablated the interaction. Consistently, while the single-site mutants were mildly defective for the stimulation of Rad51 in vitro, the double-site mutant was severely defective. Surprisingly, I found that cells containing mutations in both sites were nevertheless completely proficient in DNA repair. However, this was because the Rad55-Rad57 complex, which was thought to comprise an independent sub-pathway of HR, was rescuing the defect of the (Swi5-Sfr1)-Rad51 interaction mutants. Consistently, Swi5-Sfr1 and Rad55-Rad57 were found to interact with each other. These results provided mechanistic insights into the activation of Rad51 by Swi5-Sfr1 and uncovered a functional interplay between Swi5-Sfr1 and Rad55-Rad57.

Research Products

• [Journal Article] Real-time tracking reveals catalytic roles for the two DNA binding sites of Rad51 (2020)
  • Author(s): Ito K, Murayama Y, Kurokawa Y, Kanamaru S, Kokabu Y, Maki T, Mikawa T, Argunhan B, Tsubouchi H, Ikeguchi M, Takahashi M, Iwasaki H
  • Journal Title: Nature Communications Volume: in press Pages: -
  • Peer Reviewed / Open Access
• [Journal Article] Two auxiliary factors promote Dmc1-driven DNA strand exchange via stepwise mechanisms. (2020)
  • Author(s): Tsubouchi H, Argunhan B, Ito K, Takahashi M, Iwasaki H
  • Journal Title: Proc. Natl.Acad Sci USA Volume: in press Pages: -
  • Peer Reviewed
• [Journal Article] Cooperative interactions facilitate stimulation of Rad51 by the Swi5-Sfr1 auxiliary factor complex (2020)
  • Author(s): Argunhan Bilge、Sakakura Masayoshi、Afshar Negar、Kurihara Misato、Ito Kentaro、Maki Takahisa、Kanamaru Shuji、Murayama Yasuto、Tsubouchi Hideo、Takahashi Masayuki、Takahashi Hideo、Iwasaki Hiroshi
  • Journal Title: eLife Volume: 9 Pages: e52566
  • DOI: 10.7554/eLife.52566
  • Peer Reviewed / Open Access