Fission yeast Swi5-Sfr1 protein complex, an activator of Rad51 recombinase, forms an extremely elongated dogleg-shaped structure

Yuichi Kokabu, Yasuto Murayama, Naoyuki Kuwabara, Tomotaka Oroguchi, Hiroshi Hashimoto, Yasuhiro Tsutsui, Naohito Nozaki, Satoko Akashi, Satoru Unzai, Toshiyuki Shimizu, Hiroshi Iwasaki, Mamoru Sato, Mitsunori Ikeguchi

Research output: Contribution to journalArticle

16 Citations (Scopus)

Abstract

In eukaryotes, DNA strand exchange is the central reaction of homologous recombination, which is promoted by Rad51 recombinases forming a right-handed nucleoprotein filament on single-stranded DNA, also known as a presynaptic filament. Accessory proteins known as recombination mediators are required for the formation of the active presynaptic filament. One such mediator in the fission yeast Schizosaccharomyces pombe is the Swi5-Sfr1 complex, which has been identified as an activator of Rad51 that assists in presynaptic filament formation and stimulates its strand exchange reaction. Here, we determined the 1:1 binding stoichiometry between the two subunits of the Swi5-Sfr1 complex using analytical ultracentrifugation and electrospray ionization mass spectrometry. Small-angle x-ray scattering experiments revealed that the Swi5-Sfr1 complex displays an extremely elongated dogleg-shaped structure in solution, which is consistent with its exceptionally high frictional ratio (f/f 0) of 2.0 ± 0.2 obtained by analytical ultracentrifugation. Furthermore, we determined a rough topology of the complex by comparing the small-angle x-ray scattering-based structures of the Swi5-Sfr1 complex and four Swi5-Sfr1-Fab complexes, in which the Fab fragments of monoclonal antibodies were specifically bound to experimentally determined sites of Sfr1. We propose a model for how the Swi5-Sfr1 complex binds to the Rad51 filament, in which the Swi5-Sfr1 complex fits into the groove of the Rad51 filament, leading to an active and stable presynaptic filament.

Original languageEnglish
Pages (from-to)43569-43576
Number of pages8
JournalJournal of Biological Chemistry
Volume286
Issue number50
DOIs
Publication statusPublished - 2011 Dec 16
Externally publishedYes

Fingerprint

Rad51 Recombinase
Schizosaccharomyces
Ultracentrifugation
Yeast
X-Rays
Scattering
X rays
Electrospray ionization
Immunoglobulin Fab Fragments
Nucleoproteins
Electrospray Ionization Mass Spectrometry
Single-Stranded DNA
Homologous Recombination
Accessories
Eukaryota
Stoichiometry
Genetic Recombination
Mass spectrometry
Proteins
Monoclonal Antibodies

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology
  • Molecular Biology

Cite this

Fission yeast Swi5-Sfr1 protein complex, an activator of Rad51 recombinase, forms an extremely elongated dogleg-shaped structure. / Kokabu, Yuichi; Murayama, Yasuto; Kuwabara, Naoyuki; Oroguchi, Tomotaka; Hashimoto, Hiroshi; Tsutsui, Yasuhiro; Nozaki, Naohito; Akashi, Satoko; Unzai, Satoru; Shimizu, Toshiyuki; Iwasaki, Hiroshi; Sato, Mamoru; Ikeguchi, Mitsunori.

In: Journal of Biological Chemistry, Vol. 286, No. 50, 16.12.2011, p. 43569-43576.

Research output: Contribution to journalArticle

Kokabu, Y, Murayama, Y, Kuwabara, N, Oroguchi, T, Hashimoto, H, Tsutsui, Y, Nozaki, N, Akashi, S, Unzai, S, Shimizu, T, Iwasaki, H, Sato, M & Ikeguchi, M 2011, 'Fission yeast Swi5-Sfr1 protein complex, an activator of Rad51 recombinase, forms an extremely elongated dogleg-shaped structure', Journal of Biological Chemistry, vol. 286, no. 50, pp. 43569-43576. https://doi.org/10.1074/jbc.M111.303339
Kokabu, Yuichi ; Murayama, Yasuto ; Kuwabara, Naoyuki ; Oroguchi, Tomotaka ; Hashimoto, Hiroshi ; Tsutsui, Yasuhiro ; Nozaki, Naohito ; Akashi, Satoko ; Unzai, Satoru ; Shimizu, Toshiyuki ; Iwasaki, Hiroshi ; Sato, Mamoru ; Ikeguchi, Mitsunori. / Fission yeast Swi5-Sfr1 protein complex, an activator of Rad51 recombinase, forms an extremely elongated dogleg-shaped structure. In: Journal of Biological Chemistry. 2011 ; Vol. 286, No. 50. pp. 43569-43576.
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AU - Oroguchi, Tomotaka

AU - Hashimoto, Hiroshi

AU - Tsutsui, Yasuhiro

AU - Nozaki, Naohito

AU - Akashi, Satoko

AU - Unzai, Satoru

AU - Shimizu, Toshiyuki

AU - Iwasaki, Hiroshi

AU - Sato, Mamoru

AU - Ikeguchi, Mitsunori

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AB - In eukaryotes, DNA strand exchange is the central reaction of homologous recombination, which is promoted by Rad51 recombinases forming a right-handed nucleoprotein filament on single-stranded DNA, also known as a presynaptic filament. Accessory proteins known as recombination mediators are required for the formation of the active presynaptic filament. One such mediator in the fission yeast Schizosaccharomyces pombe is the Swi5-Sfr1 complex, which has been identified as an activator of Rad51 that assists in presynaptic filament formation and stimulates its strand exchange reaction. Here, we determined the 1:1 binding stoichiometry between the two subunits of the Swi5-Sfr1 complex using analytical ultracentrifugation and electrospray ionization mass spectrometry. Small-angle x-ray scattering experiments revealed that the Swi5-Sfr1 complex displays an extremely elongated dogleg-shaped structure in solution, which is consistent with its exceptionally high frictional ratio (f/f 0) of 2.0 ± 0.2 obtained by analytical ultracentrifugation. Furthermore, we determined a rough topology of the complex by comparing the small-angle x-ray scattering-based structures of the Swi5-Sfr1 complex and four Swi5-Sfr1-Fab complexes, in which the Fab fragments of monoclonal antibodies were specifically bound to experimentally determined sites of Sfr1. We propose a model for how the Swi5-Sfr1 complex binds to the Rad51 filament, in which the Swi5-Sfr1 complex fits into the groove of the Rad51 filament, leading to an active and stable presynaptic filament.

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