Fission yeast Swi5-Sfr1 protein complex, an activator of Rad51 recombinase, forms an extremely elongated dogleg-shaped structure.
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.
Graduate School of Nanobioscience, Yokohama City University, 1-7-29 Suehiro-cho, Tsurumi-ku, Yokohama 230-0045, Japan.
Yuichi
Y
Kokabu
Yasuto
Y
Murayama
Naoyuki
N
Kuwabara
Tomotaka
T
Oroguchi
Hiroshi
H
Hashimoto
Yasuhiro
Y
Tsutsui
Naohito
N
Nozaki
Satoko
S
Akashi
Satoru
S
Unzai
Toshiyuki
T
Shimizu
Hiroshi
H
Iwasaki
Mamoru
M
Sato
Mitsunori
M
Ikeguchi
D004260:DNA Repair / Q000235:genetics / Q000502:physiology; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D051135:Rad51 Recombinase / Q000235:genetics / Q000378:metabolism*; D012568:Schizosaccharomyces / Q000235:genetics / Q000378:metabolism*; D029702:Schizosaccharomyces pombe Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D021241:Spectrometry, Mass, Electrospray Ionization
D029702:Schizosaccharomyces pombe Proteins; C514357:Sfr1 protein, S pombe; C483769:Swi5 protein, S pombe; D051135:Rad51 Recombinase