Journal of Biological Chemistry
Volume 286, Issue 50, 16 December 2011, Pages 43569-43576
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Protein Structure and Folding
Fission Yeast Swi5-Sfr1 Protein Complex, an Activator of Rad51 Recombinase, Forms an Extremely Elongated Dogleg-shaped Structure*

https://doi.org/10.1074/jbc.M111.303339Get rights and content
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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/f0) 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.

DNA Repair
Homologous Recombination
Mass Spectrometry (MS)
Ultracentrifugation
X-ray Scattering
Yeast
Swi5-Sfr1
Fission Yeast

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*

This work was supported by grants-in-aids for scientific research on innovative areas from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (MEXT; to S. A., T. S., H. I., M. S., and M. I.) and for scientific research (A (to H. I.) and B (to M. I.)); by the Grand Challenges in Next-Generation Integrated Simulation of Living Matter, part of the Development and Use of the Next-Generation Supercomputer Project of MEXT (to M. I.); by the Japan Society for the Promotion of Science (JSPS); and by research grants for bioscience from the Takeda Science Foundation (to H. I.).

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1–S3.