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The Swi5–Sfr1 complex stimulates Rhp51/Rad51 - and Dmc1-mediated DNA strand exchange in vitro

Nature Structural & Molecular Biology volume 13, pages 823–830 (2006)Cite this article

Abstract

Nucleoprotein filaments made up of Rad51 or Dmc1 recombinases, the core structures of recombination, engage in ATP-dependent DNA-strand exchange. The ability of recombinases to form filaments is enhanced by recombination factors termed 'mediators'. Here, we show that the Schizosaccharomyces pombe Swi5–Sfr1 complex, a conserved eukaryotic protein complex, at substoichiometric concentrations stimulates strand exchange mediated by Rhp51 (the S. pombe Rad51 homolog) and Dmc1 on long DNA substrates. Reactions mediated by both recombinases are completely dependent on Swi5–Sfr1, replication protein A (RPA) and ATP, although RPA inhibits the reaction when it is incubated with single-stranded DNA (ssDNA) before the recombinase. The Swi5–Sfr1 complex overcomes, at least partly, the inhibitory effect of RPA, representing a novel class of mediator. Notably, the Swi5–Sfr1 complex preferentially stimulates the ssDNA-dependent ATPase activity of Rhp51, and it increases the amounts of Dmc1 bound to ssDNA.

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Figure 1: Swi5–Sfr1 protein complex formation.
Figure 2: The Swi5–Sfr1 complex physically interacts with Rad51 in an Sfr1-dependent manner.
Figure 3: Swi5–Sfr1 stimulates the Rad51-mediated DNA strand exchange reaction.
Figure 4: Effect of the order of addition of components to the DNA strand exchange reaction.
Figure 5: Effects of Swi5–Sfr1 on the ssDNA-binding and ATPase activities of Rad51.
Figure 6: The Swi5–Sfr1 complex physically interacts with Dmc1 in an Sfr1-dependent manner.
Figure 7: Dmc1-mediated reactions are also stimulated by Swi5–Sfr1.
Figure 8: Effects of Swi5–Sfr1 on the ssDNA-binding and ATPase activities of Dmc1.

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Acknowledgements

We thank A. Carr and A. Mazin for critical reading of the manuscript, H. Nojima (Osaka University) for kindly providing the cloned dmc1 complementary DNA and T. Kokubo for helpful discussions and encouragement. This study was supported in part by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science, and Technology of Japan and from the Japan Society for the Promotion of Science, and by a grant for the 2005 Strategic Research Project (no. K17005) of Yokohama City University. N.H and H.I. were supported by Japan Society for the Promotion of Science fellowships for young scientists and by the Human Frontier Science Program, respectively.

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  1. Yufuko Akamatsu

    Present address: Molecular Biology Program, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, New York, 10021, USA

Authors and Affiliations

  1. Division of Molecular and Cellular Biology, International Graduate School of Arts and Sciences, Yokohama City University, 1-7-29 Suehiro-cho, Tsurumi, Yokohama, 230-0045, Kanagawa, Japan

    Nami Haruta, Yumiko Kurokawa, Yasuto Murayama, Yufuko Akamatsu & Hiroshi Iwasaki

  2. Division of Protein Design, International Graduate School of Arts and Sciences, Yokohama City University, 1-7-29 Suehiro-cho, Tsurumi, Yokohama, 230-0045, Kanagawa, Japan

    Satoru Unzai

  3. Division of Mutagenesis, Department of Molecular Genetics, National Institute of Genetics, Yata 1111, Mishima, 411-8540, Shizuoka, Japan

    Yasuhiro Tsutsui

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Contributions

N.H., Y.K., Y.M., Y.A. and Y.T. designed and performed experiments. S.U. performed ultracentrifugation analysis. H.I. designed experiments and wrote the manuscript.

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Correspondence to Hiroshi Iwasaki.

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Supplementary information

Supplementary Fig. 1

Swi5–Sfr1 does not affect the amount of ADP bound to Rad51. (PDF 57 kb)

Supplementary Methods

Overproduction and purification of proteins. (PDF 30 kb)

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Haruta, N., Kurokawa, Y., Murayama, Y. et al. The Swi5–Sfr1 complex stimulates Rhp51/Rad51 - and Dmc1-mediated DNA strand exchange in vitro. Nat Struct Mol Biol 13, 823–830 (2006). https://doi.org/10.1038/nsmb1136

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