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Formation and branch migration of Holliday junctions mediated by eukaryotic recombinases

Abstract

Holliday junctions (HJs) are key intermediates in homologous recombination and are especially important for the production of crossover recombinants1,2,3,4. Bacterial RecA family proteins promote the formation and branch migration of HJs in vitro by catalysing a reciprocal DNA-strand exchange reaction between two duplex DNA molecules, one of which contains a single-stranded DNA region that is essential for initial nucleoprotein filament formation5. This activity has been reported only for prokaryotic RecA family recombinases5, although eukaryotic homologues are also essential for HJ production in vivo6,7. Here we show that fission yeast (Rhp51) and human (hRad51) RecA homologues promote duplex–duplex DNA-strand exchange in vitro. As with RecA, a HJ is formed between the two duplex DNA molecules, and reciprocal strand exchange proceeds through branch migration of the HJ. In contrast to RecA, however, strand exchange mediated by eukaryotic recombinases proceeds in the 3′→5′ direction relative to the single-stranded DNA region of the substrate DNA. The opposite polarity of Rhp51 makes it especially suitable for the repair of DNA double-strand breaks, whose repair is initiated at the processed ends of breaks that have protruding 3′ termini1,2.

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Figure 1: Rhp51 promotes a DNA four-strand exchange reaction in vitro.
Figure 2: Formation of HJs in the Rhp51-mediated four-strand exchange reaction.
Figure 3: Effects of Swi5–Sfr1 and adenine nucleotides.
Figure 4: hRad51 promotes the DNA four-strand exchange reaction in vitro.

References

  1. 1

    Pâques, F. & Haber, J. E. Multiple pathways of recombination induced by double-strand breaks in Saccharomyces cerevisiae. Microbiol. Mol. Biol. Rev. 63, 349–404 (1999)

    PubMed  PubMed Central  Google Scholar 

  2. 2

    Symington, L. S. Role of RAD52 epistasis group genes in homologous recombination and double-strand break repair. Microbiol. Mol. Biol. Rev. 66, 630–670 (2002)

    CAS  Article  Google Scholar 

  3. 3

    Bishop, D. K. & Zickler, D. Early decision; meiotic crossover interference prior to stable strand exchange and synapsis. Cell 117, 9–15 (2004)

    CAS  Article  Google Scholar 

  4. 4

    Cromie, G. A. et al. Single Holliday junctions are intermediates of meiotic recombination. Cell 127, 1167–1178 (2006)

    CAS  Article  Google Scholar 

  5. 5

    Cox, M. M. Motoring along with the bacterial RecA protein. Nature Rev. Mol. Cell Biol. 8, 127–138 (2007)

    CAS  Article  Google Scholar 

  6. 6

    Schwacha, A. & Kleckner, N. Interhomolog bias during meiotic recombination: meiotic functions promote a highly differentiated interhomolog-only pathway. Cell 90, 1123–1135 (1997)

    CAS  Article  Google Scholar 

  7. 7

    Hunter, N. & Kleckner, N. The single-end invasion: an asymmetric intermediate at the double-strand break to double-Holliday junction transition of meiotic recombination. Cell 106, 59–70 (2001)

    CAS  Article  Google Scholar 

  8. 8

    Bianco, P. R., Tracy, R. B. & Kowalczykowski, S. C. DNA strand exchange proteins: a biochemical and physical comparison. Front. Biosci. 3, D570–D603 (1998)

    CAS  Article  Google Scholar 

  9. 9

    Masson, J. Y. & West, S. C. The Rad51 and Dmc1 recombinases: a non-identical twin relationship. Trends Biochem. Sci. 26, 131–136 (2001)

    CAS  Article  Google Scholar 

  10. 10

    Baumann, P. & West, S. C. Role of the human RAD51 protein in homologous recombination and double-stranded-break repair. Trends Biochem. Sci. 23, 247–251 (1998)

    CAS  Article  Google Scholar 

  11. 11

    Haruta, N. et al. The Swi5–Sfr1 complex stimulates Rhp51/Rad51- and Dmc1-mediated DNA strand exchange in vitro. Nature Struct. Mol. Biol. 13, 823–830 (2006)

    CAS  Article  Google Scholar 

  12. 12

    West, S. C., Cassuto, E. & Howard-Flanders, P. Postreplication repair in E. coli: strand exchange reactions of gapped DNA by RecA protein. Mol. Gen. Genet. 187, 209–217 (1982)

    CAS  Article  Google Scholar 

  13. 13

    Chow, S. A., Chiu, S. K. & Wong, B. C. RecA protein-promoted homologous pairing and strand exchange between intact and partially single-stranded duplex DNA. J. Mol. Biol. 223, 79–93 (1992)

    CAS  Article  Google Scholar 

  14. 14

    Haruta, N., Yu, X., Yang, S., Egelman, E. H. & Cox, M. M. A. DNA pairing-enhanced conformation of bacterial RecA proteins. J. Biol. Chem. 278, 52710–52723 (2003)

    CAS  Article  Google Scholar 

  15. 15

    Shinagawa, H. & Iwasaki, H. Processing the Holliday junction in homologous recombination. Trends Biochem. Sci. 21, 107–111 (1996)

    CAS  Article  Google Scholar 

  16. 16

    Dunderdale, H. J. et al. Formation and resolution of recombination intermediates by E. coli RecA and RuvC proteins. Nature 354, 506–510 (1991)

    ADS  CAS  Article  Google Scholar 

  17. 17

    Haruta, N. et al. Fission yeast Swi5 protein, a novel DNA recombination mediator. DNA Repair (Amst.) 7, 1–9 (2008)

    CAS  Article  Google Scholar 

  18. 18

    Shan, Q., Cox, M. M. & Inman, R. B. DNA strand exchange promoted by RecA K72R. Two reaction phases with different Mg2+ requirements. J. Biol. Chem. 271, 5712–5724 (1996)

    CAS  Article  Google Scholar 

  19. 19

    Kim, J. I., Cox, M. M. & Inman, R. B. On the role of ATP hydrolysis in RecA protein-mediated DNA strand exchange. II. Four-strand exchanges. J. Biol. Chem. 267, 16444–16449 (1992)

    CAS  PubMed  Google Scholar 

  20. 20

    Sung, P. & Robberson, D. L. DNA strand exchange mediated by a RAD51-ssDNA nucleoprotein filament with polarity opposite to that of RecA. Cell 82, 453–461 (1995)

    CAS  Article  Google Scholar 

  21. 21

    Kmiec, E. B. & Holloman, W. K. Heteroduplex formation and polarity during strand transfer promoted by Ustilago rec1 protein. Cell 33, 857–864 (1983)

    CAS  Article  Google Scholar 

  22. 22

    Namsaraev, E. & Berg, P. Characterization of strand exchange activity of yeast Rad51 protein. Mol. Cell. Biol. 17, 5359–5368 (1997)

    CAS  Article  Google Scholar 

  23. 23

    Namsaraev, E. A. & Berg, P. Branch migration during Rad51-promoted strand exchange proceeds in either direction. Proc. Natl Acad. Sci. USA 95, 10477–10481 (1998)

    ADS  CAS  Article  Google Scholar 

  24. 24

    Gupta, R. C., Golub, E. I., Wold, M. S. & Radding, C. M. Polarity of DNA strand exchange promoted by recombination proteins of the RecA family. Proc. Natl Acad. Sci. USA 95, 9843–9848 (1998)

    ADS  CAS  Article  Google Scholar 

  25. 25

    Baumann, P. & West, S. C. Heteroduplex formation by human Rad51 protein: effects of DNA end-structure, hRP-A and hRad52. J. Mol. Biol. 291, 363–374 (1999)

    CAS  Article  Google Scholar 

  26. 26

    Mazin, A. V., Zaitseva, E., Sung, P. & Kowalczykowski, S. C. Tailed duplex DNA is the preferred substrate for Rad51 protein-mediated homologous pairing. EMBO J. 19, 1148–1156 (2000)

    CAS  Article  Google Scholar 

  27. 27

    Cox, M. M. & Lehman, I. R. Directionality and polarity in recA protein-promoted branch migration. Proc. Natl Acad. Sci. USA 78, 6018–6022 (1981)

    ADS  CAS  Article  Google Scholar 

  28. 28

    Konforti, B. B. & Davis, R. W. 3′ homologous free ends are required for stable joint molecule formation by the RecA and single-stranded binding proteins of Escherichia coli. Proc. Natl Acad. Sci. USA 84, 690–694 (1987)

    ADS  CAS  Article  Google Scholar 

  29. 29

    Jain, S. K., Cox, M. M. & Inman, R. B. On the role of ATP hydrolysis in RecA protein-mediated DNA strand exchange. III. Unidirectional branch migration and extensive hybrid DNA formation. J. Biol. Chem. 269, 20653–20661 (1994)

    CAS  PubMed  Google Scholar 

  30. 30

    Bugreev, D. V., Mazina, O. M. & Mazin, A. V. Rad54 protein promotes branch migration of Holliday junctions. Nature 442, 590–593 (2006)

    ADS  CAS  Article  Google Scholar 

  31. 31

    Constantinou, A. & West, S. C. Holliday junction branch migration and resolution assays. Methods Mol. Biol. 262, 239–253 (2004)

    CAS  PubMed  Google Scholar 

  32. 32

    Lusetti, S. L. et al. C-terminal deletions of the Escherichia coli RecA protein. Characterization of in vivo and in vitro effects. J. Biol. Chem. 278, 16372–16380 (2003)

    CAS  Article  Google Scholar 

  33. 33

    Yoshikawa, M., Iwasaki, H., Kinoshita, K. & Shinagawa, H. Two basic residues, Lys-107 and Lys-118, of RuvC resolvase are involved in critical contacts with the Holliday junction for its resolution. Genes Cells 5, 803–813 (2000)

    CAS  Article  Google Scholar 

Download references

Acknowledgements

We thank N. Haruta-Takahashi, T. Kokubo and K. Morikawa for discussions and encouragement, and T. Miyata for her help in electron microscopy sample preparation. This study was supported in part by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science, and Technology (MECSST) of Japan and from the Japan Society for the Promotion of Science (JSPS), and by a grant from the 2007 Strategic Research Project of Yokohama City University.

Author Contributions Y.M., Y.K. and H.I. designed the experiments. Y.M. and Y.K. performed the experiments. K.M. performed the electron microscopy analysis. Y.M. and H.I. wrote the manuscript.

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Correspondence to Hiroshi Iwasaki.

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Murayama, Y., Kurokawa, Y., Mayanagi, K. et al. Formation and branch migration of Holliday junctions mediated by eukaryotic recombinases. Nature 451, 1018–1021 (2008). https://doi.org/10.1038/nature06609

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