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DNA helicase Srs2 disrupts the Rad51 presynaptic filament

Nature volume 423, pages 305–309 (2003)Cite this article

Abstract

Mutations in the Saccharomyces cerevisiae gene SRS2 result in the yeast's sensitivity to genotoxic agents, failure to recover or adapt from DNA damage checkpoint-mediated cell cycle arrest, slow growth, chromosome loss, and hyper-recombination1,2. Furthermore, double mutant strains, with mutations in DNA helicase genes SRS2 and SGS1, show low viability that can be overcome by inactivating recombination, implying that untimely recombination is the cause of growth impairment1,3,4. Here we clarify the role of SRS2 in recombination modulation by purifying its encoded product and examining its interactions with the Rad51 recombinase. Srs2 has a robust ATPase activity that is dependent on single-stranded DNA (ssDNA) and binds Rad51, but the addition of a catalytic quantity of Srs2 to Rad51-mediated recombination reactions causes severe inhibition of these reactions. We show that Srs2 acts by dislodging Rad51 from ssDNA. Thus, the attenuation of recombination efficiency by Srs2 stems primarily from its ability to dismantle the Rad51 presynaptic filament efficiently. Our findings have implications for the basis of Bloom's and Werner's syndromes, which are caused by mutations in DNA helicases and are characterized by increased frequencies of recombination and a predisposition to cancers and accelerated ageing5.

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Figure 1: Purification and characterization of Srs2.
Figure 2: Srs2 inhibits Rad51-mediated DNA pairing and strand exchange.
Figure 3: Srs2 disrupts the Rad51 presynaptic filament.
Figure 4: EM analysis of Rad51 filament disruption by Srs2.

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References

  1. Klein, H. L. A radical solution to death. Nature Genet. 25, 132–134 (2000)

    Article  CAS  Google Scholar 

  2. Vaze, M. B. et al. Recovery from checkpoint-mediated arrest after repair of a double-strand break requires Srs2 helicase. Mol. Cell 10, 373–385 (2002)

    Article  CAS  Google Scholar 

  3. Lee, S. K., Johnson, R. E., Yu, S. L., Prakash, L. & Prakash, S. Requirement of yeast SGS1 and SRS2 genes for replication and transcription. Science 286, 2339–2342 (1999)

    Article  CAS  Google Scholar 

  4. Gangloff, S., Soustelle, C. & Fabre, F. Homologous recombination is responsible for cell death in the absence of the Sgs1 and Srs2 helicases. Nature Genet. 25, 192–194 (2000)

    Article  CAS  Google Scholar 

  5. Oakley, T. J. & Hickson, I. D. Defending genome integrity during S-phase: putative roles for RecQ helicases and topoisomerase III. DNA Repair 1, 175–207 (2002)

    Article  CAS  Google Scholar 

  6. Rong, L. & Klein, H. L. Purification and characterization of the SRS2 DNA helicase of the yeast Saccharomyces cerevisiae. J. Biol. Chem. 268, 1252–1259 (1993)

    CAS  PubMed  Google Scholar 

  7. Milne, G. T., Ho, T. & Weaver, D. T. Modulation of Saccharomyces cerevisiae DNA double-strand break repair by SRS2 and RAD51. Genetics 139, 1189–1199 (1995)

    CAS  PubMed  PubMed Central  Google Scholar 

  8. Chanet, R., Heude, M., Adjiri, A., Maloisel, L. & Fabre, F. Semidominant mutations in the yeast Rad51 protein and their relationships with the Srs2 helicase. Mol. Cell. Biol. 16, 4782–4789 (1996)

    Article  CAS  Google Scholar 

  9. Schild, D. Suppression of a new allele of the yeast RAD52 gene by overexpression of RAD51, mutations in srs2 and ccr4, or mating-type heterozygosity. Genetics 140, 115–127 (1995)

    CAS  PubMed  PubMed Central  Google Scholar 

  10. Sung, P. Catalysis of ATP-dependent homologous DNA pairing and strand exchange by yeast RAD51 protein. Science 265, 1241–1243 (1994)

    Article  ADS  CAS  Google Scholar 

  11. Sung, P., Trujillo, K. M. & Van Komen, S. Recombination factors of Saccharomyces cerevisiae. Mutat. Res. 451, 257–275 (2000)

    Article  CAS  Google Scholar 

  12. Cox, M. M. Recombinational DNA repair of damaged replication forks in Escherichia coli: questions. Annu. Rev. Genet. 35, 53–82 (2001)

    Article  CAS  Google Scholar 

  13. Bianco, P. R., Tracy, R. B. & Kowalczykowski, S. C. DNA strand exchange proteins: a biochemical and physical comparison. Front. Biosci. 3, 570–603 (1998)

    Article  Google Scholar 

  14. Petukhova, G., Stratton, S. & Sung, P. Catalysis of homologous DNA pairing by yeast Rad51 and Rad54 proteins. Nature 393, 91–94 (1998)

    Article  ADS  CAS  Google Scholar 

  15. 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)

    Article  CAS  Google Scholar 

  16. Van Komen, S., Petukhova, G., Sigurdsson, S. & Sung, P. Functional cross-talk among Rad51, Rad54, and replication protein A in heteroduplex DNA joint formation. J. Biol. Chem. 277, 43578–43587 (2002)

    Article  CAS  Google Scholar 

  17. Ogawa, T., Yu, X., Shinohara, A. & Egelman, E. H. Similarity of the yeast RAD51 filament to the bacterial RecA filament. Science 259, 1896–1899 (1993)

    Article  ADS  CAS  Google Scholar 

  18. 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)

    Article  CAS  Google Scholar 

  19. Sung, P. Yeast Rad55 and Rad57 proteins form a heterodimer that functions with replication protein A to promote DNA strand exchange by Rad51 recombinase. Genes Dev. 11, 1111–1121 (1997)

    Article  CAS  Google Scholar 

  20. Sugiyama, T., Zaitseva, E. M. & Kowalczykowski, S. C. A single-stranded DNA-binding protein is needed for efficient presynaptic complex formation by the Saccharomyces cerevisiae Rad51 protein. J. Biol. Chem. 272, 7940–7945 (1997)

    Article  CAS  Google Scholar 

  21. Veaute, X. et al. The Srs2 helicase prevents recombination by disrupting Rad51 nucleoprotein filaments. Nature 423, 309–312 (2003)

    Article  ADS  CAS  Google Scholar 

  22. Aboussekhra, A. et al. RADH, a gene of Saccharomyces cerevisiae encoding a putative DNA helicase involved in DNA repair. Characteristics of radH mutants and sequence of the gene. Nucleic Acids Res. 17, 7211–7219 (1989)

    Article  CAS  Google Scholar 

  23. Aboussekhra, A., Chanet, R., Adjiri, A. & Fabre, F. Semidominant suppressors of Srs2 helicase mutations of Saccharomyces cerevisiae map in the RAD51 gene, whose sequence predicts a protein with similarities to procaryotic RecA proteins. Mol. Cell. Biol. 12, 3224–3234 (1992)

    Article  CAS  Google Scholar 

  24. Liberi, G. et al. Srs2 DNA helicase is involved in checkpoint response and its regulation requires a functional Mec1-dependent pathway and Cdk1 activity. EMBO J. 19, 5027–5038 (2000)

    Article  CAS  Google Scholar 

  25. Palladino, F. & Klein, H. L. Analysis of mitotic and meiotic defects in Saccharomyces cerevisiae SRS2 DNA helicase mutants. Genetics 132, 23–37 (1992)

    CAS  PubMed  PubMed Central  Google Scholar 

  26. Adams, M. D., McVey, M. & Sekelsky, J. J. Drosophila BLM in double-strand break repair by synthesis-dependent strand annealing. Science 299, 265–267 (2003)

    Article  ADS  CAS  Google Scholar 

  27. Wu, L., Davies, S. L., Levitt, N. C. & Hickson, I. D. Potential role for the BLM helicase in recombinational repair via a conserved interaction with RAD51. J. Biol. Chem. 276, 19375–19381 (2001)

    Article  CAS  Google Scholar 

  28. Mankouri, H. W., Craig, T. J. & Morgan, A. SGS1 is a multicopy suppressor of srs2: functional overlap between DNA helicases. Nucleic Acids Res. 30, 1103–1113 (2002)

    Article  CAS  Google Scholar 

  29. Petukhova, G., Stratton, S. A. & Sung, P. Single strand DNA binding and annealing activities in the yeast recombination factor Rad59. J. Biol. Chem. 274, 33839–33842 (1999)

    Article  CAS  Google Scholar 

  30. Krejci, L., Damborsky, J., Thomsen, B., Duno, M. & Bendixen, C. Molecular dissection of interactions between Rad51 and members of the recombination-repair group. Mol. Cell. Biol. 21, 966–976 (2001)

    Article  CAS  Google Scholar 

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Acknowledgements

We thank M. Sehorn and K. Trujillo for reading the manuscript. This work was supported by research grants from the NIH (H.K., T.E. and P.S.). S.V.K. was supported in part by a predoctoral fellowship from the US Department of Defense, and Y.L. was supported by a NIH postdoctoral fellowship. The molecular electron microscopy facility at Harvard Medical School was established by a donation from the Giovanni Armeise Harvard Center for Structural Biology, and is maintained through a NIH grant.

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  1. Lumir Krejci, Stephen Van Komen and Hannah Klein: These authors contributed equally to the work

Authors and Affiliations

  1. Institute of Biotechnology and Department of Molecular Medicine, University of Texas Health Science Center at San Antonio, 15355 Lambda Drive, San Antonio, Texas, 78245, USA

    Lumir Krejci, Stephen Van Komen, Jana Villemain, Mothe Sreedhar Reddy & Patrick Sung

  2. Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts, 02115, USA

    Ying Li & Thomas Ellenberger

  3. Department of Biochemistry, New York University School of Medicine, New York, New York, 10016, USA

    Hannah Klein

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  1. Lumir Krejci
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Correspondence to Lumir Krejci or Patrick Sung.

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Krejci, L., Van Komen, S., Li, Y. et al. DNA helicase Srs2 disrupts the Rad51 presynaptic filament. Nature 423, 305–309 (2003). https://doi.org/10.1038/nature01577

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