Visualization of Human Dmc1 Presynaptic Filaments

  • Michael G. SehornEmail author
  • Hilarie A. Sehorn
Part of the Methods in Molecular Biology book series (MIMB, volume 745)


Meiosis is initiated by the programmed formation of DNA double-strand breaks (DSBs). These DSBs are repaired by homologous recombination to promote crossover formation that ensures proper chromosomal segregation in meiosis. hRad51 and hDmc1 are two human recombinases present during meiosis that are homologous to the RecA recombinase from Escherichia coli. The hRad51 and hDmc1 recombinases bind the nucleolytically processed ends of the DSB forming a presynaptic filament. Formation of the presynaptic filament is necessary for the search for homology and the progression of recombination. In this chapter, we provide a method to purify hDmc1 and prepare samples for visualizing hDmc1 nucleoprotein presynaptic filaments via transmission electron microscopy.

Key words

Meiosis homologous recombination presynaptic filament transmission electron microscopy protein purification human Dmc1 



homologous recombination


single stranded


DNA double-strand break


nitro triacetic acid



This work was supported by National Science Foundation/EPSCoR grant 2004 RII-EPS-0447660 and Clemson University.


  1. 1.
    Keeney, S., Giroux, C.N., and Kleckner, N. (1997) Meiosis-specific DNA double-strand breaks are catalyzed by Spo11, a member of a widely conserved protein family. Cell 88, 375–384.PubMedCrossRefGoogle Scholar
  2. 2.
    Passy, S.I., Yu, X., Li, Z., Radding, C.M., Masson, J.Y., West, S.C., and Egelman, E.H. (1999) Human Dmc1 protein binds DNA as an octameric ring. Proc Natl Acad Sci USA 96, 10684–10688.PubMedCrossRefGoogle Scholar
  3. 3.
    Masson, J.Y., Davies, A.A., Hajibagheri, N., Van Dyck, E., Benson, F.E., Stasiak, A.Z., Stasiak, A., and West, S.C. (1999) The meiosis-specific recombinase hDmc1 forms ring structures and interacts with hRad51. EMBO J 18, 6552–6560.PubMedCrossRefGoogle Scholar
  4. 4.
    Sehorn, M.G., Sigurdsson, S., Bussen, W., Unger, V.M., and Sung, P. (2004) Human meiotic recombinase Dmc1 promotes ATP-dependent homologous DNA strand exchange. Nature 429, 433–437.PubMedCrossRefGoogle Scholar
  5. 5.
    Bugreev, D.V., Golub, E.I., Stasiak, A.Z., Stasiak, A., and Mazin, A.V. (2005) Activation of human meiosis-specific recombinase Dmc1 by Ca2+. J Biol Chem 280, 26886–26895.PubMedCrossRefGoogle Scholar
  6. 6.
    Benson, F.E., Stasiak, A., and West, S.C. (1994) Purification and characterization of the human Rad51 protein, an analogue of E. coli RecA. EMBO J 13, 5764–5771.PubMedGoogle Scholar
  7. 7.
    Sung, P., and Robberson, D.L. (1995) DNA strand exchange mediated by a RAD51-ssDNA nucleoprotein filament with polarity opposite to that of RecA. Cell 82, 453–461.PubMedCrossRefGoogle Scholar
  8. 8.
    Baumann, P., Benson, F.E., Hajibagheri, N., and West, S.C. (1997) Purification of human Rad51 protein by selective spermidine precipitation. Mutat Res 384, 65–72.PubMedGoogle Scholar
  9. 9.
    Yu, X., Jacobs, S.A., West, S.C., Ogawa, T., and Egelman, E.H. (2001) Domain structure and dynamics in the helical filaments formed by RecA and Rad51 on DNA. Proc Natl Acad Sci USA 98, 8419–8424.PubMedCrossRefGoogle Scholar
  10. 10.
    Yang, S., VanLoock, M.S., Yu, X., and Egelman, E.H. (2001) Comparison of bacteriophage T4 UvsX and human Rad51 filaments suggests that RecA-like polymers may have evolved independently. J Mol Biol 312, 999–1009.PubMedCrossRefGoogle Scholar
  11. 11.
    Chi, P., Van Komen, S., Sehorn, M.G., Sigurdsson, S., and Sung, P. (2006) Roles of ATP binding and ATP hydrolysis in human Rad51 recombinase function. DNA Repair (Amst) 5, 381–391.CrossRefGoogle Scholar
  12. 12.
    Sheridan, S.D., Yu, X., Roth, R., Heuser, J.E., Sehorn, M.G., Sung, P., Egelman, E.H., and Bishop, D.K. (2008) A comparative analysis of Dmc1 and Rad51 nucleoprotein filaments. Nucleic Acids Res 36, 4057–4066.PubMedCrossRefGoogle Scholar
  13. 13.
    Dupaigne, P., Lavelle, C., Justome, A., Lafosse, S., Mirambeau, G., Lipinski, M., Pietrement, O., and Le Cam, E. (2008) Rad51 polymerization reveals a new chromatin remodeling mechanism. PLoS One 3, e3643.PubMedCrossRefGoogle Scholar
  14. 14.
    Davies, A.A., Masson, J.Y., McIlwraith, M.J., Stasiak, A.Z., Stasiak, A., Venkitaraman, A.R., and West, S.C. (2001) Role of BRCA2 in control of the RAD51 recombination and DNA repair protein. Mol Cell 7, 273–282.PubMedCrossRefGoogle Scholar
  15. 15.
    Galkin, V.E., Esashi, F., Yu, X., Yang, S., West, S.C., and Egelman, E.H. (2005) BRCA2 BRC motifs bind RAD51-DNA filaments. Proc Natl Acad Sci USA 102, 8537–8542.PubMedCrossRefGoogle Scholar
  16. 16.
    San Filippo, J., Chi, P., Sehorn, M.G., Etchin, J., Krejci, L., and Sung, P. (2006) Recombination mediator and Rad51 targeting activities of a human BRCA2 polypeptide. J Biol Chem 281, 11649–11657.PubMedCrossRefGoogle Scholar
  17. 17.
    Esashi, F., Galkin, V.E., Yu, X., Egelman, E.H., and West, S.C. (2007) Stabilization of RAD51 nucleoprotein filaments by the C-terminal region of BRCA2. Nat Struct Mol Biol 14, 468–474.PubMedCrossRefGoogle Scholar
  18. 18.
    Davies, O.R., and Pellegrini, L. (2007) Interaction with the BRCA2 C terminus protects RAD51-DNA filaments from disassembly by BRC repeats. Nat Struct Mol Biol 14, 475–483.PubMedGoogle Scholar
  19. 19.
    Shivji, M.K., Mukund, S.R., Rajendra, E., Chen, S., Short, J.M., Savill, J., Klenerman, D., and Venkitaraman, A.R. (2009) The BRC repeats of human BRCA2 differentially regulate RAD51 binding on single- versus double-stranded DNA to stimulate strand exchange. Proc Natl Acad Sci USA 106(32), 13254–13259.Google Scholar
  20. 20.
    Van Dyck, E., Hajibagheri, N.M., Stasiak, A., and West, S.C. (1998) Visualisation of human rad52 protein and its complexes with hRad51 and DNA. J Mol Biol 284, 1027–1038.PubMedCrossRefGoogle Scholar
  21. 21.
    McIlwraith, M.J., Van Dyck, E., Masson, J.Y., Stasiak, A.Z., Stasiak, A., and West, S.C. (2000) Reconstitution of the strand invasion step of double-strand break repair using human Rad51 Rad52 and RPA proteins. J Mol Biol 304, 151–164.PubMedCrossRefGoogle Scholar
  22. 22.
    Kiianitsa, K., Solinger, J.A., and Heyer, W.D. (2002) Rad54 protein exerts diverse modes of ATPase activity on duplex DNA partially and fully covered with Rad51 protein. J Biol Chem 277, 46205–46215.PubMedCrossRefGoogle Scholar
  23. 23.
    Li, X., Zhang, X.P., Solinger, J.A., Kiianitsa, K., Yu, X., Egelman, E.H., and Heyer, W.D. (2007) Rad51 and Rad54 ATPase activities are both required to modulate Rad51-dsDNA filament dynamics. Nucleic Acids Res 35, 4124–4140.PubMedCrossRefGoogle Scholar
  24. 24.
    Hu, Y., Raynard, S., Sehorn, M.G., Lu, X., Bussen, W., Zheng, L., Stark, J.M., Barnes, E.L., Chi, P., Janscak, P., Jasin, M., Vogel, H., Sung, P., and Luo, G. (2007) RECQL5/Recql5 helicase regulates homologous recombination and suppresses tumor formation via disruption of Rad51 presynaptic filaments. Genes Dev 21, 3073–3084.PubMedCrossRefGoogle Scholar
  25. 25.
    Bugreev, D.V., Yu, X., Egelman, E.H., and Mazin, A.V. (2007) Novel pro- and anti-recombination activities of the Bloom’s syndrome helicase. Genes Dev 21, 3085–3094.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  1. 1.Department of Genetics and BiochemistryClemson UniversityClemsonUSA

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