FRET-Based Assays to Monitor DNA Binding and Annealing by Rad52 Recombination Mediator Protein

  • Jill M. Grimme
  • Maria SpiesEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 745)


During homologous recombination and homology-directed repair of broken chromosomes, proteins that mediate and oppose recombination form dynamic complexes on damaged DNA. Quantitative analysis of these nucleoprotein assemblies requires a robust signal, which reports on the association of a recombination mediator with its substrate and on the state of substrate DNA within the complex. Eukaryotic Rad52 protein mediates recombination, repair, and restart of collapsed replication forks by facilitating replacement of ssDNA binding protein replication protein A (RPA) with Rad51 recombinase and by mediating annealing of two complementary DNA strands protected by RPA. The characteristic binding mode whereby ssDNA is wrapped around the Rad52 ring allowed us to develop robust and sensitive FRET-based assays for monitoring Rad52 interactions with protein-free DNA and ssDNA–RPA complexes. By reporting on the configuration of ssDNA dually labeled with Cy3 and Cy5 fluorescent dyes, solution-based FRET is used to analyze Rad52–RPA–DNA interactions under equilibrium binding conditions. Finally, FRET between Cy3 and Cy5 dyes incorporated into two homologous ssDNA molecules can be used to analyze interplay between Rad52-mediated DNA strand annealing and duplex DNA destabilization by RPA.

Key words

Annealing protein fluorescence Föster resonance energy transfer (FRET) homologous recombination Rad52 recombination mediator RPA 


  1. 1.
    Couedel, C., Mills, K.D., Barchi, M., Shen, L., Olshen, A., Johnson, R.D., Nussenzweig, A., Essers, J., Kanaar, R., Li, G.C., Alt, F.W., and Jasin, M. (2004) Collaboration of homologous recombination and nonhomologous end-joining factors for the survival and integrity of mice and cells. Genes Dev 18, 1293–1304.PubMedCrossRefGoogle Scholar
  2. 2.
    Sung, P., and Klein, H. (2006) Mechanism of homologous recombination: mediators and helicases take on regulatory functions. Nat Rev Mol Cell Biol 7, 739–750.PubMedCrossRefGoogle Scholar
  3. 3.
    Krogh, B.O., and Symington, L.S. (2004) Recombination proteins in yeast. Annu Rev Genet 38, 233–271.PubMedCrossRefGoogle Scholar
  4. 4.
    Mortensen, U.H., Lisby, M., and Rothstein, R. (2009) Rad52. Curr Biol 19, R676–R77.PubMedCrossRefGoogle Scholar
  5. 5.
    Mortensen, U.H., Erdeniz, N., Feng, Q., and Rothstein, R. (2002) A molecular genetic dissection of the evolutionarily conserved N terminus of yeast Rad52. Genetics 161, 549–562.PubMedGoogle Scholar
  6. 6.
    Sugiyama, T., New, J.H., and Kowalczykowski, S.C. (1998) DNA annealing by RAD52 protein is stimulated by specific interaction with the complex of replication protein A and single-stranded DNA. Proc Natl Acad Sci USA 95, 6049–6054.PubMedCrossRefGoogle Scholar
  7. 7.
    Bugreev, D.V., Hanaoka, F., and Mazin, A.V. (2007) Rad54 dissociates homologous recombination intermediates by branch migration. Nat Struct Mol Biol 14, 746–753.PubMedCrossRefGoogle Scholar
  8. 8.
    Miyazaki, T., Bressan, D.A., Shinohara, M., Haber, J.E., and Shinohara, A. (2004) In vivo assembly and disassembly of Rad51 and Rad52 complexes during double-strand break repair. EMBO J 23, 939–949.PubMedCrossRefGoogle Scholar
  9. 9.
    Sugiyama, T., Kantake, N., Wu, Y., and Kowalczykowski, S.C. (2006) Rad52-mediated DNA annealing after Rad51-mediated DNA strand exchange promotes second ssDNA capture. EMBO J 25, 5539–5548.PubMedCrossRefGoogle Scholar
  10. 10.
    McIlwraith, M.J., and West, S.C. (2008) DNA repair synthesis facilitates RAD52-mediated second-end capture during DSB repair. Mol Cell 29, 510–516.PubMedCrossRefGoogle Scholar
  11. 11.
    Nimonkar, A.V., Sica, R.A., and Kowalczykowski, S.C. (2009) Rad52 promotes second-end DNA capture in double-stranded break repair to form complement-stabilized joint molecules. Proc Natl Acad Sci USA 106, 3077–3082.PubMedCrossRefGoogle Scholar
  12. 12.
    Paques, F., and Haber, J.E. (1999) Multiple pathways of recombination induced by double-strand breaks in Saccharomyces cerevisiae. Microbiol Mol Biol Rev 63, 349–404.PubMedGoogle Scholar
  13. 13.
    Stark, J.M., Pierce, A.J., Oh, J., Pastink, A., and Jasin, M. (2004) Genetic steps of mammalian homologous repair with distinct mutagenic consequences. Mol Cell Biol 24, 9305–9316.PubMedCrossRefGoogle Scholar
  14. 14.
    Shinohara, A., Shinohara, M., Ohta, T., Matsuda, S., and Ogawa, T. (1998) Rad52 forms ring structures and co-operates with RPA in single-strand DNA annealing. Genes Cells 3, 145–156.PubMedCrossRefGoogle Scholar
  15. 15.
    Stasiak, A.Z., Larquet, E., Stasiak, A., Muller, S., Engel, A., Van Dyck, E., West, S.C., and Egelman, E.H. (2000) The human Rad52 protein exists as a heptameric ring. Curr Biol 10, 337–340.PubMedCrossRefGoogle Scholar
  16. 16.
    Singleton, M.R., Wentzell, L.M., Liu, Y., West, S.C., and Wigley, D.B. (2002) Structure of the single-strand annealing domain of human RAD52 protein. Proc Natl Acad Sci USA 99, 13492–13497.PubMedCrossRefGoogle Scholar
  17. 17.
    Kagawa, W., Kurumizaka, H., Ishitani, R., Fukai, S., Nureki, O., Shibata, T., and Yokoyama, S. (2002) Crystal structure of the homologous-pairing domain from the human Rad52 recombinase in the undecameric form. Mol Cell 10, 359–371.PubMedCrossRefGoogle Scholar
  18. 18.
    Lloyd, J.A., McGrew, D.A., and Knight, K.L. (2005) Identification of residues important for DNA binding in the full-length human Rad52 protein. J Mol Biol 345, 239–249.PubMedCrossRefGoogle Scholar
  19. 19.
    Kagawa, W., Kagawa, A., Saito, K., Ikawa, S., Shibata, T., Kurumizaka, H., and Yokoyama, S. (2008) Identification of a second DNA binding site in the human Rad52 protein. J Biol Chem 283, 24264–24273.PubMedCrossRefGoogle Scholar
  20. 20.
    Petukhova, G., Stratton, S.A., and Sung, P. (1999) Single strand DNA binding and annealing activities in the yeast recombination factor Rad59. J Biol Chem 274, 33839–33842.PubMedCrossRefGoogle Scholar
  21. 21.
    Wu, Y., Sugiyama, T., and Kowalczykowski, S.C. (2006) DNA annealing mediated by Rad52 and Rad59 proteins. J Biol Chem 281, 15441–15449.PubMedCrossRefGoogle Scholar
  22. 22.
    Ploquin, M., Bransi, A., Paquet, E.R., Stasiak, A.Z., Stasiak, A., Yu, X., Cieslinska, A.M., Egelman, E.H., Moineau, S., and Masson, J.-Y. (2008) Functional and structural basis for a bacteriophage homolog of human RAD52. Curr Biol 18, 1142–1146.PubMedCrossRefGoogle Scholar
  23. 23.
    Pant, K., Shokri, L., Karpel, R.L., Morrical, S.W., and Williams, M.C. (2008) Modulation of T4 gene 32 protein DNA binding activity by the recombination mediator protein UvsY. J Mol Biol 380, 799–811.PubMedCrossRefGoogle Scholar
  24. 24.
    Erler, A., Wegmann, S., Elie-Caille, C., Bradshaw, C.R., Maresca, M., Seidel, R., Habermann, B., Muller, D.J., and Stewart, A.F. (2009) Conformational adaptability of red[beta] during DNA annealing and implications for its structural relationship with Rad52. J Mol Biol 391, 586–598.PubMedCrossRefGoogle Scholar
  25. 25.
    Grimme, J.M., Honda, M., Wright, R., Okuno, Y., Rothenberg, E., Mazin, A.V., Ha, T., and Spies, M. (2010) Human Rad52 binds and wraps single-stranded DNA and mediates annealing via two hRad52-ssDNA complexes. Nucleic Acids Res 38, 2917–2930.PubMedCrossRefGoogle Scholar
  26. 26.
    Jackson, D., Dhar, K., Wahl, J.K., Wold, M.S., and Borgstahl, G.E. (2002) Analysis of the human replication protein A:Rad52 complex: evidence for crosstalk between RPA32, RPA70, Rad52 and DNA. J Mol Biol 321, 133–148.PubMedCrossRefGoogle Scholar
  27. 27.
    de Vries, F.A., Zonneveld, J.B., de Groot, A.J., Koning, R.I., van Zeeland, A.A., and Pastink, A. (2007) Schizosaccharomyces pombe Rad22A and Rad22B have similar biochemical properties and form multimeric structures. Mutat Res 615, 143–152.PubMedGoogle Scholar
  28. 28.
    Majka, J., and Speck, C. (2007) Analysis of protein-DNA interactions using surface plasmon resonance. Adv Biochem Eng Biotechnol 104, 13–36.PubMedGoogle Scholar
  29. 29.
    Clegg, R.M. (2002) FRET tells us about proximities, distances, orientations and dynamic properties. J Biotechnol 82, 177–179.PubMedGoogle Scholar
  30. 30.
    Rothenberg, E., Grimme, J.M., Spies, M., and Ha, T. (2008) Human Rad52-mediated homology search and annealing occurs by continuous interactions between overlapping nucleoprotein complexes. Proc Natl Acad Sci USA 105, 20274–20279.PubMedCrossRefGoogle Scholar
  31. 31.
    Henricksen, L.A., Umbricht, C.B., and Wold, M.S. (1994) Recombinant replication protein A: expression, complex formation, and functional characterization. J Biol Chem 269, 11121–11132.PubMedGoogle Scholar
  32. 32.
    Benson, F.E., Baumann, P., and West, S.C. (1998) Synergistic actions of Rad51 and Rad52 in recombination and DNA repair. Nature 391, 401–404.PubMedCrossRefGoogle Scholar
  33. 33.
    Reddy, G., Golub, E.I., and Radding, C.M. (1997) Human Rad52 protein promotes single-strand DNA annealing followed by branch migration. Mutat Res 377, 53–59.PubMedGoogle Scholar
  34. 34.
    Fanning, E., Klimovich, V., and Nager, A.R. (2006) A dynamic model for replication protein A (RPA) function in DNA processing pathways. Nucleic Acids Res 34, 4126–4137.PubMedCrossRefGoogle Scholar
  35. 35.
    Gomes, X.V., Henricksen, L.A., and Wold, M.S. (1996) Proteolytic mapping of human replication protein A: evidence for multiple structural domains and a conformational change upon interaction with single-stranded DNA. Biochemistry 35, 5586–5595.PubMedCrossRefGoogle Scholar
  36. 36.
    Gomes, X.V., and Wold, M.S. (1996) Functional domains of the 70-kilodalton subunit of human replication protein A. Biochemistry 35, 10558–10568.PubMedCrossRefGoogle Scholar
  37. 37.
    Kim, C., Snyder, R.O., and Wold, M.S. (1992) Binding properties of replication protein A from human and yeast cells. Mol Cell Biol 12, 3050–3059.PubMedGoogle Scholar
  38. 38.
    Kim, C., Paulus, B.F., and Wold, M.S. (1994) Interactions of human replication protein A with oligonucleotides. Biochemistry 33, 14197–14206.PubMedCrossRefGoogle Scholar
  39. 39.
    Parsons, C.A., Baumann, P., Van Dyck, E., and West, S.C. (2000) Precise binding of single-stranded DNA termini by human RAD52 protein. EMBO J 19, 4175–4181.PubMedCrossRefGoogle Scholar
  40. 40.
    Fischer, C.J., Maluf, N.K., and Lohman, T.M. (2004) Mechanism of ATP-dependent translocation of E. coli UvrD monomers along single-stranded DNA. J Mol Biol 344, 1287–1309.PubMedCrossRefGoogle Scholar
  41. 41.
    Luo, G., Wang, M., Konigsberg, W.H., and Xie, X.S. (2007) Single-molecule and ensemble fluorescence assays for a functionally important conformational change in T7 DNA polymerase. Proc Natl Acad Sci USA 104, 12610–12615.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  1. 1.US Army Engineer Research Development Center, Construction Engineering Research LaboratoryChampaignUSA
  2. 2.Department of BiochemistryHoward Hughes Medical Institute, University of IllinoisUrbana-Champaign, UrbanaUSA

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