Abstract
Homologous recombination (HR) is a high-fidelity DNA repair pathway that maintains genome integrity, by repairing double strand breaks (DSBs) and single-stranded DNA (ssDNA) gaps and by supporting stalled/collapsed replication forks. The RecA/Rad51 family of proteins are the key enzymes in this homology-directed repair pathway, as they perform DNA strand invasion and exchange, in concert with a host of ancillary factors. In vitro, the RecA/Rad51 family of proteins share similar enzymatic activities including catalyzing ssDNA-stimulated ATP hydrolysis, formation of displacement loops (D-loops), and DNA strand exchange. After successful DNA strand invasion, DNA synthesis restores the lost genetic information using an undamaged DNA template. In this chapter, we describe two well-established biochemical assays to investigate the signature DNA strand transfer activity of RecA/Rad51 family of proteins: the D-loop assay and the DNA strand exchange reaction. Moreover, we describe a D-loop extension assay coupling D-loop formation with DNA synthesis, which can be used to define the roles of DNA polymerases in HR. Additionally, we present a protocol to investigate protein-mediated DNA annealing, a critical step in the synthesis-dependent strand annealing (SDSA) and double-Holliday junction (dHJ) pathways as well as the single-strand annealing (SSA) pathway. The quality of supercoiled plasmid DNA is critical in reconstituted HR reactions, and a protocol describing the preparation of this DNA substrate is included.
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References
Li, X., and Heyer, W.D. (2008) Homologous recombination in DNA repair and DNA damage tolerance. Cell Res 18, 99–113.
Krogh, B.O., and Symington, L.S. (2004) Recombination proteins in yeast. Annu Rev Genet 38, 233–271.
Solinger, J.A., Kiianitsa, K., and Heyer, W.-D. (2002) Rad54, a Swi2/Snf2-like recombinational repair protein, disassembles Rad51:dsDNA filaments. Mol Cell 10, 1175–1188.
Prakash, R., Satory, D., Dray, E., Papusha, A., Scheller, J., Kramer, W., Krejci, L., Klein, H., Haber, J.E., Sung, P., and Ira, G. (2009) Yeast Mph1 helicase dissociates Rad51-made D-loops: implications for crossover control in mitotic recombination. Genes Dev 23, 67–79.
New, J.H., Sugiyama, T., Zaitseva, E., and Kowalczykowski, S.C. (1998) Rad52 protein stimulates DNA strand exchange by Rad51 and replication protein A. Nature 391, 407–410.
Li, X., Stith, C.M., Burgers, P.M., and Heyer, W.-D. (2009) PCNA is required for initiating recombination-associated DNA synthesis by DNA polymerase . Mol Cell 36, 704–713.
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.
Wu, Y., Kantake, N., Sugiyama, T., and Kowalczykowski, S.C. (2008) Rad51 protein controls Rad52-mediated DNA annealing. J Biol Chem 283, 14883–14892.
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.
Shinohara, A., and Ogawa, T. (1998) Stimulation by Rad52 of yeast Rad51-mediated recombination. Nature 391, 404–407.
Sung, P. (1997) Function of yeast Rad52 protein as a mediator between replication protein A and the Rad51 recombinase. J Biol Chem 272, 28194–28197.
Bugreev, D.V., Hanaoka, F., and Mazin, A.V. (2007) Rad54 dissociates homologous recombination intermediates by branch migration. Nature Struct Mol Biol 14, 746–753.
Kapuscinski, J., and Szer, W. (1979) Interactions of 4′, 6-diamidine-2-phenylindole with synthetic polynucleotides. Nucleic Acids Res 6, 3519–3534.
Kubista, M., Akerman, B., and Norden, B. (1987) Characterization of interaction between DNA and 4′,6-diamidino-2-phenylindole by optical spectroscopy. Biochemistry 26, 4545–4553.
Bugreev, D.V., and Mazin, A.V. (2004) Ca2+ activates human homologous recombination protein Rad51 by modulating its ATPase activity. Proc Natl Acad Sci USA 101, 9988–9993.
Acknowledgments
We thank Kirk Ehmsen, William Wright, Clare Fasching, Ryan Janke, Erin Schwartz, Shannon Ceballos, Damon Meyer, Xiao-Ping Zhang, and Margarita Alexeeva for helpful comments on the manuscript. Our work is supported by the NIH (GM58015, CA92276), the DoD (BC083684), an NIH training grant fellowship (5T32CA108459) to J.S., and a TRDRP Postdoctoral fellowship (17FT-0046) to J.L.
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Liu, J., Sneeden, J., Heyer, WD. (2011). In Vitro Assays for DNA Pairing and Recombination-Associated DNA Synthesis. In: Tsubouchi, H. (eds) DNA Recombination. Methods in Molecular Biology, vol 745. Humana Press. https://doi.org/10.1007/978-1-61779-129-1_21
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DOI: https://doi.org/10.1007/978-1-61779-129-1_21
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