title: In vitro assays for DNA pairing and recombination-associated DNA synthesis.
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.
authors.affiliation: Department of Microbiology, University of California, Davis, CA 95616-8665, USA. jtliu@ucdavis.edu
authors.forename: Jie
authors.initials: J
authors.lastname: Liu
authors.forename: Jessica
authors.initials: J
authors.lastname: Sneeden
authors.forename: Wolf-Dietrich
authors.initials: WD
authors.lastname: Heyer
mesh_terms: D004247:DNA / Q000235:genetics*; D004258:DNA Polymerase III / Q000378:metabolism; D010957:Plasmids; D051135:Rad51 Recombinase; D051721:Rad52 DNA Repair and Recombination Protein / Q000378:metabolism; D011938:Rec A Recombinases / Q000378:metabolism; D011995:Recombination, Genetic / Q000235:genetics*
chemical_list: D051721:Rad52 DNA Repair and Recombination Protein; D004247:DNA; C065639:DNA polymerase A; D051135:Rad51 Recombinase; D011938:Rec A Recombinases; D004258:DNA Polymerase III