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• W7299969 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." @default.
• W7299969 created "2016-06-24" @default.
• W7299969 creator A5038712886 @default.
• W7299969 creator A5051968023 @default.
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• W7299969 date "2011-01-01" @default.
• W7299969 modified "2023-09-23" @default.
• W7299969 title "In Vitro Assays for DNA Pairing and Recombination-Associated DNA Synthesis" @default.
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• W7299969 doi "https://doi.org/10.1007/978-1-61779-129-1_21" @default.
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