In Vitro Assays for DNA Pairing and Recombination-Associated DNA Synthesis
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.
Key wordsD-loop DNA polymerase DNA strand exchange DNA strand annealing DNA synthesis homologous recombination Rad51 Rad52 RecA supercoiled plasmid DNA
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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