DNA Recombination pp 363-383

Part of the Methods in Molecular Biology book series (MIMB, volume 745) | Cite as

In Vitro Assays for DNA Pairing and Recombination-Associated DNA Synthesis

Protocol

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.

Key words

D-loop DNA polymerase DNA strand exchange DNA strand annealing DNA synthesis homologous recombination Rad51 Rad52 RecA supercoiled plasmid DNA 

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Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  1. 1.Department of MicrobiologyUniversity of CaliforniaDavisUSA
  2. 2.Department of Microbiology and Department of Molecular and Cellular BiologyUniversity of CaliforniaDavisUSA

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