title: Real-time solution measurement of RAD51- and RecA-mediated strand assimilation without background annealing.
abstract: RAD51 is the central strand exchange recombinase in somatic homologous recombination, providing genomic stability and promoting resistance to DNA damage. An important tool for mechanistic studies of RAD51 is the D-loop or strand assimilation assay, which measures the ability of RAD51-coated single-stranded DNA (ssDNA) to search for, invade and exchange ssDNA strands with a homologous duplex DNA target. As cancer cells generally overexpress RAD51, the D-loop assay has also emerged as an important tool in oncologic drug design programs for targeting RAD51. Previous studies have adapted the traditional gel-based D-loop assay by using fluorescence-based substrates, which in principle allow for use in high-throughput screening platforms. However, these existing D-loop methods depend on linear oligonucleotide DNA duplex targets, and these substrates enable recombinase-independent ssDNA annealing that can obscure the recombinase-dependent strand assimilation signal. This compelled us to fundamentally re-design this assay, using a fluorescent target substrate that consists of a covalently closed linear double-hairpin dsDNA. This new microplate-based method represents a fast, inexpensive and non-radioactive alternative to existing D-loop assays. It provides accurate kinetic analysis of strand assimilation in high-throughput and performs well with human RAD51 and Escherichia coli RecA protein. This advance will aid in both mechanistic studies of homologous recombination and drug screening programs.
authors.affiliation: Department of Radiation and Cellular Oncology, University of Chicago, Chicago, IL 60637, USA. bbudke@uchicago.edu
authors.forename: Brian
authors.initials: B
authors.lastname: Budke
authors.forename: Yuen-Ling
authors.initials: YL
authors.lastname: Chan
authors.forename: Douglas K
authors.initials: DK
authors.lastname: Bishop
authors.forename: Philip P
authors.initials: PP
authors.lastname: Connell
mesh_terms: D029968:Escherichia coli Proteins / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D005470:Fluorometry / Q000379:methods; D059765:Homologous Recombination; D006801:Humans; D051135:Rad51 Recombinase / Q000378:metabolism*; D011938:Rec A Recombinases / Q000378:metabolism*
chemical_list: D029968:Escherichia coli Proteins; C495153:RAD51 protein, human; D051135:Rad51 Recombinase; D011938:Rec A Recombinases