FRET-Based Assays to Monitor DNA Binding and Annealing by Rad52 Recombination Mediator Protein
During homologous recombination and homology-directed repair of broken chromosomes, proteins that mediate and oppose recombination form dynamic complexes on damaged DNA. Quantitative analysis of these nucleoprotein assemblies requires a robust signal, which reports on the association of a recombination mediator with its substrate and on the state of substrate DNA within the complex. Eukaryotic Rad52 protein mediates recombination, repair, and restart of collapsed replication forks by facilitating replacement of ssDNA binding protein replication protein A (RPA) with Rad51 recombinase and by mediating annealing of two complementary DNA strands protected by RPA. The characteristic binding mode whereby ssDNA is wrapped around the Rad52 ring allowed us to develop robust and sensitive FRET-based assays for monitoring Rad52 interactions with protein-free DNA and ssDNA–RPA complexes. By reporting on the configuration of ssDNA dually labeled with Cy3 and Cy5 fluorescent dyes, solution-based FRET is used to analyze Rad52–RPA–DNA interactions under equilibrium binding conditions. Finally, FRET between Cy3 and Cy5 dyes incorporated into two homologous ssDNA molecules can be used to analyze interplay between Rad52-mediated DNA strand annealing and duplex DNA destabilization by RPA.
Key wordsAnnealing protein fluorescence Föster resonance energy transfer (FRET) homologous recombination Rad52 recombination mediator RPA
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