Single-molecule studies of yeast Rad51 paralogs

Homologous recombination (HR) is a conserved mechanism essential for the accurate repair of DNA double stranded breaks and the exchange of genetic information during meiosis. The key steps in HR are carried out by the RecA/Rad51 class of recombinases, which form a helical filament on single-stranded DNA (ssDNA) and catalyze homology search and strand exchange with a complementary duplex DNA target. In eukaryotes, assembly of the Rad51-ssDNA filament requires regulatory factors called mediators, including Rad51 paralogs. A mechanistic understanding of the role of Rad51 paralogs in HR has been hampered by the transient and diverse nature of intermediates formed with the Rad51-ssDNA filament, which cannot be resolved by traditional ensemble methods. The biochemical characterization of Rad51 paralogs, including the S. cerevisiae complex Rad55-Rad57 has also been limited by their propensity to aggregate. Here we describe the preparation of monodisperse GFP-tagged Rad55-Rad57 complex and the methodology for its analysis in our single-molecule DNA curtain assay.