An In Vitro Assay for Monitoring the Formation and Branch Migration of Holliday Junctions Mediated by a Eukaryotic Recombinase

Abstract

DNA strand exchange is a core reaction of homologous recombination directly catalyzed by Rad51/Dmc1 RecA family recombinases in eukaryotes. This reaction proceeds through the formation of several DNA intermediates. The X-shaped four-way DNA structure known as a Holliday junction (HJ) is a central intermediate in homologous recombination. Genetic and biochemical studies indicate that the HJ is important for the production of crossover-type recombinants, which are reciprocal exchange products. According to a recombination model for the repair of DNA double-strand breaks, the formation of HJs requires a reciprocal duplex–duplex DNA exchange known as the DNA four-strand exchange reaction. In vitro analyses using purified recombination proteins and model DNA substrates provide a mechanistic insight into the DNA strand exchange reaction, including the steps leading to the formation and branch migration of Holliday junctions.