Monitoring 5'-End Resection at Site-Specific Double-Strand Breaks by Southern Blot Analysis

DNA double-strand break (DSB) is one of the most deleterious types of DNA lesions threatening genome integrity. Cells have evolved several exquisite pathways to repair these breaks. Homologous recombination (HR) is an essential DSB repair mechanism that utilizes an intact homologous sequence as a template to repair DSBs with high fidelity. To initiate the HR repair, the 5'-ends of DSBs have to be nucleolytically cleaved by nucleases to generate 3'-single-strand DNA (ssDNA). Exposed 3'-ssDNA recruits the ssDNA binding protein complex RPA to activate the DNA damage checkpoint. RPA is subsequently replaced by Rad51 recombinase to form Rad51 nucleoprotein filament that catalyzes strand invasion and formation of the D-loop. Processing of 5'-ends (called resection) is a crucial step that determines the choice of repair pathways. Here we introduce an assay for monitoring the dynamics of resection at different locations from a site-specific DSB in yeast.