A Proximity Ligation Method to Detect Proteins Bound to Single-Stranded DNA after DNA End Resection at DNA Double-Strand Breaks

Abstract

After a DNA double-strand break, cells utilize either non-homologous end joining or homologous recombination to repair the broken DNA ends. Homologous recombination requires extensive nucleolytic processing of one of the DNA strands, resulting in long stretches of 3′ single-strand DNA overhangs. Typically, single-stranded DNA is measured using immunofluorescence microscopy to image the foci of replication protein A, a single-stranded DNA-binding protein. Microscopy analysis of bromodeoxyuridine foci under nondenaturing conditions has also been used to measure single-stranded DNA. Here, we describe a proximity ligation assay which uses genome-wide bromodeoxyuridine incorporation to label single-stranded DNA in order to measure the association of a protein of interest with single-stranded DNA. This method is advantageous over traditional foci analysis because it is more direct and specific than traditional foci co-localization microscopy methods, uses only one color channel, and can reveal protein-single-stranded DNA interactions that are rare and potentially undetectable using traditional microscopy methods. We show here the association of replication protein A and bromodeoxyuridine as proof-of-concept.

Keywords

• single-stranded DNA
• proximity ligation assay
• DNA end resection
• double-strand break repair