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.
Highlights
Academic Editor: Fernando AlbericioThe DNA damage response is a network of cellular pathways that sense, signal, and repair DNA lesions
replication protein A (RPA)/single-stranded DNA (ssDNA) proximity ligation assay (PLA) foci after bleomycin treatment should appear as discrete, bright foci (Figure 2A)
Another way to quantify PLA foci data is to measure the % of cells with foci, i.e., % of cells with >0 RPA/ssDNA foci—in this way, we observe a five-fold increase in the % of cells with foci (Figure 2B)
Summary
Academic Editor: Fernando AlbericioThe DNA damage response is a network of cellular pathways that sense, signal, and repair DNA lesions. Surveillance proteins that monitor DNA integrity can activate cell cycle checkpoints and DNA repair pathways in response to DNA damage to prevent the generation of potentially deleterious mutations. At DSBs, MRE11 and CtIP initiate short-range DNA end resection, which is extended by EXO1 and BLM-DNA2, leading to long stretches of 30 single-stranded DNA (ssDNA) [5,6,7,8]. This ssDNA is subsequently coated with replication protein A (RPA), which stabilizes and protects the ssDNA from degradation [9]. RPA is replaced with RAD51 recombinase, forming a nucleofilament that can scan the genome for a homologous template to achieve accurate repair [10,11]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
