A Flow Cytometry-Based Method for Analyzing DNA End Resection in G0- and G1-Phase Mammalian Cells.

TitleA Flow Cytometry-Based Method for Analyzing DNA End Resection in G0- and G1-Phase Mammalian Cells.
Publication TypeJournal Article
Year of Publication2022
AuthorsChen B-R, Tyler JK, Sleckman BP
JournalBio Protoc
Date Published2022 May 20

DNA double strand breaks (DSBs) constantly arise in cells during normal cellular processes or upon exposure to genotoxic agents, and are repaired mostly by homologous recombination (HR) and non-homologous end joining (NHEJ). One key determinant of DNA DSB repair pathway choice is the processing of broken DNA ends to generate single strand DNA (ssDNA) overhangs, a process termed DNA resection. The generation of ssDNA overhangs commits DSB repair through HR and inhibits NHEJ. Therefore, DNA resection must be carefully regulated to avoid mis-repaired or persistent DSBs. Accordingly, many approaches have been developed to monitor ssDNA generation in cells to investigate genes and pathways that regulate DNA resection. Here we describe a flow cytometric approach measuring the levels of replication protein A (RPA) complex, a high affinity ssDNA binding complex composed of three subunits (RPA70, RPA32, and RPA14 in mammals), on chromatin after DNA DSB induction to assay DNA resection. This flow cytometric assay requires only conventional flow cytometers and can easily be scaled up to analyze a large number of samples or even for genetic screens of pooled mutants on a genome-wide scale. We adopt this assay in G0- and G1- phase synchronized cells where DNA resection needs to be kept in check to allow normal NHEJ.

Alternate JournalBio Protoc
PubMed ID35813018
PubMed Central IDPMC9183964
Grant ListR01 AI074953 / AI / NIAID NIH HHS / United States
R01 CA095641 / CA / NCI NIH HHS / United States
R35 GM139816 / GM / NIGMS NIH HHS / United States
Related Faculty: 
Jessica K. Tyler, Ph.D.

Pathology & Laboratory Medicine 1300 York Avenue New York, NY 10065 Phone: (212) 746-6464
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