A high-throughput, flow cytometry-based method to quantify DNA-end resection in mammalian cells.
Replication protein A (RPA) is an essential trimeric protein complex that binds to single-stranded DNA (ssDNA) in eukaryotic cells and is involved in various aspects of cellular DNA metabolism, including replication and repair. Although RPA is ubiquitously expressed throughout the cell cycle, it localizes to DNA replication forks during S phase, and is recruited to sites of DNA damage when regions of ssDNA are exposed. During DNA double-strand break (DSB) repair by homologous recombination (HR), RPA recruitment to DNA damage sites depends on a process termed DNA-end resection. Consequently, RPA recruitment to sub-nuclear regions bearing DSBs has been used as readout for resection and for ongoing HR. Quantification of RPA recruitment by immunofluorescence-based microscopy techniques is time consuming and requires extensive image analysis of relatively small populations of cells. Here, we present a high-throughput flow-cytometry method that allows the use of RPA staining to measure cell proliferation and DNA-damage repair by HR in an unprecedented, unbiased and quantitative manner.
The Gurdon Institute and Department of Biochemistry, University of Cambridge, CB2 1QN Cambridge, United Kingdom. j.forment@gurdon.cam.ac.uk
Josep V
JV
Forment
Rachael V
RV
Walker
Stephen P
SP
Jackson
D002166:Camptothecin / Q000494:pharmacology; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D053903:DNA Breaks, Double-Stranded / Q000187:drug effects; D004249:DNA Damage; D004260:DNA Repair; D004277:DNA, Single-Stranded / Q000235:genetics / Q000378:metabolism*; D005047:Etoposide / Q000494:pharmacology; D005434:Flow Cytometry / Q000379:methods*; D057166:High-Throughput Screening Assays; D059765:Homologous Recombination / Q000187:drug effects; D006801:Humans; D051716:Replication Protein A / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D013194:Staining and Labeling
D004277:DNA, Single-Stranded; D051716:Replication Protein A; D005047:Etoposide; D002166:Camptothecin