Cargando…

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 loc...

Descripción completa

Detalles Bibliográficos
Autores principales: Forment, Josep V, Walker, Rachael V, Jackson, Stephen P
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Wiley Subscription Services, Inc., A Wiley Company 2012
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3601416/
https://www.ncbi.nlm.nih.gov/pubmed/22893507
http://dx.doi.org/10.1002/cyto.a.22155
_version_ 1782475763969163264
author Forment, Josep V
Walker, Rachael V
Jackson, Stephen P
author_facet Forment, Josep V
Walker, Rachael V
Jackson, Stephen P
author_sort Forment, Josep V
collection PubMed
description 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. © 2012 International Society for Advancement of Cytometry
format Online
Article
Text
id pubmed-3601416
institution National Center for Biotechnology Information
language English
publishDate 2012
publisher Wiley Subscription Services, Inc., A Wiley Company
record_format MEDLINE/PubMed
spelling pubmed-36014162013-03-20 A High-Throughput, Flow Cytometry-Based Method to Quantify DNA-End Resection in Mammalian Cells Forment, Josep V Walker, Rachael V Jackson, Stephen P Cytometry A Technical Notes 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. © 2012 International Society for Advancement of Cytometry Wiley Subscription Services, Inc., A Wiley Company 2012-10 2012-08-14 /pmc/articles/PMC3601416/ /pubmed/22893507 http://dx.doi.org/10.1002/cyto.a.22155 Text en Copyright © 2012 International Society for Advancement of Cytometry http://creativecommons.org/licenses/by/2.5/ Re-use of this article is permitted in accordance with the Creative Commons Deed, Attribution 2.5, which does not permit commercial exploitation.
spellingShingle Technical Notes
Forment, Josep V
Walker, Rachael V
Jackson, Stephen P
A High-Throughput, Flow Cytometry-Based Method to Quantify DNA-End Resection in Mammalian Cells
title A High-Throughput, Flow Cytometry-Based Method to Quantify DNA-End Resection in Mammalian Cells
title_full A High-Throughput, Flow Cytometry-Based Method to Quantify DNA-End Resection in Mammalian Cells
title_fullStr A High-Throughput, Flow Cytometry-Based Method to Quantify DNA-End Resection in Mammalian Cells
title_full_unstemmed A High-Throughput, Flow Cytometry-Based Method to Quantify DNA-End Resection in Mammalian Cells
title_short A High-Throughput, Flow Cytometry-Based Method to Quantify DNA-End Resection in Mammalian Cells
title_sort high-throughput, flow cytometry-based method to quantify dna-end resection in mammalian cells
topic Technical Notes
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3601416/
https://www.ncbi.nlm.nih.gov/pubmed/22893507
http://dx.doi.org/10.1002/cyto.a.22155
work_keys_str_mv AT formentjosepv ahighthroughputflowcytometrybasedmethodtoquantifydnaendresectioninmammaliancells
AT walkerrachaelv ahighthroughputflowcytometrybasedmethodtoquantifydnaendresectioninmammaliancells
AT jacksonstephenp ahighthroughputflowcytometrybasedmethodtoquantifydnaendresectioninmammaliancells
AT formentjosepv highthroughputflowcytometrybasedmethodtoquantifydnaendresectioninmammaliancells
AT walkerrachaelv highthroughputflowcytometrybasedmethodtoquantifydnaendresectioninmammaliancells
AT jacksonstephenp highthroughputflowcytometrybasedmethodtoquantifydnaendresectioninmammaliancells