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Monitoring Homologous Recombination Activity in Human Cells.

Authors
  • Vugic, Domagoj1, 2
  • Ehlén, Åsa1, 2
  • Carreira, Aura3, 4
  • 1 Institut Curie, PSL Research University, CNRS, UMR3348, F-91405, Orsay, France. , (France)
  • 2 University Paris Sud, Paris-Saclay University, CNRS, UMR3348, F-91405, Orsay, France. , (France)
  • 3 Institut Curie, PSL Research University, CNRS, UMR3348, F-91405, Orsay, France. [email protected] , (France)
  • 4 University Paris Sud, Paris-Saclay University, CNRS, UMR3348, F-91405, Orsay, France. [email protected] , (France)
Type
Published Article
Journal
Methods in molecular biology
Publisher
Clifton, N.J. : Humana Press
Publication Date
Jan 01, 2021
Volume
2153
Pages
115–126
Identifiers
DOI: 10.1007/978-1-0716-0644-5_9
PMID: 32840776
Source
Medline
Keywords
Language
English
License
Unknown

Abstract

DNA double-strand breaks (DSBs) are among the most toxic lesions. This type of DNA damage is repaired by two major pathways, homologous recombination (HR), operating only in S/G2 cell-cycle phases and nonhomologous end joining (NHEJ) which is operative throughout the cell cycle. Because HR is a template-directed repair, it is generally less prone to errors and/or translocations than NHEJ.The HR pathway involves several effector proteins and regulators that modulate the efficiency of repair and limit the repair outside S/G2 phase. Some of the genes coding for these proteins are frequently mutated in human diseases such as cancer, and pathogenic mutations or variants identified in patients often alter the HR proficiency of the cells.This chapter describes a cell-based gene-targeting reporter assay in human cells to evaluate the repair of a site-specific DSB by HR . In it, a promoter-less fluorescent protein is encoded in a plasmid flanked by two homology arms directed to a safe-harbour locus in the genome. The expression of the fluorescent protein is driven by the promoter of the endogenous locus enabling to quantify the efficiency of HR by flow cytometry. This approach can be used to determine the requirement of certain proteins, protein domains, or protein modifications for HR . It can also be used to functionally evaluate variants of the genes encoding these proteins such as BRCA1, BRCA2, RAD51C, and PALB2; which may help assess their pathogenicity. Here, we use the homologous recombination mediator BRCA2 to illustrate the assay.

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