title: Quantifying DNA double-strand breaks induced by site-specific endonucleases in living cells by ligation-mediated purification.
abstract: Recent advances in our understanding of the management and repair of DNA double-strand breaks (DSBs) rely on the study of targeted DSBs that have been induced in living cells by the controlled activity of site-specific endonucleases, usually recombinant restriction enzymes. Here we describe a protocol for quantifying these endonuclease-induced DSBs; this quantification is essential to an interpretation of how DSBs are managed and repaired. A biotinylated double-stranded oligonucleotide is ligated to enzyme-cleaved genomic DNA, allowing the purification of the cleaved DNA on streptavidin beads. The extent of cleavage is then quantified either by quantitative PCR (qPCR) at a given site or at multiple sites by genome-wide techniques (e.g., microarrays or high-throughput sequencing). This technique, named ligation-mediated purification, can be performed in 2 d. It is more accurate and sensitive than existing alternative methods, and it is compatible with genome-wide analysis. It allows the amount of endonuclease-mediated breaks to be precisely compared between two conditions or across the genome, thereby giving insight into the influence of a given factor or of various chromatin contexts on local repair parameters.
authors: 1] Université de Toulouse, Université Paul Sabatier, Laboratoire de Biologie Cellulaire et Moléculaire du Contrôle de la Prolifération (LBCMCP), Toulouse, France. [2] Centre National de la Recherche Scientifique, LBCMCP, Toulouse, France.
authors: Catherine
authors: C
authors: Chailleux
authors: 1] Université de Toulouse, Université Paul Sabatier, Laboratoire de Biologie Cellulaire et Moléculaire du Contrôle de la Prolifération (LBCMCP), Toulouse, France. [2] Centre National de la Recherche Scientifique, LBCMCP, Toulouse, France.
authors: François
authors: F
authors: Aymard
authors: 1] Université de Toulouse, Université Paul Sabatier, Laboratoire de Biologie Cellulaire et Moléculaire du Contrôle de la Prolifération (LBCMCP), Toulouse, France. [2] Centre National de la Recherche Scientifique, LBCMCP, Toulouse, France.
authors: Pierre
authors: P
authors: Caron
authors: 1] Université de Toulouse, Université Paul Sabatier, Laboratoire de Biologie Cellulaire et Moléculaire du Contrôle de la Prolifération (LBCMCP), Toulouse, France. [2] Centre National de la Recherche Scientifique, LBCMCP, Toulouse, France.
authors: Virginie
authors: V
authors: Daburon
authors: 1] Université de Toulouse, Université Paul Sabatier, Laboratoire de Biologie Cellulaire et Moléculaire du Contrôle de la Prolifération (LBCMCP), Toulouse, France. [2] Centre National de la Recherche Scientifique, LBCMCP, Toulouse, France.
authors: Céline
authors: C
authors: Courilleau
authors: 1] Université de Toulouse, Université Paul Sabatier, Laboratoire de Biologie Cellulaire et Moléculaire du Contrôle de la Prolifération (LBCMCP), Toulouse, France. [2] Centre National de la Recherche Scientifique, LBCMCP, Toulouse, France.
authors: Yvan
authors: Y
authors: Canitrot
authors: 1] Université de Toulouse, Université Paul Sabatier, Laboratoire de Biologie Cellulaire et Moléculaire du Contrôle de la Prolifération (LBCMCP), Toulouse, France. [2] Centre National de la Recherche Scientifique, LBCMCP, Toulouse, France. [3].
authors: Gaëlle
authors: G
authors: Legube
authors: 1] Université de Toulouse, Université Paul Sabatier, Laboratoire de Biologie Cellulaire et Moléculaire du Contrôle de la Prolifération (LBCMCP), Toulouse, France. [2] Centre National de la Recherche Scientifique, LBCMCP, Toulouse, France. [3].
authors: Didier
authors: D
authors: Trouche
mesh_terms: D001483:Base Sequence; D004247:DNA / Q000302:isolation & purification* / Q000378:metabolism; D053903:DNA Breaks, Double-Stranded; D004260:DNA Repair / Q000502:physiology*; D004720:Endonucleases / Q000378:metabolism*; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000235:genetics / Q000378:metabolism; D019809:Streptavidin
chemical_list: D009841:Oligonucleotides; D004247:DNA; D019809:Streptavidin; D004720:Endonucleases