Quantification and genome-wide mapping of DNA double-strand breaks.
DNA double-strand breaks (DSBs) represent a major threat to the genetic integrity of the cell. Knowing both their genome-wide distribution and number is important for a better assessment of genotoxicity at a molecular level. Available methods may have underestimated the extent of DSBs as they are based on markers specific to those undergoing active repair or may not be adapted for the large diversity of naturally occurring DNA ends. We have established conditions for an efficient first step of DNA nick and gap repair (NGR) allowing specific determination of DSBs by end labeling with terminal transferase. We used DNA extracted from HeLa cells harboring an I-SceI cassette to induce a targeted nick or DSB and demonstrated by immunocapture of 3'-OH that a prior step of NGR allows specific determination of loci-specific or genome wide DSBs. This method can be applied to the global determination of DSBs using radioactive end labeling and can find several applications aimed at understanding the distribution and kinetics of DSBs formation and repair.
Department of Biochemistry, Université de Sherbrooke, Sherbrooke, Quebec, Canada.
Marie-Chantal
MC
Grégoire
Department of Biochemistry, Université de Sherbrooke, Sherbrooke, Quebec, Canada.
Julien
J
Massonneau
Department of Biochemistry, Université de Sherbrooke, Sherbrooke, Quebec, Canada.
Frédéric
F
Leduc
Department of Biochemistry, Université de Sherbrooke, Sherbrooke, Quebec, Canada.
Mélina
M
Arguin
Department of Biochemistry, Université de Sherbrooke, Sherbrooke, Quebec, Canada.
Marc-André
MA
Brazeau
Department of Biochemistry, Université de Sherbrooke, Sherbrooke, Quebec, Canada. Electronic address: guylain.boissonneault@usherbrooke.ca.
Guylain
G
Boissonneault
D002874:Chromosome Mapping / Q000379:methods*; D004247:DNA / Q000235:genetics* / Q000378:metabolism; D053903:DNA Breaks, Double-Stranded; D004253:DNA Nucleotidylexotransferase / Q000235:genetics* / Q000378:metabolism; D004260:DNA Repair; D015252:Deoxyribonucleases, Type II Site-Specific / Q000235:genetics* / Q000378:metabolism; D056426:Genetic Loci; D015894:Genome, Human; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D010761:Phosphorus Radioisotopes / Q000378:metabolism; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics* / Q000378:metabolism; D014162:Transfection
D010761:Phosphorus Radioisotopes; D029701:Saccharomyces cerevisiae Proteins; D004247:DNA; D004253:DNA Nucleotidylexotransferase; C037725:SCEI protein, S cerevisiae; D015252:Deoxyribonucleases, Type II Site-Specific
DNA double-strand break; DNA strand break quantification; Gap; Genome-wide mapping; Nick; Single-strand break repair; Terminal deoxynucleotidyl transferase