title: Immuno-capture of UVDE generated 3'-OH ends at UV photoproducts.
abstract: A strategy amenable to the genome-wide study of DNA damage and repair kinetics is described. The ultraviolet damage endonuclease (UVDE) generates 3'-OH ends at the two major UV induced DNA lesions, cyclobutane pyrimidine dimers (CPDs) and 6,4 pyrimidine-pyrimidone dimers (6,4 PPs), allowing for their capture after biotin end-labeling. qPCR amplification of biotinylated DNA enables parallel measuring of DNA damage in several loci, which can then be combined with high-throughput screening of cell survival to test genotoxic reagents. Alternatively, a library of captured sequences could be generated for a genome wide study of damage sites and large-scale assessment of repair kinetics in different regions of the genome, using next-generation sequencing. The assay is suitable to study any DNA lesion that can be converted into 3'-OH by UVDE, or other enzymes. Toward these goals, we compared UVDE with the classical T4 endonuclease V (T4V) assay. We showed that there is a linear correlation between UV dose, 3'-OH formation and capture by immunoprecipitation, together with its potential application for in vivo studies.
authors.affiliation: Département de Microbiologie et Infectiologie, Faculté de Médecine, Université de Sherbrooke, Sherbrooke, QC J1E 4K8, Canada.
authors.forename: François
authors.initials: F
authors.lastname: Peyresaubes
authors.affiliation: Département de Microbiologie et Infectiologie, Faculté de Médecine, Université de Sherbrooke, Sherbrooke, QC J1E 4K8, Canada; Département de Biochimie, Faculté de Médecine, Université de Sherbrooke, Sherbrooke, QC J1E 4K8, Canada.
authors.forename: Annie
authors.initials: A
authors.lastname: D'Amours
authors.affiliation: Département de Biochimie, Faculté de Médecine, Université de Sherbrooke, Sherbrooke, QC J1E 4K8, Canada.
authors.forename: Frédéric
authors.initials: F
authors.lastname: Leduc
authors.affiliation: Département de Biochimie, Faculté de Médecine, Université de Sherbrooke, Sherbrooke, QC J1E 4K8, Canada.
authors.forename: Marie-Chantal
authors.initials: MC
authors.lastname: Grégoire
authors.affiliation: Département de Biochimie, Faculté de Médecine, Université de Sherbrooke, Sherbrooke, QC J1E 4K8, Canada. Electronic address: Guylain.Boissonneault@usherbrooke.ca.
authors.forename: Guylain
authors.initials: G
authors.lastname: Boissonneault
authors.affiliation: Département de Microbiologie et Infectiologie, Faculté de Médecine, Université de Sherbrooke, Sherbrooke, QC J1E 4K8, Canada. Electronic address: Antonio.Conconi@usherbrooke.ca.
authors.forename: Antonio
authors.initials: A
authors.lastname: Conconi
mesh_terms: D004249:DNA Damage; D004271:DNA, Fungal / Q000737:chemistry; D004706:Endodeoxyribonucleases / Q000378:metabolism; D016681:Genome, Fungal; D047468:Immunoprecipitation; D009152:Mutagenicity Tests; D011740:Pyrimidine Dimers / Q000032:analysis*; D012441:Saccharomyces cerevisiae / Q000235:genetics; D029702:Schizosaccharomyces pombe Proteins / Q000378:metabolism
chemical_list: D004271:DNA, Fungal; D011740:Pyrimidine Dimers; D029702:Schizosaccharomyces pombe Proteins; D004706:Endodeoxyribonucleases; C105073:uve1 protein, S pombe
keywords: 6,4 Pyrimidine-pyrimidone dimers; Cyclobutane pyrimidine dimers; T4 endonuclease V; Ultraviolet damage endonuclease (UVDE)