- Date de publication : 2015-12-22
Peyresaubes F, D'Amours A, Leduc F, Grégoire MC, Boissonneault G, Conconi A. Immuno-capture of UVDE generated 3'-OH ends at UV photoproducts. DNA Repair (Amst.). 2015;36:156-61. doi: 10.1016/j.dnarep.2015.09.019. PubMed PMID: 26547444.
dna damage dna, fungal endodeoxyribonucleases genome, fungal immunoprecipitation mutagenicity tests pyrimidine dimers saccharomyces cerevisiae schizosaccharomyces pombe proteins
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.