Immuno-capture of UVDE generated 3’-OH ends at UV photoproducts |
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Institution: | 1. Institute of Pharmacology and Toxicology, University of Zurich-Vetsuisse, Zurich, Switzerland;2. Institute of Cancer and Genetics, School of Medicine, Cardiff University, Cardiff, UK |
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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. |
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Keywords: | Cyclobutane pyrimidine dimers 6 4 Pyrimidine-pyrimidone dimers T4 endonuclease V Ultraviolet damage endonuclease (UVDE) |
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