Development of a method for mapping oxidative damage to an entire genome in vivo with a single experiment
Bernard, Sarah Elizabeth
MetadataShow full item record
Nearly all cellular maintenance functions rely on DNA-protein interactions. Several techniques exist for mapping DNA-protein interactions in living cells on a genome-wide scale using high-throughput sequencing. Each of these methods is specific for a class of DNA-protein interactions. Although when combined, these methods provide a high-resolution picture of the chromatin landscape in vivo, there is no one method is capable of providing information on protein binding at each nucleotide in the genome in a single experiment. Hydroxyl radical footprinting is a classic method for determining DNA-protein interactions. Use of the hydroxyl radical in DNA structure determination has been limited to the study of relatively small DNA fragments. The recent development of high-throughput DNA sequencing technologies now makes it possible to increase the scope of this method to the scale of an entire genome. Presented here is the development of a technique for genome-wide mapping of oxidative damage to genomic DNA in the yeast Saccharomyces cerevisiae that results from exposure to hydroxyl radicals in vivo. Sequencing libraries have been prepared through careful optimization of each step in the methodology. This technique will have numerous applications in decoding the structural biology of the genome.