Genome-wide mapping of meiosis-induced DNA double-strand breaks

Abstract

The major applications of DNA microarray technology to date have been genome-wide analysis of gene expression and analysis of DNA variation. Our goal is to develop an approach using DNA microarray technology to quantitatively study the distribution of DNA-associated proteins at the whole-genome level. An initial approach will determine the genomic distribution of DNA double-strand breaks (DSBs) that initiate meiotic recombination in Saccharomyces cerevisiae. DSB formation involves an intermediate stage where DNA ends are covalently linked to Spo11p. In a rad50S mutant background these intermediates accumulate throughout meiosis. Our approach is to purify Spo11p::His6/DNA intermediates by immobilized metal-affinity chromatography. The DNA purified from these intermediates is then hybridized to DNA microarrays to map the distribution and frequency of Spo11p-linked double-strand breaks throughout the genome. With the addition of a formaldehyde cross-linking step, this approach could be used to map the distribution of any protein that interacts with the genome, either independently or as part of a protein complex. The affinity chromatography approach allows large amounts of material to be purified, and should enable the detection of minor or transient protein/DNA intermediates.