ChIP-on-chip analysis of DNA topoisomerases.

Here we describe an adapted ChIP-on-chip protocol for the analysis of DNA topoisomerase chromosomal binding in Saccharomyces cerevisiae cells. The ChIP-on-chip technique is based on the immunoprecipitation of crosslinked chromatin (ChIP, chromatin immunoprecipitation), followed by DNA amplification and hybridization to high-density oligonucleotide arrays (Chip). Comparison of the signal intensities of immunoprecipitated and control fractions provides a measurement of the protein-DNA association along entire genomes. ChIP-on-chip analysis of DNA topoisomerase binding to chromosomal DNA opens a window to the understanding of the in vivo contribution of these enzymes to the different DNA transactions taking place concomitantly within the context of the highly organized eukaryotic genome. Chromosomal binding profiles obtained from synchronized cells allow scoring the temporal and spatial restriction of these enzymes at different cell cycle stages. By using this approach, novel aspects of DNA topoisomerase function in chromosome metabolism might be unmasked.

Top1- and Top2-mediated topological transitions at replication forks ensure fork progression and stability and prevent DNA damage checkpoint activation.

DNA topoisomerases solve topological problems during chromosome metabolism. We investigated where and when Top1 and Top2 are recruited on replicating chromosomes and how their inactivation affects fork integrity and DNA damage checkpoint activation. We show that, in the context of replicating chromatin, Top1 and Top2 act within a 600-base-pair (bp) region spanning the moving forks. Top2 exhibits additional S-phase clusters at specific intergenic loci, mostly containing promoters. TOP1 ablation does not affect fork progression and stability and does not cause activation of the Rad53 checkpoint kinase. top2 mutants accumulate sister chromatid junctions in S phase without affecting fork progression and activate Rad53 at the M-G1 transition. top1 top2 double mutants exhibit fork block and processing and phosphorylation of Rad53 and gamma H2A in S phase. The exonuclease Exo1 influences fork processing and DNA damage checkpoint activation in top1 top2 mutants. Our data are consistent with a coordinated action of Top1 and Top2 in counteracting the accumulation of torsional stress and sister chromatid entanglement at replication forks, thus preventing the diffusion of topological changes along large chromosomal regions. A failure in resolving fork-related topological constrains during S phase may therefore result in abnormal chromosome transitions, DNA damage checkpoint activation, and chromosome breakage during segregation.