Topological and Chromatin Alterations Influencing Genome Integrity

DNA topoisomerases Top1 and Top2 have redundant functions in resolving topological alterations arising during replication and transcription processes. Topoisomerases assist replication forks encountering transcription units, preventing chromosome fragility by minimizing the aberrant topological events. We investigated the role of topoisomerases in supercoil accumulation across the yeast genome using biotin tagged psoralen immunoprecipitation. We found that DNA is under-wound at gene boundaries and over-wound at transcribed regions. Top1 is associated with positively supercoiled chromatin as it accompanies RNA Polymerase II (Pol2) and its chromatin association is influenced by transcription levels of the individual genes. Top2 is associated with stable negative supercoiled chromatin at the gene boundaries, and its association is not dependent on transcription. Top2 promotes transcription efficiencies by forming gene loop structure and restricts Top1 and Pol2 leakage at gene boundaries. Ablation of Top2 protein decreases the negative supercoil accumulation at gene boundaries. Expression of E.coli TopA in topoisomerases double mutant in yeast (top2-1top1Δ) significantly resolves only the negative supercoil of gene boundaries and increases the accumulation of positive supercoil. The supercoil state at gene boundaries and ORFs are crucial for nucleosome occupancy. Using Hi-C techniques, we show that, centromeres are prominently interacting with other centromeres and the inter-chromosomal centromere interactions are depleted along with cohesin protein in top2-1top1Δ mutant expressing E.coli TopA. This work therefore summarizes the role of supercoil structures in preserving higher order architecture including nucleosome formation and chromosome organization

Sen1 and Rrm3 ensure permissive topological conditions for replication termination

Summary: Replication forks terminate at TERs and telomeres. Forks that converge or encounter transcription generate topological stress. Combining genetics, genomics, and transmission electron microscopy, we find that Rrm3hPif1 and Sen1hSenataxin helicases assist termination at TERs; Sen1 specifically acts at telomeres. rrm3 and sen1 genetically interact and fail to terminate replication, exhibiting fragility at termination zones (TERs) and telomeres. sen1rrm3 accumulates RNA-DNA hybrids and X-shaped gapped or reversed converging forks at TERs; sen1, but not rrm3, builds up RNA polymerase II (RNPII) at TERs and telomeres. Rrm3 and Sen1 restrain Top1 and Top2 activities, preventing toxic accumulation of positive supercoil at TERs and telomeres. We suggest that Rrm3 and Sen1 coordinate the activities of Top1 and Top2 when forks encounter transcription head on or codirectionally, respectively, thus preventing the slowing down of DNA and RNA polymerases. Hence Rrm3 and Sen1 are indispensable to generate permissive topological conditions for replication termination