Topological and Chromatin Alterations Influencing Genome Integrity

Mohammed Iqbal, Mohamood Adhil (2022). Topological and Chromatin Alterations Influencing Genome Integrity. PhD thesis The Open University.



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.

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