Genome binding/occupancy profiling by high throughput sequencing
Summary
DNA topological stress inhibits DNA replication fork (RF) progression and contributes to DNA replication stress. In Saccharomyces cerevisiae we demonstrate that centromeric DNA and the rDNA array are especially vulnerable to DNA topological stress during replication. The activity of the SMC complexes cohesin and condensin are linked to both the generation and repair of DNA topological stress linked damage in these regions. At cohesin enriched centromeres cohesin activity causes the accumulation of DNA damage, RF rotation and precatenation, confirming that cohesin dependent DNA topological stress impacts on normal replication progression. In contrast, at the rDNA cohesin and condensin activity inhibit the repair of damage caused by DNA topological stress. We propose that as well as generally acting to ensure faithful genetic inheritance, SMCs can disrupt genome stability by trapping DNA topological stress.
Overall design
To identify the chromosomal contexts where DNA topological stress leads to DNA damage during DNA replication, we examined cells where endogenous DNA topological stress was elevated by the depletion of Top2 in wt and various mutants. Specifically, we identified regions where DNA replication was disrupted by chromatin immunoprecipitation of H2AS129P or RPA1, followed by next generation sequencing (ChIP-SEQ). For RPA1 we used strand specific sequencing. Each experiment was repeated at least two times.
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