ChIP-Seq to map DNA double stranded breaks in WT S. cerevisiae post treatment with five different replication inhibitors

Replication–transcription conflicts have long been identified as a source of genome instability, leading to DNA double-strand break (DSBs) formation at the site of encounter. These encounter between replication and transcription is significantly increased when cells encounter stress, such as by treatment with agents that alter either of these processes. Alterations in either of these processes increase the probability of their encounter, ultimately inducing DSBs. Previous studies with hydroxyurea (HU)-treated synchronous cell cultures showed that HU-induced DSBs are a result of replication-transcription collisions. Here, we sought to determine whether replication-transcription collision is a general phenomenon used by replication inhibitors to induce DSBs. In our study, we used six different replication inhibitors, HU, methyl methane sulfonate (MMS), camptothecin (CPT), actinomycin D (AD), doxorubicin (DOX), and methotrexate (MTX), each of which acts differently. The replication inhibitor-specific DSBs tend to be enriched at gene exons and are also well correlated with the origins of replication. Gene expression around these DSBs tends to be upregulated, thus increasing the probability of encounter between the two. These results support our hypothesis that DSBs result from replication-transcription conflicts. We also observed enrichment in the transcription-dependent histone mark deposition-H3K4me3 and H3K36me3 at one DSB locus. Taken together, these results suggest that replication inhibitor treatment induces the expression of certain genes, depositing active histone marks, which upon encountering the incoming replication fork result in collision. Overall, our study maps the genome-wide location of DSBs as well as the gene expression profile induced by different replication inhibitors and provides insight into the role of chromatin architecture in regulating genome integrity. Overall design: Asynchronously grown WT cells from the W303 background were treated with five different replication (HU,MMS,CPT,DOX,MTX) inhibitors at their IC50 concentration. ChIP-seq for transcription deposited histone marks (H3K4me3 and H3K36me3) were mapped in each of the replication inhibitor treatment as well as its vehicle control. Two independent experiments were performed for each (2 total experiments per each replication inihibitor and vehivle control).