Break-Seq to map DNA double stranded breaks in WT S. cerevisiae WT post treatment with six 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. The 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 six different replication (HU,MMS,CPT, AD,DOX,MTX) inhibitors at their IC50 concentration. DSBs were mapped in each of the replication inhibitor treatment as well as its vehicle control. Two independent experiments were performed for each with each having a technical replicate (4 total experiments per each replication inihibitor and vehicle control).

复制-转录冲突长期以来被认定为基因组不稳定性的诱因之一，会在两者相遇的位点引发DNA双链断裂（double-strand break, DSBs）的形成。当细胞遭遇应激条件（如使用可改变复制或转录任一过程的试剂进行处理）时，复制与转录的相遇事件会显著增多。任一过程的异常都会提升两者相遇的概率，最终诱导DSB的产生。此前针对经羟基脲（hydroxyurea, HU）处理的同步化细胞培养物的研究证实，HU诱导的DSB源于复制-转录碰撞。本研究旨在探究复制-转录碰撞是否为复制抑制剂诱导DSB产生的普遍机制。

在本研究中，我们选用了6种作用机制各不相同的复制抑制剂：羟基脲（HU）、甲基甲烷磺酸酯（MMS）、喜树碱（CPT）、放线菌素D（AD）、多柔比星（DOX）以及甲氨蝶呤（MTX）。由各复制抑制剂诱导的特异性DSB倾向于富集于基因外显子区域，同时与复制起点（origin of replication）呈现显著相关性。这些DSB位点周边的基因表达往往会上调，进而进一步提升两者相遇的概率。上述结果支持了我们的假说：DSB源于复制-转录冲突。我们还在某一DSB位点处观察到了转录依赖性组蛋白修饰（histone mark）沉积——H3K4me3与H3K36me3的富集。

综上，本研究结果提示：复制抑制剂处理会诱导特定基因的表达，沉积活性组蛋白修饰，当这些区域遭遇前进中的复制叉时便会引发碰撞。总体而言，本研究绘制了不同复制抑制剂诱导的DSB全基因组定位图谱以及相应的基因表达谱，并为染色质结构（chromatin architecture）在调控基因组完整性（genome integrity）中的作用提供了新的认知。

整体实验设计：将W303背景下的异步生长野生型（wild type, WT）细胞以各自的半最大效应浓度（half maximal inhibitory concentration, IC50）分别用6种复制抑制剂（HU、MMS、CPT、AD、DOX、MTX）处理，并设置对应的溶剂对照。我们对每种抑制剂处理组与溶剂对照组均开展了两次独立实验，每次实验均设置技术重复（即每种抑制剂与对照共进行4次实验）。