Topoisomerase II-induced Chromosome Breakage and Translocation Is Determined by Chromosome Architecture and Transcriptional Activity [END-seq]

Topoisomerase II (TOP2) relieves torsional stress during transcription, DNA replication and chromosome segregation, by forming transient cleavage complex intermediates (TOP2ccs) that contain TOP2-linked DNA breaks. While TOP2ccs are normally reversible they can be 'trapped' by chemotherapeutic drugs such as etoposide, and subsequently converted into irreversible TOP2-linked DSBs that threaten genome stability. Here, using genomics approaches, we have quantified the etoposide-induced trapping of TOP2ccs, their conversion into irreversible TOP2-linked DSBs, and their processing during DNA repair genome-wide, as a function of time. We find that while TOP2 trapping is independent of transcription it requires pre-existing binding of cohesin to DNA. In contrast, the conversion of trapped TOP2ccs to irreversible DSBs during DNA repair is accelerated two-fold at transcribed loci, relative to non-transcribed loci. This conversion is dependent on proteasomal degradation and TDP2 phosphodiesterase activity. Quantitative modeling shows that only two critical features of pre-existing chromatin structure- namely, cohesin binding and transcriptional activity- can be used to accurately predict the kinetics of TOP2-induced DSBs. Thus, our study permits a mechanistic understanding of TOP2 induced genome instability. Overall design: END-seq profile in B, MEFs, pre-B, T, and Neurons untreated and upon etoposide treatment, and upon treatment with transcription inhibition drugs

拓扑异构酶II（Topoisomerase II，TOP2）可通过形成包含TOP2共价连接DNA断裂的瞬时切割复合物中间体（TOP2ccs），在转录、DNA复制及染色体分离过程中缓解DNA扭转张力。尽管TOP2ccs通常可逆转，但它们可被依托泊苷等化疗药物“捕获”，随后转化为威胁基因组稳定性的不可逆TOP2共价连接DNA双链断裂（DSBs）。本研究借助基因组学技术手段，定量分析了依托泊苷诱导的TOP2ccs捕获过程、其向不可逆TOP2共价连接DNA双链断裂的转化，以及全基因组范围内DNA修复过程中此类复合物的加工动态，并以时间作为观测变量展开研究。研究发现，TOP2捕获过程与转录无关，但该过程依赖于黏连蛋白预先与DNA的结合。与之相反，相较于非转录位点，DNA修复过程中捕获的TOP2ccs向不可逆DNA双链断裂的转化在转录位点处被加速了两倍。该转化过程依赖于蛋白酶体降解途径与TDP2磷酸二酯酶活性。定量建模结果显示，仅需利用预先存在的染色质结构的两个关键特征——即黏连蛋白结合与转录活性——即可精准预测TOP2诱导的DNA双链断裂的动力学特征。因此，本研究为阐释TOP2诱导的基因组不稳定性提供了机制层面的认知。实验整体设计：在未处理、依托泊苷处理以及转录抑制药物处理的B细胞、小鼠胚胎成纤维细胞（mouse embryonic fibroblasts, MEFs）、前B细胞、T细胞和神经元中开展END-seq测序分析。