Cohesin maintains replication timing to suppress DNA damage on cancer genes [HU117_CBE_PEM-seq]

Cohesin loss-of-function mutations are frequently observed in tumors, but the mechanism is unclear. Here, we found that depletion of RAD21, a core subunit of cohesin, leads to massive genome-wide DNA breaks, up to five-fold, and 147 translocation hotspot genes that are co-mutated with cohesin in multiple cancers. Increased DNA damages are independent of RAD21-loss-induced transcription alteration and loop anchor elimination. However, damage-induced chromosomal translocations coincide with the asymmetrically distributed Okazaki fragments of DNA replication, suggesting that RAD21 depletion causes replication stresses evidenced by the slower replication speed and increased stalled forks. Mechanistically, approximately 30% of the human genome exhibits an earlier replication timing after RAD21 depletion, caused by the early initiation of >900 extra dormant origins. Correspondingly, most translocation hotspot genes lie in timing-altered regions. Therefore, we conclude that cohesin dysfunction causes replication stresses induced by excessive DNA replication initiation, resulting in gross DNA damages that may promote tumorigenesis. Overall design: We employed primer-extension-mediated sequencing (PEM-seq) to study the level of genome instability under replication stresses.

黏连蛋白（cohesin）功能丧失突变在肿瘤中频繁出现，但其具体分子机制尚不明确。本研究发现，敲减黏连蛋白核心亚基RAD21可引发全基因组范围内的大规模DNA断裂，断裂水平最高可达对照组的五倍，同时鉴定出147个易位热点基因，这些基因在多种癌症中与黏连蛋白存在共突变现象。DNA损伤水平的升高并不依赖于RAD21缺失所诱导的转录改变与染色质环锚点消除。然而，损伤诱导的染色体易位与DNA复制过程中不对称分布的冈崎片段（Okazaki fragments）相吻合，提示RAD21耗竭会引发复制应激——这一现象可通过复制速度减慢、停滞复制叉数量增加得到验证。从机制层面来看，RAD21缺失后，人类基因组约30%的区域复制时序提前，这是由于超过900个额外的休眠复制起点提前激活所导致。相应地，大多数易位热点基因均位于复制时序发生改变的区域内。综上，我们认为黏连蛋白功能异常会因过度激活DNA复制起始而引发复制应激，最终导致大范围DNA损伤，这一过程可能会促进肿瘤发生。
实验总体设计：本研究采用引物延伸介导测序（primer-extension-mediated sequencing，PEM-seq）技术，探究复制应激状态下的基因组不稳定性水平。