PDS5 proteins are required for proper cohesin dynamics and participate in replication fork protection
PDS5 proteins are required for proper cohesin dynamics and participate in replication fork protection
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DOI：
10.1074/jbc.ra119.011099
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发表时间：
2020-01-03
影响因子：
4.8
通讯作者：
Losada, Ana
中科院分区：
文献类型：
--
作者：
Morales, Carmen;Ruiz-Torres, Miguel;Losada, Ana
粘附素是一种染色质结合的复合体，通过DNA环调节姐妹染色单体的凝聚力并促进远距离的相互作用。转录和复制机制如何处理染色质上粘附素的存在仍不清楚。粘附素与染色质的动态结合依赖于WAPL粘附素释放因子(WAPL)和Pds5粘附素相关因子(Pds5)，Pds5存在于脊椎动物细胞中的两个版本：Pds5A和Pds5B。利用小鼠胚胎成纤维细胞中的基因缺失，以及CRISPR介导的基因编辑和RNAi介导的人类细胞基因沉默的组合，我们分析了Pds5缺失对DNA复制的影响。我们发现，Pds5A或Pds5B足以实现适当的粘附素动力学，并且它们的同时去除增加了粘附素在染色质上的停留时间，并减缓了DNA复制。在WAPL耗竭的细胞中也观察到了类似的表型。在Pds5缺失的细胞中，粘附素下调恢复了正常的复制分叉率，这表明染色质结合的粘附素阻碍了复制体的推进。我们进一步证明，Pds5蛋白需要将WRN解旋酶相互作用蛋白1(WRNIP1)、RAD51重组酶(RAD51)和BRCA2 DNA修复相关(BRCA2)招募到停滞的叉子上，在没有它们的情况下，未受保护的叉子上的新生DNA链会被Mre11同源双链断裂修复核酸酶(Mre11)降解。这些发现表明，Pds5蛋白参与复制分叉保护，并提供了对粘附素及其调节因子如何对复制应激做出反应的洞察，复制应激是癌细胞的共同特征。
Cohesin is a chromatin-bound complex that mediates sister chromatid cohesion and facilitates long-range interactions through DNA looping. How the transcription and replication machineries deal with the presence of cohesin on chromatin remains unclear. The dynamic association of cohesin with chromatin depends on WAPL cohesin release factor (WAPL) and on PDS5 cohesin-associated factor (PDS5), which exists in two versions in vertebrate cells, PDS5A and PDS5B. Using genetic deletion in mouse embryo fibroblasts and a combination of CRISPR-mediated gene editing and RNAi-mediated gene silencing in human cells, here we analyzed the consequences of PDS5 depletion for DNA replication. We found that either PDS5A or PDS5B is sufficient for proper cohesin dynamics and that their simultaneous removal increases cohesin's residence time on chromatin and slows down DNA replication. A similar phenotype was observed in WAPL-depleted cells. Cohesin down-regulation restored normal replication fork rates in PDS5-deficient cells, suggesting that chromatin-bound cohesin hinders the advance of the replisome. We further show that PDS5 proteins are required to recruit WRN helicase-interacting protein 1 (WRNIP1), RAD51 recombinase (RAD51), and BRCA2 DNA repair associated (BRCA2) to stalled forks and that in their absence, nascent DNA strands at unprotected forks are degraded by MRE11 homolog double-strand break repair nuclease (MRE11). These findings indicate that PDS5 proteins participate in replication fork protection and also provide insights into how cohesin and its regulators contribute to the response to replication stress, a common feature of cancer cells.