PDF(Pubmed)
Abstract：
It is widely accepted that DNA replication fork stalling is a common occurrence during cell proliferation, but there are robust mechanisms to alleviate this and ensure DNA replication is completed prior to chromosome segregation. The SMC5/6 complex has consistently been implicated in the maintenance of replication fork integrity. However, the essential role of the SMC5/6 complex during DNA replication in mammalian cells has not been elucidated. In this study, we investigate the molecular consequences of SMC5/6 loss at the replication fork in mouse embryonic stem cells (mESCs), employing the auxin-inducible degron (AID) system to deplete SMC5 acutely and reversibly in the defined cellular contexts of replication fork stall and restart. In SMC5-depleted cells, we identify a defect in the restart of stalled replication forks, underpinned by excess MRE11-mediated fork resection and a perturbed localization of fork protection factors to the stalled fork. Previously, we demonstrated a physical and functional interaction of SMC5/6 with the COP9 signalosome (CSN), a cullin deneddylase that enzymatically regulates cullin ring ligase (CRL) activity. Employing a combination of DNA fiber techniques, the AID system, small-molecule inhibition assays, and immunofluorescence microscopy analyses, we show that SMC5/6 promotes the localization of fork protection factors to stalled replication forks by negatively modulating the COP9 signalosome (CSN). We propose that the SMC5/6-mediated modulation of the CSN ensures that CRL activity and their roles in DNA replication fork stabilization are maintained to allow for efficient replication fork restart when a replication fork stall is alleviated.
摘要：
人们普遍认为，DNA复制叉失速是细胞增殖过程中常见的现象，但是有强大的机制可以缓解这种情况，并确保DNA复制在染色体分离之前完成。SMC5/6复合物一直与维持复制叉的完整性有关。然而,SMC5/6复合物在哺乳动物细胞中DNA复制过程中的重要作用尚未阐明。在这项研究中,我们研究了SMC5/6在小鼠胚胎干细胞（mESCs）复制叉丢失的分子后果，在定义的复制叉失速和重启的细胞环境中，采用生长素诱导的Degron（AID）系统急剧且可逆地耗尽SMC5。在SMC5耗尽的细胞中，我们在停止的复制叉的重新启动中发现了一个缺陷，由过量的MRE11介导的叉子切除和叉子保护因子对停滞的叉子的扰动定位支撑。以前,我们证明了SMC5/6与COP9信号体(CSN)的物理和功能相互作用，一种酶促调节cullin环连接酶（CRL）活性的cullindeddase。采用DNA纤维技术的组合，AID系统，小分子抑制试验，和免疫荧光显微镜分析，我们发现SMC5/6通过负向调节COP9信号体（CSN）来促进叉保护因子对停滞复制叉的定位。我们建议SMC5/6介导的CSN调节可确保保持CRL活性及其在DNA复制叉稳定中的作用，以在缓解复制叉失速时允许有效的复制叉重启。
