The Intrinsically Disordered Region in the Human STN1 OB-Fold Domain Is Important for Protecting Genome Stability.
The Intrinsically Disordered Region in the Human STN1 OB-Fold Domain Is Important for Protecting Genome Stability.
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DOI：
10.3390/biology10100977
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发表时间：
2021-09-28
期刊：
影响因子：
4.2
通讯作者：
Wang Y
中科院分区：
文献类型：
--
作者：
Chai W;Chastain M;Shiva O;Wang Y
人CTC1-STN1-TEN1 （CST）复合物是一种ssdna结合蛋白复合物，被认为与RPA70/RPA32/RPA14复合物有关。虽然最近的研究表明，CST在多种基因组维持途径中发挥关键作用，包括保护受干扰复制下的叉稳定性，促进难以复制的DNA的有效复制，修复DNA双链断裂，以及维持端粒完整性，但对CST功能在基因组维持中的调节机制知之甚少。在这项研究中，我们在STN1的OB结构域中发现了一个内在无序区（IDR），并分析了癌症相关IDR变异的功能以及该IDR中个别极性或亲水性残基的一些丙氨酸取代。我们观察到这些变异赋予复制相关的基因组不稳定性、细胞活力降低和HU敏感性增加。利用IDR变异和IDR缺失对蛋白质相互作用的分析表明，IDR对STN1-POLα相互作用至关重要，而不是CST - rad51相互作用或CST复合物的形成。总之，我们的研究结果确定了STN1-OB中的IDR是调节CST功能在复制胁迫下保护基因组稳定性的重要因素。哺乳动物CTC1-STN1-TEN1 （CST）复合物是一种ssdna结合蛋白复合物，在保护基因组稳定性和保持端粒完整性方面发挥着重要作用。研究表明，CST定位于停滞的复制叉，对保护新生链DNA的稳定性至关重要。最近的低温电镜分析表明，CST亚基具有多个OB-fold结构域，可以形成十元超配合物。虽然被认为是RPA样的，但CST的作用与RPA不同，以保护基因组的稳定性。在这里，我们报道了虽然STN1的OB结构域与RPA32的OB结构域具有结构相似性，但STN1-OB结构域包含一个内在无序区（IDR），该区域对于在复制胁迫下维持基因组稳定性至关重要。该IDR中多个位置的单个突变，包括癌症相关突变，会导致基因组不稳定，这种不稳定会因复制应激而升高，并表现出细胞活力降低和HU敏感性增加。虽然IDR突变不会影响CST复合物的形成或CST与其结合伙伴RAD51的相互作用，但当复制受到干扰时，它们会减少RAD51病灶的形成。有趣的是，IDR对STN1-POLα相互作用至关重要。总之，我们的研究结果确定了STN1 IDR是调节CST功能在基因组稳定性维持中的重要因素。
The human CTC1–STN1–TEN1 (CST) complex is an ssDNA-binding protein complex that is thought to be related to the RPA70/RPA32/RPA14 complex. While recent studies have shown that CST plays key roles in multiple genome maintenance pathways, including protecting fork stability under perturbed replication, promoting efficient replication of difficult-to-replicate DNA, repairing DNA double-stranded breaks, and maintaining telomere integrity, it is poorly understood how CST function is regulated in genome maintenance. In this study, we identify an intrinsically disordered region (IDR) in the OB domain of STN1 and analyze the functions of cancer-associated IDR variants and a number of alanine substitutions of individual polar or hydrophilic residues in this IDR. We observe that these variants confer replication-associated genome instability, reduced cellular viability, and increased HU sensitivity. Analysis of protein–protein interactions using IDR variants and IDR deletion shows that the IDR is critical for STN1–POLα interaction, but not CST–RAD51 interaction or CST complex formation. Together, our results identify the IDR in STN1-OB as an important element modulating CST function in protecting genome stability under replication stress. The mammalian CTC1–STN1–TEN1 (CST) complex is an ssDNA-binding protein complex that has emerged as an important player in protecting genome stability and preserving telomere integrity. Studies have shown that CST localizes at stalled replication forks and is critical for protecting the stability of nascent strand DNA. Recent cryo-EM analysis reveals that CST subunits possess multiple OB-fold domains that can form a decameric supercomplex. While considered to be RPA-like, CST acts distinctly from RPA to protect genome stability. Here, we report that while the OB domain of STN1 shares structural similarity with the OB domain of RPA32, the STN1-OB domain contains an intrinsically disordered region (IDR) that is important for maintaining genome stability under replication stress. Single mutations in multiple positions in this IDR, including cancer-associated mutations, cause genome instabilities that are elevated by replication stress and display reduced cellular viability and increased HU sensitivity. While IDR mutations do not impact CST complex formation or CST interaction with its binding partner RAD51, they diminish RAD51 foci formation when replication is perturbed. Interestingly, the IDR is critical for STN1–POLα interaction. Collectively, our results identify the STN1 IDR as an important element in regulating CST function in genome stability maintenance.
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