STN1 OB Fold Mutation Alters DNA Binding and Affects Selective Aspects of CST Function.
STN1 OB Fold Mutation Alters DNA Binding and Affects Selective Aspects of CST Function.
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DOI：
10.1371/journal.pgen.1006342
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发表时间：
2016-09
期刊：
影响因子：
4.5
通讯作者：
Price CM
中科院分区：
文献类型：
--
作者：
Bhattacharjee A;Stewart J;Chaiken M;Price CM
哺乳动物CST （CTC1-STN1-TEN1）参与端粒复制和全基因组从复制应激中恢复的多个方面。CST与复制蛋白A （RPA）相似，它结合ssDNA， STN1和TEN1在结构上与RPA2和RPA3相似。CTC1和RPA1之间的保守性不太明显。目前，CST作用的机制在很大程度上是未知的。在这里，我们通过使用DNA结合突变体（STN1 OB-fold突变体，STN1- obm）来研究CST机制，以研究DNA结合与CST功能之间的关系。在体内，STN1-OBM影响内源复制胁迫和端粒双工复制的分解，但外源复制胁迫后端粒c链填充和新起点激发不受影响。这些选择性效应表明，在解决不同的复制问题时，CST的作用机制存在差异。体外结合研究表明，STN1直接结合短和长ssDNA寡核苷酸，但STN1- obm优先破坏与短底物的结合。STN1-OBM只影响与某些底物的结合，这一发现开始解释了在表达STN1-OBM的细胞中观察到的体内功能分离。CST预计将涉及不同长度和结构的DNA底物，因为它可以解决不同的复制问题。正如在STN1-OBM细胞中观察到的那样，由于STN1-OBM只会改变CST与其中一些底物的结合，突变体应该只会影响复制问题的一小部分。体外研究也为CST的结合机制提供了新的思路。与RPA一样，CST可能通过多个OB折叠与DNA接触。然而，STN1结合短底物的重要性表明CST和RPA dna -蛋白复合物的结构存在差异。基于我们的研究结果，我们提出了一个动态DNA结合模型，该模型提供了CST在不同形式复制胁迫下作用的一般机制。哺乳动物CST （CTC1/STN1/TEN1）是一个三蛋白复合物，在端粒复制的几个步骤中起辅助作用，并在复制叉停滞的恢复过程中发挥全基因组作用。CST缺失导致端粒结构异常、基因组不稳定和染色体分离缺陷。目前，我们不了解CST如何确保解决非常不同类型的复制问题。我们着手通过研究CST的突变形式来解决这个问题，该突变形式被预测会改变DNA结合。突变发生在STN1亚基上。在体内，STN1突变体（STN1- obm）影响CST功能的某些方面，而其他方面是正常的。STN1-OBM的作用并不与CST的端粒和非端粒作用一致，而是在端粒和全基因组中分离出CST功能的不同方面。体外结合研究表明，STN1-OBM仅破坏与短DNA底物的结合。由于CST在体内可能会遇到不同长度和结构的DNA底物，因为它有助于解决不同的复制问题，这一发现开始解释为什么STN1-OBM只影响CST功能的某些方面。我们的体外结合研究也揭示了CST实际上是如何与DNA结合的，并提出了一种新的“动态结合模型”，为CST如何帮助解决各种复制问题以保持基因组稳定性提供了机制解释。
Mammalian CST (CTC1-STN1-TEN1) participates in multiple aspects of telomere replication and genome-wide recovery from replication stress. CST resembles Replication Protein A (RPA) in that it binds ssDNA and STN1 and TEN1 are structurally similar to RPA2 and RPA3. Conservation between CTC1 and RPA1 is less apparent. Currently the mechanism underlying CST action is largely unknown. Here we address CST mechanism by using a DNA-binding mutant, (STN1 OB-fold mutant, STN1-OBM) to examine the relationship between DNA binding and CST function. In vivo, STN1-OBM affects resolution of endogenous replication stress and telomere duplex replication but telomeric C-strand fill-in and new origin firing after exogenous replication stress are unaffected. These selective effects indicate mechanistic differences in CST action during resolution of different replication problems. In vitro binding studies show that STN1 directly engages both short and long ssDNA oligonucleotides, however STN1-OBM preferentially destabilizes binding to short substrates. The finding that STN1-OBM affects binding to only certain substrates starts to explain the in vivo separation of function observed in STN1-OBM expressing cells. CST is expected to engage DNA substrates of varied length and structure as it acts to resolve different replication problems. Since STN1-OBM will alter CST binding to only some of these substrates, the mutant should affect resolution of only a subset of replication problems, as was observed in the STN1-OBM cells. The in vitro studies also provide insight into CST binding mechanism. Like RPA, CST likely contacts DNA via multiple OB folds. However, the importance of STN1 for binding short substrates indicates differences in the architecture of CST and RPA DNA-protein complexes. Based on our results, we propose a dynamic DNA binding model that provides a general mechanism for CST action at diverse forms of replication stress. Mammalian CST (CTC1/STN1/TEN1) is a three protein complex that aids in several steps during telomere replication and has genome-wide roles during recovery from replication fork stalling. Loss of CST leads to abnormalities in telomere structure, genomic instability and defects in chromosome segregation. Currently, we do not understand how CST acts to ensure the resolution of very diverse types of replication problem. We set out to address this question by studying a mutant form of CST that was predicted to alter DNA binding. The mutations are in the STN1 subunit. In vivo, the STN1 mutant (STN1-OBM) affects some aspects of CST function while others are normal. The effects of STN1-OBM do not align with the telomeric versus non-telomeric roles of CST but instead separate out different aspects of CST function at telomeres and genome-wide. In vitro binding studies indicate that STN1-OBM disrupts binding to only short DNA substrates. Since CST is likely to encounter DNA substrates of varied length and structure in vivo as it helps resolve different replication problems, this finding starts to explain why STN1-OBM affects only certain aspects of CST function. Our in vitro binding studies also shed light on how CST actually binds to DNA and they suggest a novel “dynamic binding model” that provides a mechanistic explanation for how CST helps resolve a diverse array of replication problems to preserve genome stability.
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影响因子：
11.4
作者：
Bochkarev, A;Bochkareva, E;Edwards, AM
通讯作者：
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影响因子：
64.8
作者：
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DOI：
10.1073/pnas.0909203106
发表时间：
2009-11-17
影响因子：
11.1
作者：
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通讯作者：
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44.1
作者：
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通讯作者：
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影响因子：
21.3
作者：
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通讯作者：
Hickson, Ian D.