Bhattacharjee Anukana, Wang Yongyao, Diao Jiajie, Price Carolyn M
Department of Cancer Biology, University of Cincinnati, Cincinnati, OH 45267, USA.
School of Life Science and Technology, Xi'an Jiaotong University, Xi'an, China.
Nucleic Acids Res. 2017 Dec 1;45(21):12311-12324. doi: 10.1093/nar/gkx878.
Human CST (CTC1-STN1-TEN1) is a ssDNA-binding complex that helps resolve replication problems both at telomeres and genome-wide. CST resembles Replication Protein A (RPA) in that the two complexes harbor comparable arrays of OB-folds and have structurally similar small subunits. However, the overall architecture and functions of CST and RPA are distinct. Currently, the mechanism underlying CST action at diverse replication issues remains unclear. To clarify CST mechanism, we examined the capacity of CST to bind and resolve DNA structures found at sites of CST activity. We show that CST binds preferentially to ss-dsDNA junctions, an activity that can explain the incremental nature of telomeric C-strand synthesis following telomerase action. We also show that CST unfolds G-quadruplex structures, thus providing a mechanism for CST to facilitate replication through telomeres and other GC-rich regions. Finally, smFRET analysis indicates that CST binding to ssDNA is dynamic with CST complexes undergoing concentration-dependent self-displacement. These findings support an RPA-based model where dissociation and re-association of individual OB-folds allow CST to mediate loading and unloading of partner proteins to facilitate various aspects of telomere replication and genome-wide resolution of replication stress.
人类CST（CTC1-STN1-TEN1）是一种单链DNA结合复合物，有助于解决端粒和全基因组范围内的复制问题。CST与复制蛋白A（RPA）相似，因为这两种复合物都含有类似的OB折叠阵列，并且具有结构相似的小亚基。然而，CST和RPA的整体结构和功能是不同的。目前，CST在各种复制问题上发挥作用的机制仍不清楚。为了阐明CST的机制，我们研究了CST结合和解决在CST活性位点发现的DNA结构的能力。我们发现CST优先结合单链-双链DNA连接点，这一活性可以解释端粒酶作用后端粒C链合成的渐进性质。我们还发现CST能解开G-四链体结构，从而为CST促进通过端粒和其他富含GC区域的复制提供了一种机制。最后，单分子荧光共振能量转移分析表明，CST与单链DNA的结合是动态的，CST复合物会发生浓度依赖性的自我置换。这些发现支持了一种基于RPA的模型，即单个OB折叠的解离和重新结合使CST能够介导伴侣蛋白的加载和卸载，以促进端粒复制的各个方面以及全基因组范围内复制应激的解决。
