CST蛋白复合体在端粒复制中对端粒酶移除与C链填补调控的分子机制研究

端粒作为真核生物染色体末端的保护结构，其长度缩短与细胞衰老息息相关，因此端粒维持机制的研究是理解细胞衰老机理的重要方向。端粒复制是一个多步骤串联的过程，包括双链区域半保留复制、核酸酶外切、G链延伸、端粒酶移除以及C链填补。人类细胞中，单链DNA结合蛋白复合体CST（CTC1-STN1-TEN1）参与调控了端粒复制过程中包括端粒酶移除和C链填补的多个步骤，但其中机制尚不清楚。由于CST复合体所有功能的同时缺失会导致非常复杂的细胞表型，为机制研究带来了很大困难。本项目中，我们将利用构建CST不同组分或功能结构域的缺失突变体，对其复杂功能进行分解，分别建立单独研究端粒酶移除或C链填补机制的细胞模型。利用上述两个细胞模型，我们还将对CST复合体在不同功能中的蛋白相互作用网络进行对比分析，找到各功能相关的蛋白通路，从而揭示CST蛋白复合体在两个功能中作用的分子机制。

Telomeres are specialized DNA/protein complexes at the ends of eukaryotic chromosomes that function to protect genome integrity. In humans, telomere shortening due to incomplete DNA replication is an important biomarker of aging. Telomere replication involves multiple steps: replication of the telomeric duplex DNA, nuclease resection, G-strand extension by telomerase, telomerase removal, and finally synthesis of the complimentary C-strand (C-strand fill-in). In human cells, the ssDNA binding complex CST (CTC1-STN1-TEN1) is required for multiple aspects of telomere replication including telomerase removal and engagement of DNA polymerase for C-strand fill-in. However, the mechanism of CST action in these processes is still unclear. Complete disruption of the CST complex leads to complex phenotypes because multiple aspects of telomere replication are affected. Thus to better investigate the molecular mechanism of CST function in specific steps of telomere replication, we plan to utilize cell lines expressing mutant CST that retains partial activity. We have already identified a CTC1 OB fold deletion mutation that affects telomerase removal but not C-strand fill-in. We have also generated TEN1 knockout cells that disrupt C-strand fill-in but not telomerase removal. Together these two cell lines will enable us to dissect the mechanism by which CST terminates telomerase action and then engages DNA polymerase for C-strand synthesis.