Structure and function of factors mediating Tetrahymena telomerase action at telomeres

真核生物中线性染色体的末端由称为端粒的DNA-蛋白质复合物组成。端粒蛋白特异性结合端粒DNA的双链和单链富含G的重复序列，以保护这些末端免于重组和降解。细胞DNA复制机制无法完全复制端粒DNA导致端粒逐渐缩短，这部分是由专门的逆转录酶端粒酶拯救的。端粒酶活性与端粒长度维持直接相关，端粒长度维持调节细胞的正常寿命。由于端粒酶在肿瘤细胞中的异常上调，端粒酶已经成为治疗干预的主要靶点，这是肿瘤细胞永生表型的原因。在Juli Feigon的宿主实验室中，最近通过电子显微镜研究解决了纤毛四膜虫端粒酶全酶的整体结构和亚基排列。这些研究的结果表明，与人类相比，四膜虫端粒自我更新的调节机制存在差异。在人类中，端粒酶向其细胞底物的募集是由端粒的亚基（TPP 1）介导的，而在四膜虫中提出的端粒对应物（Tpt 1）似乎仅起端粒保护剂的作用，而TPP 1的募集作用被分配给端粒酶全酶（p50）的组成部分。在这种亚基的排列中，Tpt 1与单链端粒结合蛋白Pot 1复合占据并保护端粒重复序列，或者端粒酶结合并催化重复序列的合成。这两个多亚基复合物的交替结合是负责端粒长度regulation.The总体目标的项目在这里提出的是调查的结构和功能的主要是uncharacterized端粒酶抑制剂复合物Tpt 1-Pot 1a的端粒酶全酶（p50-TEB）内的“激活”亚基相比。为了实现这一目标，第一个结构和动力学的Tpt 1在隔离和复杂的Pot 1a将使用NMR光谱和X射线晶体学作为补充方法进行研究。结构数据可以直接与宿主实验室中正在进行的端粒酶亚基工作进行比较，这些工作旨在改善这些竞争对手的结构。此外，生物化学研究将解决ssDNA结合特性的Tpt 1-Pot 1a与端粒酶活性测定在直接竞争底物DNA结合。从这里提出的项目中获得的见解应该有助于提高对Tpt 1-Pot 1a和端粒酶的结构和端粒DNA结合特性差异的理解，最终协调端粒长度的维持。

The ends of linear chromosomes in eukaryotic organisms consist of DNA-protein complexes termed telomeres. Telomere proteins bind specifically to double- and single-stranded G-rich repeats of telomeric DNA to protect these ends from recombination and degradation. The inability of the cellular DNA replication machinery to fully replicate telomeric DNA leads to gradual telomere shortening, which is rescued, in part, by the specialized reverse transcriptase telomerase. Telomerase activity is directly coupled to telomere length maintenance, which regulates the normal life span of cells. Telomerase has emerged as a major target for therapeutic intervention due to the aberrant upregulation of telomerase in tumor cells, which is responsible for their immortal phenotype. The overall architecture and subunit arrangement of the telomerase holoenzyme from the ciliate Tetrahymena was recently solved by electron microscopy studies in the host laboratory of Juli Feigon. The results of these studies propose a difference in the regulatory mechanism underlying telomere self-renewal in Tetrahymena compared to humans. While in humans, recruitment of telomerase to its cellular substrate is mediated by a subunit of the telomere (TPP1), the proposed telomere counterpart in Tetrahymena (Tpt1) appears to function only as a protector of telomeres, while the recruitment role of TPP1 is assigned to an integral part of the telomerase holoenzyme (p50). In this arrangement of subunits, either Tpt1 in complex with the single-stranded telomere binding protein Pot1 occupies and protects telomeric repeats, or telomerase binds and catalyzes repeat synthesis. Alternate binding of these two multisubunit complexes is responsible for telomere length regulation.The overall objective of the project presented here is to investigate structure and function of the largely uncharacterized telomerase inhibitor complex Tpt1-Pot1a in comparison to the ‘activating’ subunits within the telomerase holoenzyme (p50-TEB). To achieve this goal, first structure and dynamics of Tpt1 in isolation and in complex with Pot1a will be investigated using NMR-spectroscopy and X-ray crystallography as complementary methods. Structural data can be directly compared to ongoing work on the telomerase subunits in the host laboratory, which is directed towards improving the structure of these competing counterparts. In addition, biochemical studies will address ssDNA binding properties of Tpt1-Pot1a together with telomerase activity assays in direct competition for substrate DNA binding. The insights derived from the project proposed here should contribute significantly to improve understanding regarding differences in structure and telomeric DNA binding properties of Tpt1-Pot1a and telomerase that ultimately coordinate telomere length maintenance.