Structure and function of factors mediating Tetrahymena telomerase action at telomeres

在真核生物中，线性染色体的末端由称为端粒的dna -蛋白质复合物组成。端粒蛋白与端粒DNA的双链和单链富含g的重复序列特异性结合，以保护这些端粒免受重组和降解。细胞DNA复制机制无法完全复制端粒DNA，导致端粒逐渐缩短，这在一定程度上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，从而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，端粒酶已成为治疗干预的主要靶点，这是肿瘤细胞不朽表型的原因。最近在julie Feigon的宿主实验室通过电子显微镜研究解决了纤毛虫四膜虫端粒酶全酶的总体结构和亚基排列。这些研究的结果表明，四膜动物端粒自我更新的调控机制与人类不同。而在人类中，端粒酶向其细胞底物的募集是由端粒亚基（TPP1）介导的，四膜虫中提出的端粒对应物（Tpt1）似乎仅作为端粒的保护者起作用，而TPP1的募集作用被分配给端粒酶全酶的一个组成部分（p50）。在这种亚基排列中，要么Tpt1与单链端粒结合蛋白Pot1复合物占据并保护端粒重复序列，要么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负责端粒长度调节。这里提出的项目的总体目标是研究端粒酶抑制剂复合物Tpt1-Pot1a的结构和功能，并与端粒酶全酶（p50-TEB）中的“激活”亚基进行比较。为了实现这一目标，首先将使用核磁共振光谱和x射线晶体学作为补充方法研究分离Tpt1和与Pot1a配合的Tpt1的结构和动力学。结构数据可以直接与宿主实验室正在进行的端粒酶亚基研究进行比较，后者旨在改善这些竞争对手的结构。此外，生化研究将研究Tpt1-Pot1a的ssDNA结合特性，以及端粒酶活性测定，以直接竞争底物DNA结合。本项目提出的见解将有助于提高对Tpt1-Pot1a和端粒酶的结构差异和端粒DNA结合特性的理解，这些特性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.