Functional Interactions of Telomere Protein with Human Telomerase

描述（由申请人提供）： 小结 端粒是染色体末端的 DNA-蛋白质复合物，其功能显然相反：端粒提供保护性染色体帽，确保基因组稳定性，但端粒 DNA 也必须易于与染色体末端复制酶端粒酶相互作用。最近的研究表明，人类端粒蛋白 TPP1 与 POT1 形成异二聚体，并结合染色体末端的单链 DNA 延伸，不仅有助于染色体加帽，还有助于招募端粒酶并刺激其持续合成能力，即在引物结合后添加多个端粒 DNA 重复序列的能力。本研究的主要目标是了解 POT1-TPP1 负责端粒酶激活的分子相互作用，以及它们对体内端粒维持的贡献程度。最近发现的 TPP1 功能分离突变体保留了形成 POT1-TPP1-DNA 复合物的全部能力，但在刺激端粒酶持续合成能力方面存在缺陷，使这些实验成为可能。具体目标 1 测试了 TPP1 的特定表面介导端粒酶持续合成能力的假设，并确定同一表面是否参与体外端粒酶招募。这些方法包括使用与 POT1-TPP1 和功能分离突变体结合的引物直接测定端粒酶活性，以及​​评估结合的下拉测定。具体目标 2 涉及另一个伙伴（端粒酶本身）的相互作用元素。利用小鼠和人类端粒酶 RNA 之间的序列差异，定点诱变和活性测定将用于鉴定有助于 POT1-TPP1 相互作用的 RNA 元件。此外，TERT 表面的 G100 对于与 POT1-TPP1 的功能相互作用是必需的，并且将鉴定其他氨基酸决定簇。具体目标 3 解决了一个关键问题：体外酶测定中观察到的 POT1-TPP1 增强端粒酶活性对体内端粒维持有多大贡献？功能分离的 TPP1 突变体将以单拷贝整合到特殊 HeLa 细胞系的表达位点中，使用短发夹 RNA 敲低内源 TPP1，并评估端粒长度、端粒酶募集和染色体加帽。具体目标 4 针对 hTERT 基因的非同义 SNP（单核苷酸多态性），据报道，这些非同义 SNP 与先天性角化不良、再生障碍性贫血、特发性肺纤维化和癌症等疾病相关。突变的TERT将在人体细胞中与端粒酶RNA组装、免疫纯化，并测试POT1-TPP1刺激端粒酶活性的缺陷，这有可能揭示新的疾病机制。这项工作的长期目标是了解人类端粒 DNA 结合蛋白促进端粒酶募集和端粒重复合成的机制，并评估扰乱这些过程的突变如何导致人类疾病。 公共卫生相关性： 叙述端粒是 DNA 和蛋白质的复合物，它封闭人类染色体的末端，确保其稳定性。它们还调节端粒酶，这是一种分子机器，是端粒 DNA 完全复制所必需的，并且与癌症和过早衰老疾病有关。该项目将揭示端粒结合结构蛋白调节人类端粒酶的新机制。

DESCRIPTION (provided by applicant): Summary Telomeres, the DNA-protein complexes at the ends of chromosomes, serve apparently opposing functions: telomeres provide protective chromosome caps that ensure genome stability, yet telomeric DNA must also be accessible to interact with the chromosome end-replicating enzyme, telomerase. Recent work indicates that the human telomeric protein TPP1, which forms a heterodimer with POT1 and binds the single- stranded DNA extensions at the very ends of chromosomes, not only contributes to chromosome capping but also helps recruit telomerase and stimulates its processivity - the ability to add multiple telomeric DNA repeats after primer binding. The broad goal of the proposed research is to understand the molecular interactions responsible for telomerase activation by POT1-TPP1 and how much they contribute to telomere maintenance in vivo. Recently discovered separation-of-function mutants of TPP1, which retain full ability to form the POT1-TPP1-DNA complex but are defective in stimulating telomerase processivity, enable these experiments. Specific Aim 1 tests the hypothesis that a specific surface of TPP1 mediates telomerase processivity and determines whether the same surface is involved in telomerase recruitment in vitro. The approaches include direct assays of telomerase activity with the primer bound to POT1-TPP1 and separation-of-function mutants, and pull-down assays to assess binding. Specific Aim 2 addresses the interacting elements of the other partner, the telomerase itself. Exploiting sequence differences between the mouse and human telomerase RNAs, site-specific mutagenesis and activity assays will be used to identify RNA elements that contribute to POT1-TPP1 interaction. In addition, G100 on the surface of TERT is necessary for functional interaction with POT1-TPP1, and additional amino acid determinants will be identified. Specific Aim 3 addresses a key question: how much does the enhancement of telomerase activity by POT1-TPP1 seen in enzyme assays in vitro contribute to telomere maintenance in vivo? The separation- of-function TPP1 mutants will be integrated at single copy into an expression locus in a special HeLa cell line, the endogenous TPP1 will be knocked down using short hairpin RNAs, and telomere length, telomerase recruitment, and chromosome capping will be assessed. Specific Aim 4 addresses nonsynonymous SNPs (single nucleotide polymorphisms) of the hTERT gene that have been reported to be associated with diseases including dyskeratosis congenita, aplastic anemia, idiopathic pulmonary fibrosis, and cancer. The mutant TERTs will be assembled with telomerase RNA in human cells, immunopurified, and tested for defects in POT1-TPP1 stimulation of telomerase activity, which has the potential to reveal new disease mechanisms. The long-term goals of this work are to understand the mechanisms by which human telomeric DNA-binding proteins contribute to telomerase recruitment and telomeric repeat synthesis and to assess how mutations that perturb these processes contribute to human disease. PUBLIC HEALTH RELEVANCE: Narrative Telomeres are complexes of DNA and protein that cap off the ends of human chromosomes, ensuring their stability. They also regulate telomerase, a molecular machine that is necessary for complete replication of telomeric DNA and is involved in both cancer and diseases of premature aging. This project will reveal new mechanisms by which the telomere-bound structural proteins regulate human telomerase.