Functional Interactions of Telomere Protein with Human Telomerase

描述(由申请人提供)： 摘要端粒是位于染色体末端的DNA-蛋白质复合体，具有明显相反的功能：端粒提供保护染色体帽以确保基因组稳定，但端粒DNA也必须能够与染色体末端复制酶-端粒酶相互作用。最近的工作表明，人类端粒蛋白TPP1与POT1形成异源二聚体，并结合染色体末端的单链DNA延伸部分，不仅有助于染色体封顶，还有助于招募端粒酶并刺激其处理能力--在引物结合后增加多个端粒DNA重复的能力。这项研究的主要目标是了解POT1-TPP1激活端粒酶的分子相互作用，以及它们在体内对端粒维持有多大贡献。最近发现的TPP1功能分离突变体，保留了完全形成POT1-TPP1-DNA复合体的能力，但在刺激端粒酶活性方面存在缺陷，使这些实验成为可能。特异性目标1验证了TPP1的一个特定表面介导端粒酶活性的假设，并确定同一表面是否参与了体外端粒酶的募集。这些方法包括直接检测与POT1-TPP1和功能分离突变体结合的引物的端粒酶活性，以及评估结合的下拉试验。特定目标2解决了另一个伙伴的相互作用元素，端粒酶本身。利用小鼠和人类端粒酶RNA之间的序列差异，定点突变和活性分析将被用于识别有助于POT1-TPP1相互作用的RNA元件。此外，TERT表面的G100是与POT1-TPP1功能相互作用所必需的，其他氨基酸决定因素将被确定。特定目标3解决了一个关键问题：体外酶分析中发现的POT1-TPP1增强端粒酶活性对体内端粒维持有多大贡献？将功能分离的TPP1突变体以单拷贝的形式整合到一个特殊的HeLa细胞系中的一个表达位点，使用短发夹状RNA击倒内源性TPP1，并对端粒长度、端粒酶募集和染色体封顶进行评估。具体目标4针对hTERT基因的非同义SNPs(单核苷酸多态)，已有报道与先天性角化不良、再生障碍性贫血、特发性肺纤维化和癌症等疾病有关。突变的TERT将在人类细胞中与端粒酶RNA组装，免疫纯化，并检测POT1-TPP1刺激端粒酶活性的缺陷，这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招募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： 叙述性端粒是DNA和蛋白质的复合体，覆盖在人类染色体的末端，确保它们的稳定性。它们还调节端粒酶，端粒酶是完成端粒DNA复制所必需的分子机器，与癌症和过早衰老的疾病有关。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.