Functional Interactions of Telomere Protein with Human Telomerase

端粒蛋白与人端粒酶的功能相互作用

• 批准号: 8788935
• 负责人: THOMAS ROBERT CECH
• 金额: $ 13.16万
• 依托单位: UNIVERSITY OF COLORADO
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-05-01 至 2016-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8788935
• 关键词: Address; Affect; Alleles; Amino Acid Substitution; Amino Acids; Aplastic Anemia; Binding; Biochemical; Biological Assay; Cell Line; Cells; Chromatin; Chromosomes; Complex; DNA; DNA Binding; DNA-Binding Proteins; Defect; Disease; Dyskeratosis Congenita; Elements; Ensure; Enzymes; Event; Failure; Genes; Genetic; Genome Stability; Goals; Hamman-Rich syndrome; Hela Cells; Human; Human Chromosomes; Immunofluorescence Immunologic; In Vitro; Length; Life; Malignant Neoplasms; Measurement; Measures; Mediating; Molecular; Molecular Machines; Mus; Mutation; Nucleotides; Pathology; Population; Premature aging syndrome; Process; Proteins; RNA; RNA Sequences; RNA-Directed DNA Polymerase; Reagent; Recruitment Activity; Reporting; Research; Single Nucleotide Polymorphism; Single-Stranded DNA; Site-Directed Mutagenesis; Structural Protein; Structure; Surface; TERT gene; Tail; Telomerase; Telomerase RNA Component; Telomere Maintenance; Telomere Shortening; Testing; Time; Tissues; Work; base; human disease; in vitro Assay; in vitro activity; in vivo; knock-down; mutant; protein complex; research study; small hairpin RNA; telomerase reverse transcriptase; telomere; tool; transcriptome sequencing

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.

描述（由申请人提供）： 端粒是染色体末端的DNA-蛋白质复合物，具有明显相反的功能：端粒提供保护性染色体帽，确保基因组稳定性，但端粒DNA也必须与染色体末端复制酶端粒酶相互作用。最近的工作表明，与POT 1形成异二聚体并结合染色体末端单链DNA延伸的人端粒蛋白TPP 1不仅有助于染色体加帽，而且有助于募集端粒酶并刺激其持续合成能力-在引物结合后添加多个端粒DNA重复的能力。拟议研究的广泛目标是了解POT 1-TPP 1激活端粒酶的分子相互作用，以及它们在体内对端粒维持的贡献。最近发现的TPP 1的功能分离突变体使这些实验成为可能，所述突变体保留形成POT 1-TPP 1-DNA复合物的全部能力，但在刺激端粒酶持续合成能力方面有缺陷。特异性目的1检验TPP 1的特异性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募集。这些方法包括用与POT 1-TPP 1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。特异性目标2针对另一个伴侣的相互作用元件，端粒酶本身。利用小鼠和人端粒酶RNA之间的序列差异，将使用位点特异性诱变和活性测定来鉴定有助于POT 1-TPP 1相互作用的RNA元件。此外，TERT表面上的G100对于与POT 1-TPP 1的功能性相互作用是必需的，并且将鉴定另外的氨基酸决定簇。具体目标3解决了一个关键问题：在体外酶测定中观察到的POT 1-TPP 1对端粒酶活性的增强在多大程度上有助于体内端粒的维持？将功能分离TPP 1突变体以单拷贝整合到特定HeLa细胞系中的表达基因座中，使用短发夹RNA敲低内源性TPP 1，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），其已被报道与包括先天性角化不良、再生障碍性贫血、特发性肺纤维化和癌症在内的疾病相关。突变的TERTs将在人类细胞中与端粒酶RNA组装，免疫纯化，并测试POT 1-TPP 1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结合蛋白有助于端粒酶募集和端粒重复序列合成的机制，并评估干扰这些过程的突变如何导致人类疾病。