In Vitro Reconstitution and Biochemical Characterization of Yeast Telomerase

酵母端粒酶的体外重建和生化表征

• 批准号: 8122869
• 负责人: Karen Adell Lewis
• 金额: $ 5.13万
• 依托单位: UNIVERSITY OF COLORADO
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-06-01 至 2013-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8122869
• 关键词: Affect; Aging; Aplastic Anemia; Baculoviruses; Binding; Biochemical; Biological Assay; C-terminal; Calorimetry; Cell Aging; Cells; Chromosomes; Chronic; Competitive Binding; Complex; Crystallography; Cyclin B; DNA; DNA Binding; DNA Damage; DNA Sequence; DNA biosynthesis; Data; Dependence; Disease; Dissection; Dyskeratosis Congenita; Elements; Enzymes; Evaluation; Event; Exhibits; Genetic; Genome; Genome Stability; Hamman-Rich syndrome; Hand; Health; Holoenzymes; Human; In Vitro; Individual; Insecta; Length; Malignant Neoplasms; Mass Spectrum Analysis; Mediating; Modeling; Molecular; Molecular Models; Mutation; Nature; Nucleoproteins; Phosphorylation; Play; Proliferating; Protein Subunits; Proteins; Proteolysis; RNA; RNA Binding; Reagent; Recombinant Proteins; Recombinants; Recruitment Activity; Regulation; Ribonucleoproteins; Role; Saccharomyces cerevisiae; Single-Stranded DNA; Structure; System; Tandem Repeat Sequences; Techniques; Telomerase; Telomere Maintenance; Telomere Shortening; Telomere-Binding Proteins; Testing; Time; Tumor Suppression; Yeast Model System; Yeasts; base; cell growth; ds-DNA; genetic regulatory protein; human disease; in vitro activity; in vivo; insight; light scattering; molecular modeling; mutant; novel; reconstitution; research study; telomerase reverse transcriptase; telomere; time use; yeast genetics

DESCRIPTION (provided by applicant): Eukaryotic chromosomes terminate in structures called telomeres, which contain tandem sequence repeats. With each cell replication cycle, telomeres become progressively shorter due to the inability of the DNA replication machinery to completely synthesize the opposing strand. Shortened telomeres induce replicative senescence, which is avoided in rapidly proliferating cells by the activation of the reverse transcriptase telomerase. Telomerase is a ribonucleoprotein that adds tandem repeats to the ends of telomeres to maintain length, and is both positively and negatively regulated in vivo. Dysregulation of telomerase leads to several human diseases, including cancer, aplastic anemia, dyskeratosis congenita, and idiopathic pulmonary fibrosis. Several models of the molecular mechanisms of telomerase regulation have been developed based in vivo experiments. The proposed studies will use standard biochemical and biophysical techniques to evaluate the structures of the telomere-associated proteins Cdc13 and Est1, which will be used to develop a novel in vitro system of reconstituted yeast telomerase. This system will be used to rigorously study the mechanisms of telomerase activity and regulation that have been genetically identified in vivo. Yeast model systems have previously provided considerable insight into human telomere maintenance, and a wealth of yeast genetic data is available to rigorously evaluate the function of individual proteins in the system. In Aim 1, we will determine the structure of the telomerase-recruitment domain of the telomere-binding protein Cdc13, and assay the role of this domain in DNA binding by full-length Cdc13. Aim 2 will characterize the biochemical activities of the telomerase regulatory subunit Est1, which will be recombinantly produced in insect cells to generate large yields of a high-quality reagent that is critically needed in the field. Aim 3 will test the central element of the recruitment model of telomerase activation by determining if a direct physical interaction between Cdc13 and Est1 exists, and then evaluating interactions between genetically identified mutants of both proteins. Aim 4 will reconstitute the entire telomerase complex in vitro for the first time, using recombinant reagents that have been extensively characterized. Activity assays will be performed using this novel system to fully test the Cdc13/Est1 recruitment model of telomerase activation. These in vitro studies of the molecular mechanisms of yeast telomerase activity will increase our general understanding of telomerase activity and regulation, and will specifically provide a framework for the dissection of the molecular mechanisms of human telomere maintenance that are disrupted in disease. PUBLIC HEALTH RELEVANCE: The ends of linear chromosomes comprise specialized structures, called telomeres, that have essential roles in aging, genomic stability, and cancer. The length of the telomere is a marker of cellular aging, and sufficiently shortened telomeres arrest cell growth. Nearly all human cancers evade that checkpoint by activating the enzyme telomerase, which restores telomere length through the addition of DNA sequence. However, insufficient telomerase activity can also cause chronic fatal diseases such as aplastic anemia. The proper maintenance of telomeres and regulation of telomerase are therefore essential for human health.

描述（由申请人提供）：真核染色体终止于称为端粒的结构，该结构包含串联序列重复。在每个细胞复制周期中，由于 DNA 复制机器无法完全合成相反的链，端粒逐渐变短。缩短的端粒会诱导复制性衰老，而快速增殖的细胞可以通过激活逆转录酶端粒酶来避免这种情况。端粒酶是一种核糖核蛋白，可在端粒末端添加串联重复序列以维持长度，并且在体内受到正向和负向调节。端粒酶失调会导致多种人类疾病，包括癌症、再生障碍性贫血、先天性角化不良和特发性肺纤维化。已经基于体内实验开发了几种端粒酶调节分子机制的模型。 拟议的研究将使用标准的生化和生物物理技术来评估端粒相关蛋白 Cdc13 和 Est1 的结构，这将用于开发重组酵母端粒酶的新型体外系统。该系统将用于严格研究体内基因鉴定的端粒酶活性和调节机制。酵母模型系统之前已经为人类端粒维持提供了相当多的见解，并且大量的酵母遗传数据可用于严格评估系统中单个蛋白质的功能。 在目标 1 中，我们将确定端粒结合蛋白 Cdc13 的端粒酶招募结构域的结构，并通过全长 Cdc13 分析该结构域在 DNA 结合中的作用。目标 2 将表征端粒酶调节亚基 Est1 的生化活性，该亚基将在昆虫细胞中重组生产，以产生该领域急需的大量高质量试剂。目标 3 将通过确定 Cdc13 和 Est1 之间是否存在直接物理相互作用，然后评估两种蛋白质的遗传鉴定突变体之间的相互作用，来测试端粒酶激活招募模型的核心要素。目标 4 将首次使用经过广泛表征的重组试剂在体外重建整个端粒酶复合物。将使用该新型系统进行活性测定，以全面测试端粒酶激活的 Cdc13/Est1 招募模型。 这些对酵母端粒酶活性分子机制的体外研究将增加我们对端粒酶活性和调节的一般理解，并将专门为解析疾病中破坏的人类端粒维持的分子机制提供框架。 公共健康相关性：线性染色体的末端包含称为端粒的特殊结构，它在衰老、基因组稳定性和癌症中发挥着重要作用。端粒的长度是细胞衰老的标志，充分缩短的端粒会阻止细胞生长。几乎所有人类癌症都通过激活端粒酶来逃避该检查点，端粒酶通过添加 DNA 序列来恢复端粒长度。然而，端粒酶活性不足也会导致再生障碍性贫血等慢性致命疾病。因此，端粒的正确维护和端粒酶的调节对于人类健康至关重要。