In Vitro Reconstitution and Biochemical Characterization of Yeast Telomerase

描述（由申请人提供）：真核染色体终止于称为端粒的结构，其包含串联序列重复。随着每个细胞复制周期，由于DNA复制机制无法完全合成相对链，端粒逐渐变短。缩短的端粒诱导复制性衰老，这在快速增殖的细胞中通过逆转录酶端粒酶的激活来避免。端粒酶是一种核糖核蛋白，其在端粒末端添加串联重复以维持长度，并且在体内受到正调控和负调控。端粒酶的失调导致几种人类疾病，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。基于体内实验，已经开发了几种端粒酶调节的分子机制的模型。 拟议的研究将使用标准的生物化学和生物物理技术来评估端粒相关蛋白Cdc 13和Est 1的结构，这将用于开发一种新的重组酵母端粒酶体外系统。该系统将用于严格研究端粒酶活性和调节的机制，已在体内遗传鉴定。酵母模型系统以前提供了相当多的洞察人类端粒的维护，和丰富的酵母遗传数据可用于严格评估系统中的单个蛋白质的功能。 在目的1中，我们将确定端粒结合蛋白Cdc 13的端粒酶募集结构域的结构，并分析全长Cdc 13在DNA结合中的作用。目的2将表征端粒酶调节亚基Est 1的生化活性，其将在昆虫细胞中重组产生以产生该领域急需的大量高质量试剂。目的3将测试端粒酶激活的募集模型的中心元素，确定是否存在Cdc 13和Est 1之间的直接物理相互作用，然后评估两种蛋白质的遗传鉴定的突变体之间的相互作用。目的4将首次在体外重组整个端粒酶复合物，使用重组试剂，已被广泛表征。将使用这种新的系统进行活性测定，以充分测试端粒酶激活的Cdc 13/Est 1募集模型。 酵母端粒酶活性的分子机制的这些体外研究将增加我们的端粒酶活性和调节的一般理解，并将特别提供一个框架，解剖的人端粒的维护，在疾病中被破坏的分子机制。

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.