In Vitro Reconstitution and Biochemical Characterization of Yeast Telomerase

描述(申请人提供)：真核染色体终止在称为端粒的结构中，其中包含串联序列重复。在每个细胞复制周期中，由于DNA复制机制无法完全合成相反的链，端粒逐渐变短。端粒缩短会导致复制性衰老，在快速增殖的细胞中，这是通过激活逆转录酶端粒酶来避免的。端粒酶是一种核糖核蛋白，在端粒末端增加串联重复序列以保持长度，在体内既受正向调控，也受负向调控。端粒酶的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。在活体实验的基础上，已经建立了几种端粒酶调控的分子机制模型。这项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，这将被用于开发一种新的体外重组酵母端粒酶系统。这一系统将被用来严格研究体内已被遗传鉴定的端粒酶活性和调节机制。酵母模型系统已经为人类端粒的维持提供了相当大的洞察力，并且有丰富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，并分析该结构域在全长CDC13与DNA结合中的作用。目的2将表征端粒酶调节亚基EST1的生化活性，该亚单位将在昆虫细胞中重组生产，以产生该领域迫切需要的高质量试剂的大量产量。目的3将通过确定cdc13和est1之间是否存在直接物理相互作用，然后评估这两种蛋白的基因识别突变体之间的相互作用，来测试端粒酶激活的招募模型的中心元素。目标4将首次在体外重建整个端粒酶复合体，使用已被广泛表征的重组试剂。将使用这一新系统进行活性分析，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的分子机制的体外研究将增加我们对端粒酶活性和调控的一般理解，并将专门为剖析疾病中人类端粒维持的分子机制提供一个框架。 与公共健康相关：线性染色体的末端由称为端粒的特殊结构组成，它们在衰老、基因组稳定和癌症方面起着至关重要的作用。端粒的长度是细胞老化的标志，而端粒足够短会阻止细胞生长。几乎所有的人类癌症都通过激活端粒酶来逃避这个检查点，端粒酶通过增加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，端粒的适当维持和端粒酶的调节对人类健康至关重要。

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.