Structure and Function of Telomerase

端粒酶核糖核蛋白逆转录酶将端粒重复序列添加到染色体末端。这 DNA合成的不寻常模式抵消了基因组上发生的末端序列丢失 以DNA为模板的DNA聚合酶复制。大多数真核细胞的染色体很少，很少 端粒和少量端粒酶。纤毛原生动物是个例外：它们拥有数千到数百万只 每个细胞的端粒和高水平的端粒酶。这项提议充分利用了纤毛虫的优势。 嗜热四膜虫用于端粒酶研究。端粒酶的丰富促进了直接生化 内源组装酶的检测。四膜虫端粒酶的研究也得到了 无与伦比的体外重建分析和强大的方法，在体内的分子遗传操作。 内源组装的四膜虫和人类端粒酶表现出惊人的相似生化 性质，特别是与其他模式生物的端粒酶相比。因此，对 四膜虫端粒酶将继续为实验上较难处理的人类酶的研究提供信息。 我们对端粒酶的了解受到酶结构知识的匮乏和端粒酶的困难的限制 确定和表征内源酶功能所需的因素。填写这些知识 差距是这项提议的长期目标。根据我们之前的体外重建研究，Aim I 描述了利用定点特定的交联剂、荧光法研究重组RNA和蛋白质的结构 共振能量转移和结晶学。根据我们之前对一个 内源性组装的端粒酶全酶，AIMS II，III和IV在体外和体内的应用 对四种新的全酶蛋白进行生化和生物活性鉴定。 对端粒酶结构和功能的洞察直接关系到人类的进步 健康。大多数人类体细胞缺乏功能性端粒酶，因此具有分裂能力，即 受到端粒侵蚀的限制。在以过早端粒丢失为特征的疾病中，临时端粒酶 激活可以恢复细胞更新的潜力。癌细胞异常激活端粒酶以获得 延长了细胞分裂能力，需要端粒酶才能继续存活。因为这个原因， 端粒酶抑制剂可能是广泛有效的癌症治疗药物。

The telomerase ribonucleoprotein reverse transcriptase adds telomeric repeats to chromosome ends. This unusual mode of DMAsynthesis counteracts the terminal sequence loss that occurs upon genome replication by DNA-templated DNA polymerases. Most eukaryotic cells have few chromosomes, few telomeres and little telomerase. Ciliated protozoa are the exception: they possess thousands to millions of telomeres per cell and high levels of telomerase. This proposal capitalizes on advantages of the ciliate Tetrahymena thermophila for studies of telomerase. Telomerase abundance facilitates direct biochemical assays of endogenously assembled enzyme. Studies of Tetrahymena telomerase are also aided by unparalleled in vitro reconstitution assays and robust methods for molecular genetic manipulation in vivo. Endogenously assembled Tetrahymena and human telomerases show remarkably similar biochemical properties, particularly when compared to telomerases from other model organisms. Thus, studies of Tetrahymena telomerase will continue to inform studies of the less experimentally tractable human enzyme. Our understanding of telomerase is limited by scant knowledge of enzyme structure and by the difficulty of identifying and characterizing factors required for endogenous enzyme function. Filling these knowledge gaps is the long-term goal of this proposal. Following from our previous in vitro reconstitution studies, Aim I describes recombinant RNA and protein structural studies using site-specific cross-linking, fluorescence resonance energy transfer and crystallography. Following from our previous affinity purification of an endogenously assembled telomerase holoenzyme, Aims II, III and IV use in vitro and in vivo reconstitution assays to characterize the biochemical and biological activities of four new holoenzyme proteins. Insights about telomerase structure and function have direct relevance for the improvement of human health. Most human somatic cells lack functional telomerase and therefore have a capacity for division that is limited by telomere erosion. In diseases characterized by premature telomere loss, temporary telomerase activation could restore cellular renewal potential. Cancer cells aberrantly activate telomerase to gain extended cell division capacity and require telomerase for their continued viability. For this reason, telomerase inhibitors could be broadly effective cancer therapeutics.