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 一种不寻常的DMA合成方式抵消了基因组末端序列的丢失 DNA模板DNA聚合酶复制。大多数真核细胞的染色体很少， 端粒和少量的端粒酶。有纤毛的原生动物是个例外：它们拥有数千到数百万的 端粒和高水平的端粒酶。这个建议利用了纤毛虫的优势 嗜热四膜虫用于端粒酶的研究。端粒酶丰度促进直接生化 内源性组装酶的测定。四膜虫端粒酶的研究也有助于 无与伦比的体外重建测定和体内分子遗传操作的稳健方法。 内源性组装的四膜虫和人类端粒酶显示出非常相似的生化特性， 这些特性，特别是当与来自其他模式生物的端粒酶相比时。因此，研究 四膜虫端粒酶将继续为研究这种在实验上不易处理的人类酶提供信息。 我们对端粒酶的理解受到酶结构知识的限制， 鉴定和表征内源酶功能所需的因素。把这些知识 消除差距是本提案的长期目标。根据我们之前的体外重建研究，Aim I 描述了重组RNA和蛋白质结构的研究，使用位点特异性交联，荧光 共振能量转移和晶体学。从我们先前的亲和纯化一种 内源性组装的端粒酶全酶，目的II、III和IV用于体外和体内重建 分析，以表征四种新的全酶蛋白的生物化学和生物活性。 对端粒酶结构和功能的了解对于改善人类的生物学行为具有直接的意义。 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， 受到端粒侵蚀的限制在以端粒过早丢失为特征的疾病中， 激活可以恢复细胞更新潜力。癌细胞异常激活端粒酶， 延长的细胞分裂能力，并且需要端粒酶来维持它们的持续活力。基于这个原因， 端粒酶抑制剂可能是广泛有效的癌症治疗剂。

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.