Structure and Function of Telomerase

端粒酶的结构和功能

基本信息

批准号：
7933115
负责人：
Kathleen Collins
金额：
$ 17.14万
依托单位：
UNIVERSITY OF CALIFORNIA BERKELEY
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-09-30 至 2010-09-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7933115
关键词：
Affinity Chromatography Animal Model Biochemical Biological Biological Assay Biological Models Caspase-1 Cell division Cells Chromosomes Chromosomes, Human, Pair 3 Complex Crystallography DNA biosynthesis DNA-Directed DNA Polymerase Disease Enzymes Epitopes Eukaryotic Cell Fluorescence Resonance Energy Transfer Funding Genetic Genome Goals Health Holoenzymes Human In Vitro Knowledge Laboratories Malignant Neoplasms Methods Molecular Molecular Genetics Process Property Proteins Protozoa RNA-Directed DNA Polymerase Regulation Ribonucleoproteins Role Short Tandem Repeat Site Somatic Cell Staging Structural Protein Structure Telomerase Telomerase Inhibitor Telomerase RNA Component Telomere Length Maintenance Tetrahymena Tetrahymena thermophila Therapeutic cancer cell crosslink enzyme mechanism enzyme structure genetic manipulation in vivo insight novel p65 premature recombinant RNA reconstitution telomerase reverse transcriptase telomere transcriptional coactivator p75

项目摘要

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.

端粒酶核糖核蛋白逆转录酶为染色体末端增加了端粒重复。这 dmasynsiss的异常模式抵消了基因组上发生的末端序列损失通过DNA-模拟的DNA聚合酶复制。大多数真核细胞几乎没有染色体，很少端粒和小端粒酶。纤毛原生动物是例外：它们拥有数千至数百万每个细胞和高水平端粒酶的端粒。该提案资本利用了纤毛的优势四心苯胺热嗜热酶研究端粒酶。端粒酶丰度有助于直接生化内源组装酶的测定。四心肌端粒酶的研究也得到了帮助无与伦比的体外重构测定法和体内分子遗传操作的鲁棒方法。内源组装的四膜虫和人端粒酶显示出非常相似的生化特性，特别是与其他模型生物的端粒酶相比。因此，研究四心肌端粒酶将继续为研究较少的实验性人类酶提供信息。我们对端粒酶的理解受到酶结构知识的知识的限制和识别和表征内源性酶功能所需的因素。填写这些知识差距是该提议的长期目标。从以前的体外重构研究中，目标我描述使用位点特异性交联，荧光的重组RNA和蛋白质结构研究共振能量传递和晶体学。从我们以前的亲和力净化之后内源组装的端粒酶全酶，AIMS II，III和IV在体外和体内重建表征四种新全酶蛋白的生化和生物学活性的测定。关于端粒酶结构和功能的见解与人类的改善具有直接相关性健康。大多数人类体细胞缺乏功能端粒酶，因此具有分裂的能力受端粒侵蚀的限制。在以端粒损失为特征的疾病中，临时端粒酶激活可以恢复细胞更新潜力。癌细胞异常激活端粒酶以获得扩展细胞分裂的能力，需要端粒酶的持续生存能力。为此原因，端粒酶抑制剂可能是广泛有效的癌症治疗剂。