Analysis of telomerase reverse transcriptase

描述（由申请人提供）：端粒酶是一种核糖核蛋白复合物，负责补充端粒末端重复序列的富g链。它采用一小段内嵌的稳定相关RNA片段作为模板，预先存在的染色体末端作为引物合成端粒束。端粒酶活性在癌细胞中被特异性激活，并通过赋予细胞无限的复制潜力来促进肿瘤转化。肿瘤细胞中端粒酶的抑制或消耗导致端粒缩短和凋亡，验证了抗端粒酶策略在癌症治疗中的潜力。本研究的目的是利用出芽酵母酿酒酵母作为模型系统来了解端粒酶的机制和调控。在过去的资助期内，我们的研究已经获得了丰富的关于催化TERT蛋白结构和功能的信息。此外，他们揭示了(i)催化TERT亚基内的两个端粒酶特异性结构域，它们负责端粒酶学的独特特征，即重复复制短RNA模板的能力；（ii） est1p亚基中的α -螺旋重复结构域（称为TPR）具有新的rna结合活性，（iii）通过atp依赖性染色质重塑复合体（INO80复合体）调节端粒结构和功能。基于这些发现，我们提出了一系列的生化、遗传和细胞生物学研究，旨在：(1)阐明端粒酶组分介导的蛋白质- DNA和蛋白质- rna相互作用的机制和生物学意义；(2)确定染色质重塑复合物在调节端粒结构和端粒酶活性中的作用。拟议研究的结果有望拓宽和深化目前对端粒酶机制的理解，并为端粒调控的新方面提供见解。由于几乎所有将被检测的因素在酵母和人类之间都是保守的，我们预计许多发现将适用于人类端粒和端粒酶，并将促进基于端粒生物学的临床应用的发展。

DESCRIPTION (provided by applicant): Telomerase is a ribonucleoprotein complex responsible for replenishing the G-rich strand of the telomere terminal repeats. It employs a small, embedded segment of a stably associated RNA component as template, and pre-existing chromosomal ends as primers to synthesize telomere tracts. Telomerase activity is specifically activated in cancer cells, and promotes neoplastic transformation by endowing cells with unlimited replicative potential. Inhibition or depletion of telomerase in tumor cells leads to telomere shortening and apoptosis, validating the potential of anti-telomerase strategies in cancer therapy. The goal of this research is to understand telomerase mechanisms and regulation using the budding yeast Saccharomyces cerevisiae as a model system. Our studies during the past funding period have yielded a wealth of information on the structure and functions of the catalytic TERT protein. In addition, they revealed (i) two telomerase-specific domains within the catalytic TERT subunit that are responsible for a unique feature of telomerase enzymology, i.e., the ability to repetitively copy a short RNA template; (ii) an alpha-helical repeat domain (named TPR) in the Est1 p subunit that possesses a novel RNA-binding activity, and (iii) regulation of telomere structure and function by an ATP-dependent chromatin remodeling complex (the INO80 complex). Based on these findings, we propose a series of biochemical, genetic and cell biological studies directed toward (1) clarifying the mechanisms and biological significance of the protein- DNA and protein-RNA interactions mediated by telomerase components, and (2) ascertaining the roles of chromatin remodeling complexes in modulating telomere structures and telomerase activity. Results from the proposed studies are expected to broaden and deepen current understandings of telomerase mechanisms and provide insights into a novel aspect of telomere regulation. Because almost all of the factors that will be examined are conserved between yeast and humans, we anticipate that many of the findings will be applicable to human telomeres and telomerase, and will facilitate the development of clinical applications based on telomere biology.