Telomerase function and regulation in a new model system

新模型系统中的端粒酶功能和调节

• 批准号: 7094239
• 负责人: NEAL F LUE
• 金额: $ 29.2万
• 依托单位: WEILL MEDICAL COLL OF CORNELL UNIV
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-08-01 至 2008-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7094239
• 关键词: Candida albicans; enzyme activity; enzyme mechanism; fungal genetics; genetic recombination; immunoprecipitation; intermolecular interaction; mutant; protein protein interaction; protein purification; protein structure function; site directed mutagenesis; telomerase; telomere

DESCRIPTION (provided by applicant): Telomerase is a ribonucleoprotein complex responsible for extending the dG-rich strand of the telomere terminal repeats. It employs a small segment of a stably associated RNA component as template, and pre-existing chromosomal ends as primers to accomplish reverse transcription. Telomerase activity isspecifically activated in cancer cells, and promotes neoplastic transformation by endowing cells with unlimited replicative potential. Selective inactivation of telomerase in tumor cells leads to telomere shortening and apoptosis, validating the potential of telomerase inhibitors in anti-cancer therapy. The goal of this research is to investigate telomerase regulation and function in Candida albicans, a relatively unexplored experimental system. In contrast to other well-studied fungi, Candida possesses longer telomere tracts, a regular extended telomere repeat, and exhibits more active recombination at telomeres. Because of these unusual features, we have been able to develop unique biochemical and molecular toolsfor addressing fundamental issues in the field. Most importantly, we have been able to demonstrate for the first time the effects of two regulatory factors on telomerase activity in vitro, and are thus in a position to approach telomerase regulation using the powerful combination of yeast genetics and biochemistry. Our preliminary studies suggest two major hypotheses: (1) the CaEstlp and CaEst3p subunit of the telomerase complex can activate telomerase function through protein-protein and/or protein-DNA interaction, and (2) some telomerase subunits can participate in the protection of telemere ends independent of their telomere extension function. To examine these hypotheses, we will utilize a series of biochemical, genetic, and cell biological assays to analyze Candida telomeres and telomerase inappropriate mutant strains. We will also develop purified in vitro systems to assess telomerase activation by regulatory factors, and to investigate relevant protein-DNA and protein-protein interactions. We anticipate that our findings will contribute to a detailed molecular understanding of telomerase function and regulation.

描述（由申请人提供）：端粒酶是一种核糖核蛋白复合物，负责延长端粒末端重复序列的富含dG的链。它采用一小段稳定相关的RNA组分作为模板，并将预先存在的染色体末端作为引物来完成逆转录。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而促进肿瘤转化。端粒酶在肿瘤细胞中的选择性失活导致端粒缩短和凋亡，验证了端粒酶抑制剂在抗癌治疗中的潜力。 本研究的目标是研究白念珠菌中的端粒酶调节和功能，这是一个相对未开发的实验系统。与其他研究充分的真菌相比，念珠菌具有较长的端粒区，有规律的端粒重复序列，并在端粒处表现出更活跃的重组。由于这些不寻常的特征，我们已经能够开发独特的生物化学和分子工具来解决该领域的基本问题。最重要的是，我们已经能够证明第一次在体外端粒酶活性的两个调节因子的影响，并因此能够接近端粒酶调节使用酵母遗传学和生物化学的强大组合。我们的初步研究提出了两个主要假设：（1）端粒酶复合物的CaEstlp和CaEst 3 p亚基可以通过蛋白质-蛋白质和/或蛋白质-DNA相互作用激活端粒酶功能;（2）某些端粒酶亚基可以参与保护端粒末端，而不依赖于它们的端粒延伸功能。为了验证这些假设，我们将利用一系列的生化，遗传和细胞生物学检测来分析念珠菌端粒和端粒酶不适当的突变株。我们也将开发纯化的体外系统，以评估端粒酶活化的调控因子，并研究相关的蛋白质-DNA和蛋白质-蛋白质的相互作用。我们期望我们的研究结果将有助于对端粒酶功能和调控的详细分子理解。