Telomerase function and regulation in a new model system

描述(申请人提供)：端粒酶是一种核糖核蛋白复合体，负责延长端粒末端重复序列中富含DG的链。它使用一小段稳定结合的RNA组分作为模板，以预先存在的染色体末端作为引物来完成反转录。端粒酶活性在癌细胞中被特异性地激活，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选择性失活导致端粒缩短和细胞凋亡，验证了端粒酶抑制剂在抗癌治疗中的潜力。 这项研究的目的是研究白念珠菌端粒酶的调节和功能，这是一个相对未被探索的实验系统。与其他研究较好的真菌相比，念珠菌具有更长的端粒束，规则的延长的端粒重复，并在端粒上表现出更活跃的重组。由于这些不同寻常的特点，我们已经能够开发出独特的生化和分子工具来解决该领域的基本问题。最重要的是，我们首次在体外证明了两种调节因子对端粒酶活性的影响，从而能够利用酵母遗传学和生物化学的强大组合来探讨端粒酶的调节。我们的初步研究提出了两个主要假设：(1)端粒酶复合体的Caestlp和Caest3p亚基可以通过蛋白质-蛋白质和/或蛋白质-DNA相互作用来激活端粒酶功能；(2)一些端粒酶亚基可以独立于其端粒延伸功能而参与端粒末端的保护。为了验证这些假设，我们将利用一系列生化、遗传学和细胞生物学分析方法来分析念珠菌端粒和端粒酶不合适的突变株。我们还将开发体外纯化系统，以评估调节因子对端粒酶的激活，并研究相关的蛋白质-DNA和蛋白质-蛋白质相互作用。我们期望我们的发现将有助于详细的分子理解端粒酶的功能和调节。

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.