Telomerace Recruitment to Telomeres Via Core Telomore Binding Proteins

描述(申请人提供)：人类染色体的末端被称为端粒的DNA-蛋白质复合体保护。在成年体细胞中，端粒逐渐变短，直到达到极短的长度，导致衰老或危机。这一过程限制了大多数细胞类型的增殖能力。端粒酶是负责合成组成端粒的重复DNA的酶。我们的实验室主要致力于了解端粒酶的核心成分：端粒酶RNA(HRT)和端粒酶逆转录酶(HTERT)在细胞内运输的分子基础。我们假设端粒酶的招募是在细胞周期的S期触发的，并通过该酶与核心端粒结合蛋白的动态结合而促进。我们实验室开发的荧光显微镜技术将被用于各种分析，以评估端粒酶以及一组核心端粒结合蛋白在促进端粒招募方面的内在作用。我们已经定义了两个特定的目标：AIM1-hTERT在端粒酶靶向端粒末端的作用将被剖析。这将通过测试一组hTERT突变体将端粒酶靶向多种细胞类型的端粒的能力来实现。此外，体外结合试验将确定hTERT的哪些结构域对结合核心端粒结合蛋白中的中心招募候选蛋白TPP1至关重要。 目的2-将确定核心端粒结合蛋白在介导端粒酶重新聚集到端粒中的作用。比较RNAi介导的耗竭和逆转录病毒载体辅助的每种核心端粒结合蛋白的过表达对hTR和hTERT募集的影响。以这种方式确定的招募候选人将成为额外突变分析的重点，以确定他们对端粒酶招募的贡献(S)。公共卫生相关性：癌症已成为对我们社会健康的严重威胁。这种疾病一视同仁，事实证明，在构成美国人口的不同种族和民族中，这种疾病是共同的主要死亡原因。在几乎所有的癌症中，端粒酶的重新激活对于延长肿瘤的维持和增殖至关重要。HTERT与核心端粒结合蛋白结合的特征将有助于我们理解通过招募和运输来调节端粒酶的动态机制。这在开发抑制端粒酶和对抗绝大多数癌症的新方法方面可能被证明是无价的。

DESCRIPTION (provided by applicant): The ends of human chromosomes are protected by DNA-protein complexes called telomeres. In adult somatic cells, telomeres progressively shorten until reaching a critically short length, resulting in senescence or crisis. This process limits the proliferative capacity of most cell types. Telomerase is the enzyme responsible for synthesizing the repetitive DNA that comprises the telomere. Our lab is primarily focusing on understanding the molecular basis for the intracellular trafficking of the core components of telomerase: telomerase RNA (hTR), and telomerase reverse transcriptase (hTERT). We hypothesize that the recruitment of telomerase is triggered during S-phase of the cell cycle and is facilitated by the dynamic association of the enzyme with core telomere binding proteins. Fluorescence microscopy techniques developed by our lab will be employed in a variety of assays to assess the intrinsic roles of telomerase, as well as those of a panel of core telomere binding proteins, in facilitating recruitment to telomeres. We have defined two specific aims: Aim1-The role of hTERT in targeting telomerase to the telomere end will be dissected. This will be accomplished by testing the ability of a panel of hTERT mutants to target telomerase to telomeres in multiple cell types. Additionally, in vitro binding assays will determine which domains of hTERT are critical for binding TPP1, the central recruitment candidate among core telomere binding proteins. Aim 2-The role of core telomere binding proteins in mediating telomerase recruitment to telomeres will be determined. The effects of RNAI mediated depletion and retroviral vector assisted overexpression of each core telomere binding protein on the recruitment of both hTR and hTERT will be contrasted. Recruitment candidates identified in this manner will become the focus of additional mutational analysis to identify their contribution(s) to telomerase recruitment. Public Health Relevance: Cancer has become a serious threat to the health of our society. This disease does not discriminate, proving to be a leading cause of death shared among the diverse racial and ethnic groups that make up the american population. Telomerase is reactivation is critical to prolonged tumor maintenance and proliferation in nearly all cancers. The characterization of hTERT association with core telomere binding proteins should contribute significantly to our understanding of the dynamic mechanism of telomerase regulation through recruitment and trafficking. This may prove invaluable in developing new ways to inhibit telomerase and combat the vast majority of cancers.