Molecular Mechanism of Telomerase Action

描述(由申请人提供)： 项目摘要端粒酶是一种特殊的逆转录酶(RT)，它只使用其固有的端粒酶RNA(TR)亚单位的一小部分作为模板，在染色体末端合成端粒DNA重复序列。端粒酶的这种高度专门化的功能依赖于一种特殊的机制，通过这种机制，模板RNA和端粒DNA在重复序列的过程中解离和重新排列。然而，端粒酶模板转位的详细机制仍有待确定。这项研究计划旨在阐明端粒酶作用的独特机制，并确定调节模板转位特定步骤的元件。虽然端粒酶使用单链端粒DNA作为其天然底物，但我们最近发现，端粒酶可以作为传统的以RNA/DNA双链为底物的RT。更令人惊讶的是，端粒酶识别双链底物具有序列特异性。这些重要的发现为端粒酶作用的分子机制提供了很好的见解。我们假设，双链结合和双链解离是端粒-重复序列合成周期中的重要步骤，并调节端粒-重复序列的添加速率和加工性。该研究计划的具体目标包括：(1)确定模板转位效率中的双重结合亲和力；(2)确定模板转位的限速步骤；(3)表征端粒酶对核苷酸加成的序列依赖性终止。我们希望这些实验的结果将极大地增加我们对端粒酶机制的理解。 公共卫生相关性： 项目叙事端粒酶是一种专门的RNA依赖的DNA聚合酶，它将DNA重复添加到染色体末端，被视为抗衰老和抗癌治疗的重要药物靶点。本研究计划的目的是了解端粒酶作用的详细机制，并确定调节端粒酶功能的重要元件和因素。了解端粒酶作用的分子机制将为寻找端粒酶相关和端粒介导性疾病的治疗方法奠定重要基础。

DESCRIPTION (provided by applicant): Project Summary Telomerase is a specialized reverse transcriptase (RT) that synthesizes telomere DNA repeats at chromosome ends, using only a very short region of its intrinsic telomerase RNA (TR) subunit as template. This highly specialized function of telomerase relies on a special mechanism whereby the template RNA and the telomeric DNA dissociate and realign during the processive synthesis of repeats. However, the detailed mechanism of telomerase template translocation remains to be determined. This research program aims to articulate the unique mechanism of telomerase action and identify elements that regulate specific steps of template translocation. Although telomerase uses the single-stranded telomeric DNA as its native substrate, we have recently discovered that telomerase can act as a conventional RT utilizing RNA/DNA duplex as substrate. More surprisingly, telomerase recognizes the duplex substrate with a sequence-specificity. These crucial findings have provided great insights into the molecular mechanism of telomerase action. We hypothesize that duplex- binding and duplex-dissociation are important steps of the telomere-repeat synthesis cycle, and regulate telomere-repeat addition rate and processivity. Specific Aims of the research program include (1) Determining the role duplex-binding affinity in template translocation efficiency, (2) Determining the rate-limiting step of template translocation, and (3) characterize the sequence- dependent termination of nucleotide addition by telomerase. We expect the outcomes of these experiments will greatly add to our understanding of telomerase mechanism. PUBLIC HEALTH RELEVANCE: Project Narrative Telomerase is a specialized RNA-dependent DNA polymerase that adds DNA repeats to chromosome ends and viewed as an important drug target for anti-ageing and anti-cancer therapies. The goal of this research program is to understand the detailed mechanism of telomerase action, and identify elements and factors important for regulating telomerase function. Comprehension of the molecular mechanism of telomerase action will lay important foundations for finding cures to telomerase-related and telomere-mediated diseases.