Molecular Mechanism of Telomerase Action

描述（由申请人提供）： 端粒酶是一种专门的逆转录酶（RT），仅使用其内在端粒酶RNA（TR）亚基的很短区域作为模板，在染色体末端合成端粒DNA重复序列。端粒酶的这种高度特化的功能依赖于一种特殊的机制，即模板RNA和端粒DNA在重复序列的进行性合成过程中解离和重新排列。然而，端粒酶模板易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，并确定调控模板易位特定步骤的元件。虽然端粒酶是以端粒DNA为底物的，但最近发现端粒酶也可以作为以RNA/DNA双链体为底物的常规RT。更令人惊讶的是，端粒酶以序列特异性识别双链体底物。这些重要的发现为端粒酶作用的分子机制提供了深刻的见解。我们假设双链体结合和端粒解离是端粒重复序列合成循环的重要步骤，并调节端粒重复序列的添加速率和持续合成能力。研究计划的具体目的包括（1）确定模板易位效率中的作用，（2）确定模板易位的限速步骤，和（3）表征端粒酶对核苷酸添加的序列依赖性终止。我们期望这些实验的结果将大大增加我们对端粒酶机制的理解。

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.