Structure and Function of Telomerase

端粒酶的结构和功能

基本信息

批准号：
8964697
负责人：
Kathleen Collins
金额：
$ 32.92万
依托单位：
UNIVERSITY OF CALIFORNIA BERKELEY
依托单位国家：
美国
项目类别：
财政年份：
1996
资助国家：
美国
起止时间：
1996-05-01 至 2019-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8964697
关键词：
Active Sites Address Architecture Back Biochemical Biological Models Catalytic Domain Cell Lineage Cell Proliferation Cell Transplantation Cell division Cells Chromatin Chromosomes, Human, Pair 3 Collaborations Crystallography DNA DNA biosynthesis DNA-Directed DNA Polymerase DNA-Protein Interaction Diagnostic Disease Down-Regulation Electron Microscopy Embryo Embryonic Development Enzymes Equilibrium Failure Fluorescence Foundations Funding Gene Mutation Genome Genome Stability Goals Holoenzymes Human In Vitro Knowledge Length Maps Mediating Modeling Molecular Natural regeneration Nucleic Acids Polymerase Protein Dynamics Proteins Publishing Pulmonary Fibrosis RNA Recruitment Activity Regulation Research Resolution Ribonucleoproteins Short Tandem Repeat Side Single-Stranded DNA Slide Specificity Structure System TNFRSF5 gene Telomerase Telomerase RNA Component Telomere Shortening Terminal Repeat Sequences Testing Tetrahymena Therapeutic Tissues base bone marrow failure syndrome cancer cell cancer therapy cell growth regulation density embryo cell enzyme activity human disease human tissue in vivo inhibitor/antagonist insight neoplastic cell premature public health relevance reconstruction single molecule telomerase reverse transcriptase telomere tissue regeneration tumorigenesis

项目摘要

DESCRIPTION (provided by applicant): The eukaryotic ribonucleoprotein reverse transcriptase telomerase elongates chromosome 3' ends de novo, adding back telomeric simple-sequence repeats that are lost with each cycle of genome replication due to incomplete end-replication by the conventional DNA synthesis machinery. Down- regulation of telomerase after early embryogenesis sets an upper limit for human somatic tissue renewal, which is encountered prematurely in human telomerase deficiencies or "telomeropathies" including a bone marrow failure syndrome, pulmonary fibrosis and other disorders. On the other hand, tumor cells over-activate telomerase to support their indefinite proliferative capacity. Telomerase is unique among polymerases in its reiterative copying of a template within the enzyme's integral RNA subunit. Instead of generating product that is RNA-DNA duplex, telomerase releases single-stranded telomeric repeat DNA. The elaborate catalytic cycle required to support this activity arises from collaboration of telomerase reverse transcriptase (TERT), telomerase RNA (TER) and the numerous other subunits of a biologically active telomerase holoenzyme. The long-term objective of research funded by this RO1 is to determine components, structures, biochemical mechanisms and cellular regulations of telomerase. These goals will inform fundamental knowledge about mechanisms of genome synthesis, genome stability, control of cellular proliferation and tumorigenesis and also specificity principles for dynamic protein-nucleic acid interaction. The Specific Aims build from and extend the Collins lab two-decade track record of insights about telomerase composition, assembly, activity, recruitment to telomeres and regulation using two enabling cellular systems studied in parallel: human cells and the ciliate Tetrahymena. In the current funding period we accomplished the first holoenzyme structure determination with resolution sufficient to place all of the subunits and derive a model of TER tertiary structure. We discovered unanticipated mechanisms underlying several steps of the telomerase catalytic cycle of repeat synthesis in vitro and subunit interactions that mediate telomerase recruitment to telomeres in vivo. We will exploit these advances and our single-molecule-level analysis of human telomerase architecture to determine structures of the human telomerase holoenzyme as well as atomic-resolution structures of Tetrahymena telomerase holoenzyme proteins (Aim 1), define the biochemical and molecular basis for the specificities of telomerase-DNA interaction (Aim 2) and test hypotheses about how telomerase finds an extreme chromosome 3' terminus and elongates it in coordinates with other DNA replication machinery (Aim 3). These studies inform strategies of telomerase modulation for boosting cellular regeneration in human tissues and cell transplantations, and provide targets for anti-cancer therapy.

描述（由申请人提供）：真核核糖核蛋白逆转录酶端粒酶从头延长染色体3'末端，添加回端粒简单序列重复序列，这些重复序列由于早期端粒酶下调后传统DNA合成机制导致的不完整末端复制而丢失。胚胎发生设定了人类体细胞组织更新的上限，在人类端粒酶缺陷或“端粒病”（包括骨骼）中过早遇到这种情况另一方面，肿瘤细胞过度激活端粒酶以支持其无限增殖能力，端粒酶在酶的整合RNA亚基内重复复制模板，这是独一无二的。作为RNA-DNA双链体的产物，端粒酶释放单链端粒重复DNA，支持这种活性所需的复杂催化循环来自于相互协作。端粒酶 RO1 资助的研究的长期目标是确定端粒酶的成分、结构、生化机制和细胞调控。将提供有关基因组合成机制、基因组稳定性、细胞增殖和肿瘤发生的控制以及动态蛋白质-核酸相互作用的特异性原理的基础知识。源自并扩展了柯林斯实验室二十年来对端粒酶组成、组装、活性、端粒募集和调节的见解，使用并行研究的两个支持细胞系统：人类细胞和纤毛虫四膜虫。在当前的资助期内，我们完成了这项研究。首次全酶结构测定，分辨率足以放置所有亚基并得出 TER 三级结构模型，我们发现了端粒酶催化几个步骤背后的意想不到的机制。我们将利用这些进展和对人类端粒酶结构的单分子水平分析来确定人类端粒酶全酶的结构以及原子分辨率结构。四膜虫端粒酶全酶蛋白（目标 1），定义端粒酶-DNA 相互作用特异性的生化和分子基础（目标 2）并测试关于端粒酶如何找到极端染色体 3' 末端并与其他 DNA 复制机制协调延长它的假设（目标 3）。这些研究为促进人体组织和细胞移植中的细胞再生的端粒酶调节策略提供了信息。并为抗癌治疗提供靶点。