Telomeric Protein Function and Regulation

端粒蛋白的功能和调控

• 批准号: 9326324
• 负责人: Jayakrishnan Nandakumar
• 金额: $ 30.49万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-08-04 至 2021-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9326324
• 关键词: Affect; Affinity; Binding; Biochemical; Biochemistry; Bioinformatics; Biological; Biology of Aging; CRISPR/Cas technology; Cell Cycle; Cell division; Cell physiology; Cells; Cellular biology; Chromosomes; Code; Complex; Crystallization; DNA; DNA Binding; DNA Repair; DNA-Directed DNA Polymerase; Data; Deficiency Diseases; Disease; Dissection; Dyskeratosis Congenita; Enzymes; Equilibrium; Event; Future; Gene Expression; Gene Silencing; Genes; Human; In Vitro; Individual; Inherited; Knock-out; Maintenance; Malignant Neoplasms; Maps; Mediating; Messenger RNA; Methods; Microscopy; Modeling; Molecular; Multiprotein Complexes; Mutagenesis; Mutation; N-terminal; Names; Nucleoproteins; Patients; Physiological; Premature aging syndrome; Prevalence; Protein Isoforms; Proteins; Proteome; RNA; RNA Biochemistry; RNA Interference; Recruitment Activity; Regulation; Repetitive Sequence; Ribonucleoproteins; SS DNA BP; Series; Somatic Cell; Stem cells; Structure; Tandem Repeat Sequences; Technology; Telomerase; Testing; Tissues; Translation Initiation; Translations; Untranslated RNA; Ursidae Family; X-Ray Crystallography; base; cancer cell; chromosome replication; crosslink; design; experimental study; gene function; genome integrity; inhibitor/antagonist; innovation; interdisciplinary approach; knockout gene; novel; overexpression; protein complex; protein function; repaired; response; self-renewal; single molecule; telomere; therapeutic development

Telomeric protein function and regulation Project Summary/Abstract Continued cell division mandates the maintenance of chromosome ends, which undergo shortening due to the inability of the replicative DNA polymerases to completely synthesize them. Telomerase is a unique ribonuclear protein enzyme that can bind chromosomes ends to extend them with repetitive sequences called telomeric DNA. Using this mechanism, telomerase can help reduce the erosion of chromosome ends and sustain the continued proliferation of actively dividing cells such as somatic stem cells, and cells that make up ~90% of human cancers. Whereas aberrant activation of telomerase in non-dividing somatic cells predisposes them to cancer, mutations in genes that reduce telomerase function in stem cells result in one of many premature aging diseases, including dyskeratosis congenita. Therefore understanding how telomerase function is tightly regulated in cells bears major implications for the biology of aging, and for diseases such as cancer. Telomeric DNA in the cells is not exposed, because this would result in its recognition and repair by the DNA damage response/repair machineries in the cell, leading to illicit end-to-end DNA-joining events at chromosome ends. Our cells therefore have in place a six-protein complex named shelterin that specifically binds telomeric DNA to protect it from illicit DNA repair events. TPP1 is a unique shelterin protein that is central to both telomerase function and chromosome end protection. However, how a single TPP1 gene facilitates the solutions of such distinct biological problems remains unknown. Using a multi-disciplinary approach that includes biochemistry, cell biology, single-molecule microscopy, X-ray crystallography, and bioinformatics, this proposal aims to understand how TPP1 upholds chromosome end protection and end replication. Aim 1 of the proposal will reveal at a molecular level how TPP1 helps protect the single-stranded regions of telomeric DNA with the help of the POT1 protein. Aim 2 of this proposal will involve a novel selective-knockout strategy using CRISPR- Cas9 technology, to ask how natural isoforms of TPP1 facilitate different aspects of end protection and end replication. Aim 3 will explore the mechanism-of-action and pervasiveness of novel RNAs that regulate TPP1 expression in human cells using RNA biochemistry, cell biology and bioinformatics approaches. These studies will reveal the mechanistic basis for how a single TPP1 gene can orchestrate both chromosome end protection and end replication, and discover previously unanticipated mechanisms by which noncoding RNAs regulate TPP1 abundance and function in human cells.

端粒蛋白的功能与调控 项目总结/摘要 持续的细胞分裂要求维持染色体末端，染色体末端由于细胞分裂而缩短。 复制型DNA聚合酶无法完全合成它们。端粒酶是一种独特的核糖核 一种蛋白酶，可以结合染色体末端，使其延伸出重复的端粒序列 DNA.利用这种机制，端粒酶可以帮助减少染色体末端的侵蚀并维持染色体的完整性。 持续增殖的活跃分裂细胞，如体干细胞，以及占约90%的细胞， 人类癌症然而，在非分裂体细胞中端粒酶的异常激活使它们倾向于 在癌症中，减少干细胞中端粒酶功能的基因突变导致许多过早的 衰老疾病，包括先天性角化不良。因此，了解端粒酶的功能与 在细胞中调节的蛋白质对衰老生物学和癌症等疾病具有重要意义。端粒 细胞中的DNA是不暴露的，因为这会导致其识别和修复的DNA损伤 细胞中的反应/修复机制，导致染色体末端的非法端到端DNA连接事件。 因此，我们的细胞中有一种名为shelterin的六蛋白复合物，它特异性地结合端粒DNA， 保护它免受非法DNA修复事件的影响。TPP 1是一种独特的shelterin蛋白，是端粒酶 功能和染色体末端保护。然而，如何一个单一的TPP 1基因促进这种解决方案， 独特的生物学问题仍然未知。使用包括生物化学在内的多学科方法， 细胞生物学，单分子显微镜，X射线晶体学和生物信息学，该提案旨在 了解TPP 1如何维持染色体末端保护和末端复制。该提案的目标1将 在分子水平上揭示TPP 1如何帮助保护端粒DNA的单链区域 POT 1蛋白质该提案的目标2将涉及使用CRISPR的新型选择性敲除策略- Cas9技术，以了解TPP 1的天然同种型如何促进末端保护和末端修饰的不同方面。 复制的目的3将探索调节TPP 1的新型RNA的作用机制和普遍性 使用RNA生物化学、细胞生物学和生物信息学方法在人细胞中表达。这些研究 将揭示单一TPP 1基因如何协调染色体末端保护的机制基础， 并终止复制，并发现以前未预料到的机制，非编码RNA调节 TPP 1在人类细胞中的丰度和功能。