Telomeric Protein Function and Regulation

端粒蛋白的功能及其调控 项目摘要/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 复制的DNA聚合酶不能完全合成它们。端粒酶是一种独特的核糖核 一种可以结合染色体末端的蛋白质酶，通过称为端粒的重复序列延长染色体的末端 DNA利用这一机制，端粒酶可以帮助减少染色体末端的侵蚀，并维持 继续增殖分裂活跃的细胞，如躯体干细胞和占~90%的细胞 人类癌症。然而，未分裂的体细胞中端粒酶的异常激活使它们容易患上 癌症，干细胞中降低端粒酶功能的基因突变导致许多早产儿之一 老年性疾病，包括先天性角化不良。因此，了解端粒酶的功能是如何紧密的 在细胞中的调控对衰老的生物学和癌症等疾病具有重大意义。端粒 细胞中的DNA不会暴露，因为这将导致DNA损伤对其进行识别和修复 细胞中的反应/修复机制，导致在染色体末端发生非法的端到端DNA连接事件。 因此，我们的细胞有一种名为Shelterin的六种蛋白质复合体，它专门将端粒DNA结合到 保护它免受非法DNA修复事件的影响。TPP1是一种独特的保护素蛋白，它对端粒酶和端粒酶 功能和染色体末端保护。然而，单个TPP1基因是如何促进解决这些问题的 不同的生物问题仍然未知。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射线结晶学和生物信息学，这项提议旨在 了解TPP1如何维护染色体末端保护和末端复制。该提案的目标1将 从分子水平揭示TPP1如何帮助保护端粒DNA的单链区域 POT1蛋白。该提案的目标2将涉及一种新的选择性击倒策略，使用CRISPR- Cas9技术，询问TPP1的天然亚型如何促进末端保护和末端保护的不同方面 复制。目标3将探索调节TPP1的新RNA的作用机制和普遍性 利用RNA生物化学、细胞生物学和生物信息学方法在人类细胞中表达。这些研究 将揭示单个TPP1基因如何协调两个染色体末端保护的机制基础 并结束复制，并发现以前未曾预料到的非编码RNA调节机制 TPP1在人类细胞中的丰度和功能。

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.