Telomere structure and function in Arabidopsis

描述（由申请人提供）：真核生物基因组的完整性部分来源于端粒，端粒是一种专门的核蛋白复合物，它在物理上覆盖染色体末端，以将其与双链断裂区分开来，从而促进其完全复制。必需的加帽蛋白或端粒维持酶端粒酶的丢失导致DNA损伤检查点的激活和端到端染色体融合，最终导致大规模基因组不稳定。端粒酶和/或端粒维持的缺陷导致与增殖相关的人类疾病，而正确的端粒酶调节对于阻止癌发生至关重要。阐明端粒复合体的成分如何保护基因组对于理解人类疾病的机制至关重要。在这里，我们建议利用拟南芥模型系统的许多优点，调查在遗传上易处理的高等真核生物的端粒帽的重要组成部分。普遍的观点是端粒被两种不同的复合物覆盖，脊椎动物中的shelterin和酵母中的CST（Cdc 13，Stn 1，Ten 1）。在之前的资助期间，我们在拟南芥中发现了一种新的端粒复合物，由STN 1和TEN 1直系同源物和一种新的端粒蛋白CTC 1组成。这种新的CST复合物是植物和人类端粒完整性所必需的。植物和人类CTC 1同源物共享相同的整体结构域结构和非特异性ssDNA结合，不同于酵母Cdc 13。然而，拟南芥CST在染色体末端保护中起着更关键的作用，类似于酵母CST。因此，拟南芥CST的研究可能为阐明高等真核生物端粒复制和染色体末端保护机制提供重要的进化桥梁。这项更新申请的中心假设是，CTC 1是一种多功能支架，可以保护染色体末端并促进端粒DNA复制。目的1将研究CST复合物内的相互作用，并探索TEN 1-STN 1关联对染色体末端保护的功能相关性。目的2将评估CST在端粒保护与端粒DNA复制中的作用。目的3将验证POT 1a通过与CTC 1和TER 1的相互作用将端粒酶募集到染色体末端的假设。最后，目标4将采用正向遗传学方法和一个创新的遗传筛选，可能只有在拟南芥分离shelterin和CST相互作用的因素和新的端粒蛋白。这些研究将有助于协调CST和shelterin如何共同促进端粒完整性。总之，从这四个目标产生的结果将协同产生更广泛的理解端粒如何稳定高等真核生物基因组。 公共卫生相关性：端粒对于基因组的完整性至关重要，因此，了解端粒复合体如何保障基因组的稳定性对于阐明调节细胞增殖和阻止癌变的基本机制至关重要。在模式生物中的研究已经建立了人类端粒生物学的范式，并继续发现新的端粒组成和调控机制。在这个传统中，我们将利用拟南芥的遗传易处理性和其对端粒功能障碍的非凡耐受性来研究多细胞生物中的新端粒帽复合物。

DESCRIPTION (provided by applicant): The integrity of eukaryote genomes is derived in part from telomeres, the specialized nucleoprotein complexes that physically cap the chromosome terminus to distinguish it from a double-strand break to facilitate its complete replication. Loss of essential capping proteins or the telomere maintenance enzyme, telomerase, results in activation of DNA damage checkpoints and end-to-end chromosome fusions that culminate in massive genome instability. Defects in telomerase and/or telomere maintenance lead to proliferation-related human diseases, while correct telomerase regulation is essential to impede carcinogenesis. Elucidating how constituents of the telomere complex safeguard the genome is essential for understanding mechanisms that underlie human disease. Here we propose to exploit the many advantages of the Arabidopsis model system to investigate essential components of the telomere cap in a genetically tractable higher eukaryote. The prevailing view has been that telomeres are capped by two distinct complexes, shelterin in vertebrates and CST (Cdc13, Stn1, Ten1) in yeast. In the previous funding period, we discovered a new telomere complex in Arabidopsis comprised of STN1 and TEN1 orthologs and a novel telomere protein, CTC1. This new CST complex is required for telomere integrity in both plants and humans. The plant and human CTC1 orthologs share the same overall domain structure and non-specific ssDNA binding, distinct from yeast Cdc13. However, Arabidopsis CST plays a more pivotal role in chromosome end protection, akin to yeast CST. Thus, studies of Arabidopsis CST may provide a critical evolutionary bridge to elucidate mechanisms of telomere replication and chromosome end protection in higher eukaryotes. The central hypothesis of this renewal application that CTC1 is a multifunctional scaffold that protects chromosome ends and promotes telomeric DNA replication. Aim 1 will investigate interactions within the CST complex and explore the functional relevance of the TEN1- STN1 association for chromosome end protection. Aim 2 will assess the role of CST in telomere protection versus telomeric DNA replication. Aim 3 will test the hypothesis that POT1a recruits telomerase to the chromosome terminus via interactions with CTC1 and TER1. Finally, Aim 4 will employ forward genetic approaches and an innovative genetic screen possible only in Arabidopsis to isolate shelterin and CST- interacting factors and new telomere proteins. The studies will help to reconcile how CST and shelterin work together to promote telomere integrity. Altogether, the results generated from these four Aims will synergize to yield a much broader understanding of how telomeres stabilize higher eukaryotic genomes. PUBLIC HEALTH RELEVANCE: Telomeres are essential for genome integrity and as a consequence, understanding how the telomere complex safeguards genome stability will be crucial for elucidating the fundamental mechanisms that regulate cell proliferation and impede carcinogenesis. Studies in model organisms have established the paradigms for human telomere biology, and continue to uncover novel telomere components and regulatory mechanisms. In this tradition, we will exploit the genetic tractability of Arabidopsis and its extraordinary tolerance to telomere dysfunction to investigate a new telomere capping complex in multicellular organisms.