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，端粒是一种特殊的核蛋白复合物，它在物理上覆盖在染色体末端，以将其与双链断裂区分开来，以促进其完全复制。必要的封顶蛋白或端粒维持酶（端粒酶）的缺失，会导致DNA损伤检查点的激活和端到端染色体融合，最终导致大量基因组不稳定。端粒酶和/或端粒维持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变至关重要。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势来研究遗传易处理的高等真核生物端粒帽的基本组成部分。主流观点认为端粒由两种不同的复合物覆盖，脊椎动物中的庇护蛋白和酵母中的CST （Cdc13, Stn1, Ten1）。在之前的资助期内，我们在拟南芥中发现了一个新的端粒复合体，由STN1和TEN1同源物以及一个新的端粒蛋白CTC1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。与酵母Cdc13不同，植物和人类CTC1同源物具有相同的整体结构域结构和非特异性ssDNA结合。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一个重要的进化桥梁。这个更新应用的中心假设是CTC1是一个多功能支架，保护染色体末端并促进端粒DNA复制。目的1将研究CST复合体内的相互作用，并探索TEN1- STN1关联与染色体末端保护的功能相关性。目的2将评估CST在端粒保护和端粒DNA复制中的作用。目的3将验证POT1a通过与CTC1和TER1的相互作用将端粒酶募集到染色体末端的假设。最后，Aim 4将采用先进的遗传方法和仅在拟南芥中可能的创新遗传筛选来分离庇护蛋白和CST相互作用因子以及新的端粒蛋白。这些研究将有助于协调CST和庇护蛋白如何共同促进端粒完整性。总的来说，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。

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.