Telomere structure and function in Arabidopsis

描述(申请人提供)：真核生物基因组的完整性部分来自端粒，端粒是一种特殊的核蛋白复合体，它物理上覆盖着染色体末端，以区分它与双链断裂，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丢失会导致DNA损伤检查点的激活和端到端的染色体融合，最终导致大量基因组的不稳定。端粒酶和/或端粒维持缺陷会导致与增殖相关的人类疾病，而正确的端粒酶调控对于阻止肿瘤的发生至关重要。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基础机制至关重要。在这里，我们建议利用拟南芥模型系统的许多优点来研究遗传上易驯化的高等真核生物端粒帽的基本成分。普遍的观点认为，端粒由两个不同的复合体组成，脊椎动物中的Shelterin和酵母中的Cst(CDc13，Stn1，Ten1)。在之前的资助期间，我们在拟南芥中发现了一个新的端粒复合体，它由STN1和TEN1同源物以及一种新的端粒蛋白CTC1组成。这种新的CST复合体对于植物和人类的端粒完整性都是必需的。植物和人类CTc1同源基因具有相同的整体结构域结构和非特异性的单链DNA结合，不同于酵母Cdc13。然而，拟南芥CST在染色体末端保护中发挥着更关键的作用，类似于酵母CST。因此，对拟南芥CST的研究可能为阐明高等真核生物的端粒复制和染色体末端保护机制提供重要的进化桥梁。这种更新应用的中心假设是，CTC1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复合体内的相互作用，并探索TEN1-STN1关联对染色体末端保护的功能相关性。目的2将评估CST在端粒保护和端粒DNA复制中的作用。目的3将验证POT1a通过与CTC1和TER1相互作用将端粒酶招募到染色体末端的假设。最后，Aim 4将使用正向遗传方法和一种只有在拟南芥中才可能的创新遗传筛选来分离Shelterin和CST相互作用因子和新的端粒蛋白。这些研究将有助于协调CST和避孕素是如何共同促进端粒完整性的。总之，这四个目标产生的结果将协同作用，产生对端粒如何稳定高等真核基因组的更广泛的理解。 与公共健康相关：端粒对于基因组的完整性是必不可少的，因此，了解端粒复合体如何保护基因组的稳定性对于阐明调控细胞增殖和阻止癌症发生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例，并继续发现新的端粒成分和调节机制。在这一传统中，我们将利用拟南芥的遗传适应性和对端粒功能障碍的非凡耐受性来研究一种新的多细胞生物体中的端粒封顶复合体。

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.