Telomere structure and function in Arabidopsis

描述(申请人提供)：端粒是高阶核蛋白结构，物理上覆盖了染色体末端，有助于保持基因组的完整性。在具有无限增殖能力的细胞中，包括95%的人类癌症，端粒由端粒酶维持。相比之下，大多数正常的胞体中没有端粒酶，端粒变短，直到染色体末端变得没有上限，与双链断裂难以区分。端到端的融合被诱导，最终导致细胞周期停滞。在这里，我们建议利用拟南芥来研究遗传上易驯化的高等真核生物端粒帽的基本成分。拟南芥对端粒功能障碍有极高的耐受性。这一发现，再加上其简单的遗传学，完全测序的基因组，以及大量的转基因工具，为研究端粒生物学中的必要基因提供了独特的机会。在这一新的应用中，我们将重点介绍最近发现的端粒1保护蛋白(POT1)，它结合在染色体的末端，在染色体末端保护和端粒长度调节中发挥核心作用。拟南芥编码三种截然不同的Pot蛋白，AtPot1、AtPot2和AtPot3，它们似乎在功能上是非冗余的。我们推测，拟南芥Pot蛋白的功能分离源于它们与端粒DNA和端粒其他蛋白质组分的不同相互作用。该提案由五个具体目标组成。前两个重点是确定Pot蛋白在体外和体内与端粒DNA的相互作用，以及在体内阐明端粒复合体中的相互作用伙伴。对于后三个目标，我们将利用遗传学和生化方法相结合的方法来研究AtPot1对端粒酶的正调控机制(目标3)，AtPot2对染色体末端保护的贡献(目标4)，以及当AtPot3影响该蛋白在端粒的功能时对其明显的发育调节(目标5)。作为目标5的一部分，我们还将测试端粒在植物生命周期的特定阶段没有上限的假设。鉴于植物和人类的端粒结构和组成具有很强的保守性，这些研究应该会揭示所有高等真核生物共有的机制。

DESCRIPTION (provided by applicant): Telomeres are higher order nucleoprotein structures that physically cap the chromosome terminus and help to preserve genome integrity. In cells with unlimited proliferative capacity, including 95% of human cancers, telomeres are maintained by telomerase. By contrast, telomerase is absent from most of the normal soma and telomeres shorten until chromosome ends become uncapped and indistinguishable from double strand breaks. End-to-end fusions are induced, ultimately leading to cell cycle arrest. Here we propose to exploit Arabidopsis to investigate essential components of the telomere cap in a genetically tractable higher eukaryote. Arabidopsis has an exceptionally high tolerance for telomere dysfunction. This finding, coupled with its facile genetics, completely sequenced genome, and arsenal of transgenic tools offer unique opportunities for investigating essential genes in telomere biology. In this renewal application, we will focus on the recently discovered Protection of telomeres 1 (Pot1) protein, which binds the extreme terminus of the chromosome and plays a central role in chromosome end protection and telomere length regulation. Arabidopsis encodes three strikingly different Pot proteins, AtPot1, AtPot2 and AtPot3, which appear to be functionally non-redundant. We hypothesize that the separation of function of Arabidopsis Pot proteins derives from their distinct interactions with telomeric DNA and with other protein components of the telomere. The proposal is comprised of five Specific Aims. The first two focus on defining the interactions of Pot proteins with telomeric DNA in vitro and in vivo, and elucidating interaction partners in the telomere complex in vivo. For the last three Aims, we will exploit a combination of genetic and biochemical approaches to examine the mechanism of positive regulation of telomerase by AtPot1 (Aim 3), the contribution of AtPot2 to chromosome end protection (Aim 4), and the apparent developmental regulation of AtPot3 as it impacts this protein's function at telomeres (Aim 5). As part of Aim 5, we will also test the hypothesis that telomeres are uncapped in a specific stage of the plant life cycle. Given the strong conservation in telomere architecture and composition in plants and humans, these studies should uncover mechanisms common to all higher eukaryotes.