Telomere structure and function in Arabidopsis

描述（由申请人提供）：端粒是高级核蛋白结构，其物理上覆盖染色体末端并有助于保持基因组完整性。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，端粒酶在大多数正常的索马中是不存在的，并且端粒缩短直到染色体末端变得脱帽并且与双链断裂难以区分。诱导端对端融合，最终导致细胞周期停滞。在这里，我们建议利用拟南芥调查的端粒帽在遗传上听话的高等真核生物的重要组成部分。拟南芥对端粒功能异常具有极高的耐受性。这一发现，加上其简单的遗传学，完全测序的基因组，和军火库的转基因工具提供了独特的机会，调查端粒生物学中的必需基因。在本次更新申请中，我们将重点关注最近发现的Protection of telomeres 1（Pot1）蛋白，该蛋白结合染色体的末端，在染色体末端保护和端粒长度调节中发挥核心作用。拟南芥编码三种明显不同的Pot蛋白，AtPot1，AtPot2和AtPot3，它们似乎在功能上是非冗余的。我们推测，拟南芥壶蛋白的功能分离来自于它们与端粒DNA和端粒的其他蛋白质组分的不同相互作用。该提案包括五个具体目标。前两个集中在定义的相互作用的壶蛋白与端粒DNA在体外和体内，并阐明在体内的端粒复合物的相互作用的合作伙伴。对于最后三个目标，我们将利用遗传和生物化学方法的组合来研究AtPot1（目标3），AtPot2对染色体末端保护的贡献（目标4），以及AtPot3的明显发育调节的端粒酶的正调控机制，因为它影响了这种蛋白质在端粒的功能（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被脱帽的假设。鉴于植物和人类端粒结构和组成的高度保守性，这些研究应该揭示所有高等真核生物共同的机制。

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.