Role of POT1 in telomere length regulation

项目摘要 我们将使用最先进的生物化学，细胞和遗传学方法来了解 ssDNA结合蛋白如何保护端粒免受DNA损伤的机制 反应，调节其长度和复制。使用新的体外生物化学测定，我们将 研究RPA和hPOT 1之间的分子差异是否是其独特的基础， 在端粒中起作用。我们将确定hPOT 1中抑制端粒所需的残基 末端保护和C-链切除（Aim 1）。我们将机械地确定POT 1b如何 促进端粒长度维持，并使用条件性耗竭的造血干细胞 内源性POT 1 a/B的表达，以确定参与端粒延长的氨基酸残基 维持干细胞所必需的（目的2）。我们将探索POT 1a如何保护端粒 复制压力。最后，我们使用BioID将Claspin鉴定为端粒相互作用蛋白 参与POT 1a缺失端粒的DNA复制（Aim 3）。拟议的研究提出 一个独特的机会来解决端粒领域的一些最重要的问题。

Project Summary We will use state-of-the-art biochemical, cellular and genetic approaches to understand mechanistically how ssDNA binding proteins protect telomeres from activating a DNA damage response, regulate its length and replication. Using novel in vitro biochemical assays, we will examine whether the molecular distinctions between RPA and hPOT1 underlie their unique functions at telomeres. We will define the residues in hPOT1 that are required to repress telomere end protection and C-strand resection (Aim 1). We will determine mechanistically how POT1b promotes telomere length maintenance, and use hematopoietic stem cells conditionally depleted of endogenous POT1a/b to define the amino acid residues involved in telomere elongation necessary for the stem cell maintenance (Aim 2). We will explore how POT1a protects telomeres from replication stress. Finally, we used BioID to identify Claspin as a telomere interacting protein involved in DNA replication at telomeres devoid of POT1a (Aim 3). The proposed studies present a unique opportunity to address some of the most important questions in the telomere field.