Structural and Functional Characterization of Telomere Protection and Maintenance

 描述(申请人提供)：真核生物中线性染色体的进化对染色体的自然末端提出了几个挑战，例如错误地识别为DNA损伤位置、DNA降解和DNA复制不完整。这些挑战的一个解决方案是形成保护帽结构，包括端粒重复序列和相关蛋白质的串联阵列。保护素复合体保护端粒免受DNA损伤修复(DDR)机制的处理。然而，保护素如何重塑端粒DNA以及区分端粒末端和受损DNA末端的分子基础仍不清楚。这些问题可以通过解析保护素结合的端粒DNA的结构和研究保护素和端粒末端的DDR信号之间的动态相互作用来解决。端粒束在每次细胞分裂时缩短，直到到达 当它们的结构失效触发复制性衰老的极限。因此，端粒被称为决定体细胞增殖寿命的内部生物钟。在生殖系和干细胞中，端粒酶可以逆转端粒束的丢失，端粒酶是一种特殊的逆转录酶，可以将端粒重复添加到染色体末端。端粒酶在端粒末端的募集及其重复加成过程受到Shelterin的严格调控。目前，我们对端粒酶的机械力化学循环和保护素成分在调节端粒酶活性中的具体作用缺乏完整的了解。通过实时监测端粒重复合成将获得进一步的见解 通过单核苷酸分辨率的端粒酶。这项建议的主要目标是发展体外结构和单分子生物物理方法，以剖析端粒维持和保护的分子机制。我们有三个具体的目标：第一，我们将重建人类避难所蛋白复合体，并使用负染色电子显微镜(EM)确定载脂蛋白和DNA结合复合体的结构。然后，我们将尝试使用冷冻EM获得该复合体的高分辨率结构，以揭示介导端粒保护和维持的蛋白质-核酸相互作用。其次，我们将在流室中的模型端粒DNA底物上组装保护素，并研究保护素如何使用单分子FRET将端粒重塑为其受保护的形式。保护素和端粒末端靶向蛋白之间的动态相互作用将在端粒末端被监测。我们的目标是解决这个概念上的两难境地，即如何在允许端粒酶招募的同时，保护端粒末端免受DDR信号蛋白RPA(复制蛋白A)的结合。第三，利用光学捕捉法，我们的目标是识别与端粒酶合成端粒重复序列相关的机械力化学转变，并为掩蔽素亚基增强端粒酶的处理能力提供机制解释。

 DESCRIPTION (provided by applicant): The evolution of linear chromosomes in eukaryotes poses several challenges for the natural ends of chromosomes, such as misrecognition as DNA damage sites, DNA degradation, and incomplete DNA replication. A solution to these challenges has been the formation of a protective cap structure comprising a tandem array of telomeric repeats and associated proteins. The shelterin complex protects telomeres against processing by the DNA damage repair (DDR) machinery. However, the molecular basis of how shelterin remodels telomeric DNA and distinguishes telomere termini from damaged DNA ends remains unclear. These questions can be addressed by resolving the structure of shelterin-bound telomeric DNA and investigating the dynamic interplay between shelterin and DDR signals at the telomeric terminus. Telomeric tracts shorten upon each cell division until reaching a limit at which their structural failure triggers replicative senescence. Thus, telomeres are referred to as internal biological clocks that determine the proliferative lifespan of somatic cell. In germ-line and stem cells, the loss of telomeric tracts can be reversed by telomerase, a special reverse transcriptase that adds telomeric repeats to chromosome ends. Recruitment of telomerase to the telomeric terminus and its repeat addition processivity are tightly regulated by shelterin. We currently lack an integrated understanding of telomerase's mechanochemical cycle and the specific roles that shelterin components have in modulating telomerase processivity. Further insights will be gained by real-time monitoring of telomeric repeat synthesis by telomerase at a single nucleotide resolution. The primary objective of this proposal is to develop structural and single-molecule biophysical approaches in vitro to dissect the molecular mechanism of telomere maintenance and protection. We have three specific aims: First, we will reconstitute the human shelterin complex and determine the structure of the apo and the DNA-bound complexes using negative stain electron microscopy (EM). We will then attempt to obtain a high resolution structure of the complex using cryo-EM to reveal the protein-nucleic acid interactions that mediate the protection and maintenance of telomeres. Second, we will assemble shelterin on a model telomeric DNA substrate in a flow chamber and study how shelterin remodels telomeres into their protected form using single-molecule FRET. The dynamic interplay between shelterin and telomere- end targeting proteins will be monitored at telomeric terminus. We aim to address the conceptual dilemma of how shelterin protects telomeric terminus against binding of a DDR signaling protein, RPA (replication protein A), while allowing the recruitment of telomerase. Third, using optical trapping, we aim to identify the mechanochemical transitions associated with telomeric repeat synthesis by telomerase and provide a mechanistic explanation for the enhancement of telomerase processivity by shelterin subunits.