Discerning the mechanism of telomere dysfunction caused by a mutant telomerase template

项目摘要/摘要 特发性肺纤维化(IPF)是一种进行性的、致命性的肺部疾病，与 端粒维持。端粒是真核生物末端保护性的非编码DNA末端 染色体。端粒序列TTAGGG在所有脊椎动物中都是保守的，并被保护性的 蛋白质复合体，以一种序列依赖的方式命名为保护素。掩体复合体的位移 导致DNA损伤反应，导致染色体融合和细胞周期停滞。随着每一轮的 由于末端复制问题和基因组压力的共同作用，细胞分裂、端粒末端缩短。 为了抵消这种缩短，细胞利用端粒酶。端粒酶由一种蛋白质组成 端粒酶逆转录酶(TERT)组件和RNA模板，端粒酶RNA组件 (Tr)。IPF中的突变通常与端粒维持所需的基因有关，例如 TERT、TERC(Tr)、RTEL1、DKC1及相关基因。TR有451个碱基对，包含11个碱基对 负责结合端粒突出部分的模板，并充当反转录模板 六个新基地。我们已经确定了一名特发性肺纤维化患者，该患者在tr模板中存在杂合子突变。 该区域被预测为编码不同的端粒序列。值得注意的是，全基因组测序 从患者身上发现，端粒显示出很大一部分不同的端粒序列。 在4亿年的进化过程中端粒序列的严格保守表明，变异 在端粒序列中是不能容忍的，因为阻止了保护素的结合。这件事的性质是 意想不到的发现将在此提案中进行检查。我们假设这个变异的端粒酶模板，和 随后的变异型端粒增加，可能通过两个独立的机制导致细胞功能障碍； 通过端粒缩短和/或保护素结合的丧失。这些机制将在 遵循目标。目标1将研究添加不同的端粒序列如何影响酶 端粒酶活性和由此产生的细胞端粒长度。目标2将检查变体的效果 序列对基因组的稳定性有影响，包括保护素的结合和细胞内的DNA损伤反应。 这一提议的完成将使我们对端粒序列退化如何影响 基因组稳定性和端粒维持。

Project Summary/Abstract Idiopathic pulmonary fibrosis (IPF) is a progressive, fatal lung disease that is strongly linked to deficits in telomere maintenance. Telomeres are protective non-coding DNA end caps at the ends of eukaryotic chromosomes. The telomere sequence, TTAGGG, is conserved in all vertebrates and is bound by a protective protein complex termed shelterin in a sequence-dependent manner. Displacement of the shelterin complex leads to a DNA damage response, resulting in chromosomal fusions and cell cycle arrest. With each round of cell division, telomere ends shorten due to a combination of the end replication problem and genomic stress. To counterbalance this shortening, cells utilize the enzyme telomerase. Telomerase is composed of a protein component telomerase reverse transcriptase (TERT) and an RNA template, the telomerase RNA component (TR). Mutations in IPF are often linked to genes required for telomere maintenance, such as mutations in TERT, TERC (TR), RTEL1, DKC1, and related genes. TR is 451 base pairs and contains an 11 base pair template responsible for binding the telomeric overhang and acting as a template for the reverse transcription of six new bases. We have identified a patient with IPF that is heterozygous for a mutation in the TR template region which is predicted to code for a variant telomere sequence. Remarkably, whole genome sequencing from the patient demonstrated that the telomeres display a significant portion of variant telomere sequences. Strict conservation of the telomere sequence throughout 400 million years of evolution suggests that variation in the telomere sequence would not be tolerated due to blocking shelterin binding. The nature of this unexpected find will be examined in this proposal. We hypothesize that this variant telomerase template, and subsequent variant telomere addition, may cause cellular dysfunction through two independent mechanisms; through telomere shortening and/or a loss of shelterin binding. These mechanisms will be explored in the following aims. Aim 1 will examine how the addition of a variant telomere sequence affects the enzymatic activity of telomerase and resulting telomere length in cells. Aim 2 will examine the effect that the variant sequence has on genomic stability, including shelterin binding and the DNA damage response in cells. Completion of this proposal will give us new insights into how telomeric sequence degeneration can affect genomic stability and telomere maintenance.