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

辨别端粒酶模板突变引起的端粒功能障碍的机制

• 批准号: 10576260
• 负责人: Angela Marie Hinchie
• 金额: $ 4.68万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-05-01 至 2024-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10576260
• 关键词: A549; Affect; Affinity; Base Pairing; Binding; Biological; Cell Aging; Cell Culture Techniques; Cell Cycle Arrest; Cell Line; Cell division; Cells; Child; Chromosomes; Code; Complex; DNA; DNA Damage; Diagnostic; Disease; Electrophoretic Mobility Shift Assay; Enzymes; Evolution; Functional disorder; Genes; Genome Stability; Genomic Instability; Genomics; Goals; Health; Heterogeneity; Human; Impairment; In Vitro; LOX gene; Lead; Length; Link; Lung diseases; Measurement; Measures; Melanoma Cell; Mutation; Nature; Nucleotides; Outcome; Pathogenicity; Patients; Persons; Proteins; RNA; RNA-Directed DNA Polymerase; Reverse Transcription; Role; Stress; TERF1 gene; Telomerase; Telomerase RNA Component; Telomere Maintenance; Telomere Maintenance Gene; Telomere Shortening; Testing; United States; Untranslated RNA; Variant; Vertebrates; base; cellular transduction; chromosome fusion; genome sequencing; idiopathic pulmonary fibrosis; insight; melanoma; mutant; novel; overexpression; proband; protein complex; response; telomere; tool; whole genome

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.

项目总结/摘要 特发性肺纤维化（IPF）是一种进行性、致死性肺部疾病，与以下疾病的缺陷密切相关： 端粒维持端粒是位于真核生物DNA末端的保护性非编码DNA端帽， 染色体端粒序列TTAGGG在所有脊椎动物中都是保守的， 蛋白质复合物称为shelterin在序列依赖性的方式。掩蔽复合体的位移 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 在细胞分裂中，由于末端复制问题和基因组应激的组合，端粒末端缩短。 为了平衡这种缩短，细胞利用端粒酶。端粒酶是由一种蛋白质 端粒酶RNA组分包括端粒酶逆转录酶（TERT）组分和RNA模板，端粒酶RNA组分 （TR）。IPF中的突变通常与端粒维持所需的基因相关，例如 TERT、TERC（TR）、RTEL 1、DKC 1和相关基因。TR是451个碱基对，包含11个碱基对， 负责结合端粒突出端并作为逆转录模板的模板 六个新基地。我们发现了一名IPF患者，其TR模板突变为杂合子 预测编码变异端粒序列的区域。值得注意的是，全基因组测序 从病人身上提取的DNA证明了端粒显示了很大一部分的变异端粒序列。 在4亿年的进化过程中，端粒序列的严格保守表明， 在端粒序列中，由于阻断shelterin结合，的性质 本提案将审查意想不到的发现。我们假设这种变异的端粒酶模板， 随后的变异端粒添加，可能通过两种独立的机制引起细胞功能障碍; 通过端粒缩短和/或shelterin结合的丧失。这些机制将在 的目标。目的1将研究如何增加一个变异的端粒序列影响酶的作用。 端粒酶活性和细胞中端粒长度。目标2将检验变量 序列具有基因组稳定性，包括shelterin结合和细胞中的DNA损伤反应。 这项提案的完成将使我们对端粒序列变性如何影响 基因组稳定性和端粒维持。