Molecular Characterization Of The Mitochondrial Dna Poly

线粒体 DNA 聚的分子表征

• 批准号: 7328482
• 负责人: William Copeland
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF ENVIRONMENTAL HEALTH SCIENCES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7328482
• 关键词: Molecular; Characterization; Mitochondrial; Dna; Poly

Mitochondrial diseases are devastating disorders for which there is no cure and no proven treatment. About 1 in 2000 individuals are at risk of developing a mitochondrial disease sometime in their lifetime. Half of those affected are children who show symptoms before age 5 and approximately 80% of these will die before age 20. The mortality rate is roughly that of cancer. The human suffering imposed by mitochondrial and metabolic diseases is enormous, yet much work is needed to understand the genetic and environmental causes of these diseases. Mitochondrial genetic diseases are characterized by alterations in the mitochondrial genome, as point mutations, deletions, rearrangements, or depletion of the mitochondrial DNA (mtDNA). The mutation rate of the mitochondrial genome is 10-20 times greater than of nuclear DNA, and mtDNA is more prone to oxidative damage than is nuclear DNA. Mutations in human mtDNA cause premature aging, severe neuromuscular pathologies and maternally inherited metabolic diseases, and influence apoptosis. The primary goal of this project is to understand the contribution of the replication apparatus in the production and prevention of mutations in mtDNA. Since the genetic stability of mitochondrial DNA depends on the accuracy of DNA polymerase gamma (pol gamma), we have focused this project on understanding the role of the human pol gamma in mtDNA mutagenesis. Human mitochondrial DNA is replicated by the two-subunit gamma, composed of a 140 kDa subunit containing catalytic activity and a 55 kDa accessory subunit. The catalytic subunit contains DNA polymerase activity, 3'-5' exonuclease proofreading activity, and 5'dRP lyase activity required for base excision repair. As the only DNA polymerase in animal cell mitochondria, pol gamma participates in DNA replication and DNA repair. The 140 kDa catalytic subunit for pol gamma is encoded by the nuclear POLG gene. To date there are nearly 90 pathogenic mutations in POLG that cause a wide spectrum of disease including Progressive external ophthalmoplegia (PEO), parkinsonism, premature menopause, Alpers syndrome, mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) or sensory ataxic neuropathy, dysarthria, and ophthalmoparesis (SANDO). PEO is a mitochondrial disorder associated with mtDNA deletions and point mutations. PEO is characterized by late onset (between 18 and 40 years of age) bilateral ptosis and progressive weakening of the external eye muscle, resulting in blepharoptosis and ophthalmoparesis, proximal muscle weakness and wasting as well as exercise intolerance. The disease is often accompanied by cataract, hypogonadism, dysphagia, hearing loss and may, within several years, lead to development of neuromuscular problems. Neurological problems may include depression or avoidant personality. Skeletal muscles of PEO patients present red ragged fibers and lowered activity of respiratory chain enzymes. PEO can be inherited in an autosomal dominant or recessive manner. Alpers syndrome is an autosomal recessive mitochondrial DNA depletion disorder that affects children and young adults. It is a devastating disease characterized by psychomotor retardation, hepatic failure and intractable seizures, as well as tissue-specific mtDNA depletion. Alpers syndrome is 100% fatal with no cure available. This syndrome has been exclusively associated with mutations in POLG. Carriers for this disease are 1:250 with presentation occurring 1:100,000 to 250,000. We and others have identified over 30 pathogenic POLG mutations that cause Alpers in over 45 probands. Ataxia/neuropathy resulting from mutations in POLG is an autosomal recessive disorder affecting patients in their mid-teens to later years usually resulting in premature death. The disease is accompanied mainly by mtDNA deletions. The ataxia usually occurs in combination with various central nervous system features including myoclonus, epilepsy, cognitive decline, nystagmus, dysarthria, thalamic and cerebellar white matter lesions on MRI, and evidence of neuronal loss in discrete gray nuclei. Presently, there are nearly 90 pathogenic disease mutations in the POLG gene that cause PEO, ataxia-neuropathy and Alpers syndrome. To rapidly characterize the effects of these mutations, we have developed a versatile system that enables the consequences of homologous mutations, introduced in situ into the yeast mtDNA polymerase gene MIP1, to be evaluated in vivo in haploid and diploid cells. Overall, distinct phenotypes for expression of each of the mip1-PEO mutations were observed, including respiration-defective cells with decreased viability, dominant-negative mutant polymerases, elevated levels of mitochondrial and nuclear DNA damage and chromosomal mutations. Mutations in the polymerase domain caused the most severe phenotype accompanied by loss of mtDNA and cell viability, whereas the mutation in the exonuclease domain showed mild dominance with loss of mtDNA. Interestingly, the linker region mutation caused elevated mitochondrial and nuclear DNA damage. The cellular processes contributing to these observations in the mutant yeast cells are potentially relevant to understanding the pathologies observed in human mitochondrial disease patients. We have also identified a new genetic locus in progressive external ophthalmoplegia and biochemically characterized the defect.. We describe a heterozygous dominant mutation (c.1352GrA/p.G451E) in POLG2, the gene encoding the p55 accessory subunit of pol gamma, that causes progressive external ophthalmoplegia with multiple mtDNA deletions and cytochrome c oxidase (COX)?deficient muscle fibers. Biochemical characterization of purified, recombinant G451E-substituted p55 protein in vitro revealed incomplete stimulation of the catalytic subunit due to compromised subunit interaction. Although G451E p55 retains a wild-type ability to bind DNA, it fails to enhance the DNA-binding strength of the p140-p55 complex. In vivo, the disease most likely arises through haplotype insufficiency or heterodimerization of the mutated and wild-type proteins, which promote mtDNA deletions by stalling the DNA replication fork. The progressive accumulation of mtDNA deletions causes COX deficiency in muscle fibers and results in the clinical phenotype. Mitochondrial mutational spectra in human cells, tissues and derived tumors for bp 10,030?10,130 are essentially identical, suggesting a predominant mutagenic role for endogenous processes. We hypothesized that errors mediated by mitochondrial DNA polymerase gamma were the primary sources of mutations. Point mutations created in this sequence by human DNA pol gamma in vitro were thus compared to the eighteen mutational hotspots, all single base substitutions, previously found in human tissues. The set of concordant hotspots accounted for 83% of these in vivo mutational events. About half of these mutations are insensitive to prolonged heating of DNA during PCR and half increase proportionally with heating time at 98◦C. Primary misincorporation errors and miscopying errors past thermal denaturing products such as deaminated cytosines (uracils) thus appear to be of approximately equal importance. For the sequence studied, these data support the conclusion that, endogenous error mediated by DNA pol gamma constitutes the primary source of mitochondrial point mutations in human tissues .

线粒体疾病是毁灭性的疾病，无法治愈，也没有经过证实的治疗方法。大约每 2000 人中就有 1 人在一生中的某个时候面临罹患线粒体疾病的风险。受影响的人中有一半是在 5 岁之前出现症状的儿童，其中大约 80% 将在 20 岁之前死亡。死亡率与癌症大致相同。线粒体和代谢疾病给人类带来了巨大的痛苦，但还需要做大量工作来了解这些疾病的遗传和环境原因。线粒体遗传病的特征是线粒体基因组的改变，如线粒体 DNA (mtDNA) 的点突变、缺失、重排或耗竭。线粒体基因组的突变率是核DNA的10-20倍，并且线粒体DNA比核DNA更容易受到氧化损伤。人类线粒体 DNA 突变会导致过早衰老、严重的神经肌肉病变和母系遗传代谢疾病，并影响细胞凋亡。 该项目的主要目标是了解复制装置在 mtDNA 突变的产生和预防中的贡献。由于线粒体 DNA 的遗传稳定性取决于 DNA 聚合酶 gamma (pol gamma) 的准确性，因此我们将本项目的重点放在了解人类 pol gamma 在 mtDNA 诱变中的作用。人类线粒体 DNA 通过双亚基 γ 进行复制，该 γ 亚基由一个具有催化活性的 140 kDa 亚基和一个 55 kDa 辅助亚基组成。催化亚基包含 DNA 聚合酶活性、3'-5' 核酸外切酶校对活性和碱基切除修复所需的 5'dRP 裂合酶活性。 pol gamma作为动物细胞线粒体中唯一的DNA聚合酶，参与DNA复制和DNA修复。 pol gamma 的 140 kDa 催化亚基由核 POLG 基因编码。迄今为止，POLG 中有近 90 种致病性突变，可引起多种疾病，包括进行性外眼肌麻痹 (PEO)、帕金森病、过早绝经、阿尔珀斯综合征、线粒体神经胃肠脑肌病 (MNGIE) 或感觉性共济失调神经病、构音障碍和眼肌轻瘫 (SANDO) ）。 PEO 是一种与 mtDNA 缺失和点突变相关的线粒体疾病。 PEO 的特点是晚发（18 至 40 岁之间）双侧上睑下垂和外眼肌进行性衰弱，导致上睑下垂和眼肌麻痹、近端肌肉无力和消瘦以及运动不耐受。该疾病通常伴有白内障、性腺功能减退、吞咽困难、听力损失，并可能在几年内导致神经肌肉问题的发展。神经系统问题可能包括抑郁或回避型人格。 PEO 患者的骨骼肌呈现红色参差不齐的纤维，呼吸链酶活性降低。 PEO 可以以常染色体显性或隐性方式遗传。 阿尔珀斯综合征是一种常染色体隐性遗传线粒体 DNA 缺失疾病，影响儿童和年轻人。这是一种毁灭性的疾病，其特征是精神运动迟缓、肝功能衰竭和顽固性癫痫发作，以及组织特异性 mtDNA 耗竭。阿尔珀斯综合征 100% 致命，无法治愈。该综合征仅与 POLG 突变相关。这种疾病的携带者比例为 1:250，发病比例为 1:100,000 至 250,000。我们和其他人已经在超过 45 个先证者中发现了超过 30 种导致 Alpers 的致病性 POLG 突变。 POLG 突变导致的共济失调/神经病是一种常染色体隐性遗传疾病，影响青少年中期至晚年的患者，通常会导致过早死亡。该病主要伴有线粒体DNA缺失。共济失调通常与各种中枢神经系统特征同时发生，包括肌阵挛、癫痫、认知能力下降、眼球震颤、构音障碍、MRI 上丘脑和小脑白质病变以及离散灰核神经元损失的证据。 目前，POLG基因中有近90种致病突变，可导致PEO、共济失调神经病和阿尔珀斯综合征。为了快速表征这些突变的影响，我们开发了一种多功能系统，能够在单倍体和二倍体细胞中对原位引入酵母线粒体 DNA 聚合酶基因 MIP1 的同源突变的后果进行评估。总体而言，观察到每种 mip1-PEO 突变表达的不同表型，包括活力下降的呼吸缺陷细胞、显性失活突变聚合酶、线粒体和核 DNA 损伤和染色体突变水平升高。聚合酶结构域的突变引起最严重的表型，并伴有 mtDNA 和细胞活力的丧失，而外切核酸酶结构域的突变则表现出轻微的优势，并伴有 mtDNA 的丧失。有趣的是，连接区突变导致线粒体和核 DNA 损伤加剧。在突变酵母细胞中促成这些观察的细胞过程可能与理解在人类线粒体疾病患者中观察到的病理学相关。 我们还在进行性外眼肌麻痹中发现了一个新的基因位点，并对这种缺陷进行了生化表征。我们描述了 POLG2 中的杂合显性突变 (c.1352GrA/p.G451E)，该基因编码 pol gamma 的 p55 辅助亚基，导致进行性眼外肌麻痹，伴有多个 mtDNA 缺失和细胞色素 C 氧化酶 (COX) 缺乏肌纤维。纯化的重组 G451E 取代的 p55 蛋白的体外生化特征表明，由于亚基相互作用受损，催化亚基的刺激不完全。尽管G451E p55保留了野生型结合DNA的能力，但它无法增强p140-p55复合物的DNA结合强度。在体内，这种疾病很可能是由于突变型和野生型蛋白的单倍型不足或异二聚化引起的，这些蛋白通过阻止 DNA 复制叉来促进 mtDNA 缺失。 mtDNA 缺失的逐渐积累导致肌纤维中 COX 缺乏，并导致临床表型。 人类细胞、组织和衍生肿瘤中 bp 10,030?10,130 的线粒体突变谱基本上相同，表明内源过程具有主要的诱变作用。我们假设由线粒体 DNA 聚合酶 γ 介导的错误是突变的主要来源。因此，将人类 DNA pol gamma 在体外在此序列中产生的点突变与先前在人体组织中发现的 18 个突变热点（所有单碱基替换）进行比较。这组一致热点占这些体内突变事件的 83%。大约一半的突变对 PCR 过程中 DNA 的长时间加热不敏感，一半突变随着 98°C 的加热时间成比例地增加。因此，原发性错误掺入错误和热变性产物（例如脱氨基胞嘧啶（尿嘧啶））的错误复制错误似乎具有大致相同的重要性。对于所研究的序列，这些数据支持这样的结论：DNA pol gamma介导的内源性错误构成了人体组织中线粒体点突变的主要来源。