Molecular Characterization Of The Mitochondrial Dna Polymerase

线粒体 DNA 聚合酶的分子表征

• 批准号: 8336584
• 负责人: William Copeland
• 金额: $ 172.13万
• 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/8336584
• 关键词: 5' -deoxyribose phosphate lyase; Active Sites; Address; Affect; Affinity; Age; Age of Onset; Aging; Alpers' Syndrome; Amino Acid Substitution; Animals; Apoptosis; Area; Ataxia; Base Excision Repairs; Behavior; Binding; Biochemical; Bypass; C-terminal; Catalytic Domain; Cells; Child; Chronic progressive external ophthalmoplegia; Complex; DNA; DNA Binding; DNA Maintenance; DNA Primers; DNA Repair; DNA Sequence Rearrangement; DNA biosynthesis; DNA polymerase gamma; DNA-Directed DNA Polymerase; Data; Defect; Deoxyribonucleotides; Diabetes Mellitus; Dimerization; Discrimination; Disease; Drops; Dysarthria; Enzymes; Epilepsy; Exclusion; Frequencies; Gene Proteins; General Population; Genes; Genetic; Genomic Instability; Goals; Guanosine Triphosphate; Hereditary Disease; Human; Individual; Inherited; Intervention; Lead; Link; Maps; Mediating; Metabolic Diseases; Microcephaly; Mitochondria; Mitochondrial DNA; Mitochondrial Diseases; Molecular; Muscle hypotonia; Mutagenesis; Mutation; Myopathy; Neuropathy; Nuclear; Nucleotides; Ophthalmopareses; Parkinsonian Disorders; Pathogenesis; Pathogenicity; Pathology; Patients; Phosphodiesterase I; Physicians; Point Mutation; Polymerase; Premature Menopause; Premature aging syndrome; Prevalence; Prevention; Production; Proteins; RNA; RNA primers; RNA-Directed DNA Polymerase; Reaction; Recombinant Proteins; Recombinants; Reporting; Reverse Transcription; Ribonucleotides; Ribose; Risk; Role; Sensory; Side; Stretching; Stroke; Symptoms; Time; Variant; Work; base; deoxyguanosine triphosphate; deoxyribonucleoside triphosphate; dimer; helicase; human DNA; human MPP1 protein; hydroxyl group; mitochondrial genome; mutant; neuromuscular; novel; oxidative damage; repaired

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 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 nearly 200 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). Presently, there are over 150 pathogenic disease mutations in the POLG gene that cause PEO, ataxia-neuropathy and Alpers syndrome. The c.1550 g→t mutation in the POLG gene causing the G517V substitution has been reported by many groups to be associated with a variety of mitochondrial diseases, including autosomal dominant and recessive forms of ataxia neuropathy, myopathy and microcephaly, progressive external ophthalmoplegia, diabetes, strokes, hypotonia, and epilepsy. However, the variable disease presentation and age of onset raises suspicion of its pathogenicity. Because of the varied reported associated symptoms and request from physicians to address the consequence of this mutation, we have carried out the biochemical analysis of the purified recombinant human DNA polymerase γ protein harboring the G517V substitution. These analyses revealed that the G517V mutant enzyme retained 80-90% of wild-type DNA polymerase activity, in addition to its functional interaction with the p55 accessory subunit. DNA binding by the mutant was also only slightly lower than the wild-type enzyme. Our data suggest that the G517V mutation by itself in pol γ most likely does not have a role in mitochondrial disorders. Defects in mitochondrial DNA (mtDNA) maintenance comprise an expanding repertoire of polymorphic diseases caused, in part, by mutations in the genes encoding the p140 mtDNA polymerase (POLG), its p55 accessory subunit (POLG2) or the mtDNA helicase (C10orf2). In an exploration of nuclear genes for mtDNA maintenance linked to mitochondrial disease, 8 heterozygous mutations (6 novel) in POLG2 were identified in 1 control and 8 patients with POLG-related mitochondrial disease that lacked POLG mutations. Of these eight mutations we biochemically characterized 7 variants (c.307G>A (G103S); c.457C>G (L153V); c.614C>G (P205R); c.1105A>G (R369G); c.1158T>G (D386E); c.1268C>A (S423Y); c.1423_1424delTT (L475DfsX2)) that were previously uncharacterized along with the wild-type protein and the G451E pathogenic variant. These seven mutations encode amino acid substitutions that map throughout the protein, including the p55 dimer interface and the C-terminal domain that interacts with the catalytic subunit. Recombinant proteins harboring these alterations were assessed for stimulation of processive DNA synthesis, binding to the p140 catalytic subunit, binding to dsDNA, and self-dimerization. Whereas the G103S, L153V, D386E and S423Y proteins displayed wild-type behavior, the P205R and R369G p55 variants had reduced stimulation of processivity and decreased affinity for the catalytic subunit. Additionally, the L475DfsX2 variant, which possesses a C-terminal truncation, was unable to bind the p140 catalytic subunit, unable to bind dsDNA and formed aberrant oligomeric complexes. Our biochemical analysis helps to explain the pathogenesis of POLG2 mutations in mitochondrial disease and emphasizes the need to quantitatively characterize the biochemical consequences of newly discovered mutations before classifying them as pathogenic. The presence of RNA in mitochondrial DNA has been known for several decades but the origin has been a topic of controversy. The existence of RNA in the mitochondrial genome provides opportunities for genomic instability, specifically mediating breaks in DNA, which can lead to deletions. Deletions in mitochondrial DNA are associated with several mitochondrial diseases as well as aging. During DNA synthesis, DNA polymerases must select against ribonucleotides, present at much higher levels compared to deoxyribonucleotides. Most DNA polymerases are equipped to exclude ribonucleotides from its active site through a bulky side chain residue that can sterically block the 2-hydroxyl group of the ribose ring. However, many nuclear replicative and repair DNA polymerases incorporate ribonucleotides into DNA, suggesting that the exclusion mechanism is not perfect. In this study, we show that the human mitochondrial DNA polymerase γ discriminates ribonucleotides efficiently but differentially based on the base identity. While, UTP is discriminated by 77,000-fold compared to dTTP, the discrimination drops to 1,100-fold for GTP versus dGTP. In addition, the efficiency of the enzyme reduced 3-14-fold depending on the identity of the incoming nucleotide when it extends from a primer containing a 3-terminal ribonucleotide. DNA polymerase γ is also proficient in performing single nucleotide reverse transcription reactions from both DNA and RNA primer terminus, although, its bypass efficiency is significantly diminished with increasing stretches of ribonucleotides in template DNA. Furthermore, we show that the E895A mutant enzyme is compromised in its ability to discriminate ribonucleotides mainly due to its defects in deoxyribonucleoside triphosphate binding and is also a poor reverse transcriptase.

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。一半的受影响者是在 5 岁之前出现症状的儿童，其中大约 80% 将在 20 岁之前死亡。线粒体和代谢疾病给人类带来巨大痛苦，但需要做大量工作来了解这些疾病的遗传和环境原因。疾病。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 200 种致病性突变，可导致多种疾病，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)。 目前，POLG基因中有超过150种致病突变，可导致PEO、共济失调神经病和阿尔珀斯综合征。 许多团体报告 POLG 基因中的 c.1550 g→t 突变导致 G517V 取代，该突变与多种线粒体疾病有关，包括常染色体显性和隐性形式的共济失调神经病、肌病和小头畸形、进行性外眼肌麻痹、糖尿病、中风、肌张力低下和癫痫。 然而，不同的疾病表现和发病年龄引起了对其致病性的怀疑。 由于相关症状的报道多种多样，并且医生要求解决该突变的后果，我们对含有 G517V 取代的纯化重组人 DNA 聚合酶 γ 蛋白进行了生化分析。 这些分析表明，G517V 突变酶除了与 p55 辅助亚基的功能性相互作用外，还保留了 80-90% 的野生型 DNA 聚合酶活性。 突变体的 DNA 结合也仅略低于野生型酶。 我们的数据表明，pol γ 中的 G517V 突变本身很可能在线粒体疾病中没有作用。 线粒体 DNA (mtDNA) 维护缺陷包括不断扩大的多态性疾病，部分原因是编码 p140 mtDNA 聚合酶 (POLG)、其 p55 辅助亚基 (POLG2) 或 mtDNA 解旋酶 (C10orf2) 的基因突变所致。 在对与线粒体疾病相关的 mtDNA 维持的核基因的探索中，在 1 名对照者和 8 名患有 POLG 相关线粒体疾病且缺乏 POLG 突变的患者中发现了 POLG2 的 8 种杂合突变（6 种新突变）。 在这八个突变中，我们对 7 个变体进行了生化鉴定（c.307G>A (G103S)；c.457C>G (L153V)；c.614C>G (P205R)；c.1105A>G (R369G)；c.1158T> G (D386E);c.1268C>A (S423Y); c.1423_1424delTT (L475DfsX2))，之前与野生型蛋白和 G451E 致病性变异一起未被表征。 这七个突变编码映射整个蛋白质的氨基酸取代，包括 p55 二聚体界面和与催化亚基相互作用的 C 端结构域。 评估含有这些改变的重组蛋白对持续 DNA 合成的刺激、与 p140 催化亚基的结合、与 dsDNA 的结合以及自二聚化。 尽管 G103S、L153V、D386E 和 S423Y 蛋白表现出野生型行为，但 P205R 和 R369G p55 变体对持续合成能力的刺激减少，并且对催化亚基的亲和力降低。 此外，具有C端截短的L475DfsX2变体无法结合p140催化亚基，无法结合dsDNA并形成异常的寡聚复合物。 我们的生化分析有助于解释线粒体疾病中 POLG2 突变的发病机制，并强调在将新发现的突变分类为致病性之前需要定量表征新发现的突变的生化后果。 几十年来人们就知道线粒体 DNA 中存在 RNA，但其起源一直是一个有争议的话题。 线粒体基因组中 RNA 的存在为基因组不稳定提供了机会，特别是介导 DNA 断裂，从而导致缺失。 线粒体 DNA 缺失与多种线粒体疾病以及衰老有关。在 DNA 合成过程中，DNA 聚合酶必须选择核糖核苷酸，与脱氧核糖核苷酸相比，核糖核苷酸的含量要高得多。 大多数 DNA 聚合酶都能够通过一个庞大的侧链残基将核糖核苷酸排除在其活性位点之外，该侧链残基可以在空间上阻断核糖环的 2-羟基。 然而，许多核复制和修复DNA聚合酶将核糖核苷酸整合到DNA中，这表明排除机制并不完美。 在这项研究中，我们证明人类线粒体 DNA 聚合酶 γ 能够有效地区分核糖核苷酸，但根据碱基身份进行差异化。 与 dTTP 相比，UTP 的歧视性高出 77,000 倍，而 GTP 与 dGTP 的歧视性则下降至 1,100 倍。 此外，当酶从含有 3 末端核糖核苷酸的引物延伸时，酶的效率会降低 3-14 倍，具体取决于引入核苷酸的身份。 DNA 聚合酶 γ 还擅长从 DNA 和 RNA 引物末端进行单核苷酸逆转录反应，但其旁路效率会随着模板 DNA 中核糖核苷酸延伸的增加而显着降低。 此外，我们表明，E895A 突变酶区分核糖核苷酸的能力受到损害，主要是由于其脱氧核糖核苷三磷酸结合的缺陷，并且也是一种较差的逆转录酶。