Kinetics of DNA polymerase gamma upon mutation and nucleoside analog exposure

突变和核苷类似物暴露后 DNA 聚合酶 γ 的动力学

• 批准号: 8538465
• 负责人: Christal Dyane Sohl
• 金额: $ 4.69万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-01 至 2014-07-06
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8538465
• 关键词: Active Sites; Adult; Affect; Affinity; Age of Onset; Alpers' Syndrome; American; Anti-HIV Agents; Ataxia; Binding; Biological Assay; Catalytic Domain; Cause of Death; Cessation of life; Chronic progressive external ophthalmoplegia; Circular Dichroism; Clinical Trials; DNA; DNA biosynthesis; DNA polymerase gamma; DNA-Directed DNA Polymerase; Defect; Development; Disease; Disease Progression; Dissociation; Enzyme Inhibition; Enzyme Kinetics; Enzymes; Excision; Exhibits; Exposure to; HIV; HIV Infections; Holoenzymes; Human; Human Activities; In Vitro; Kinetics; Lactic Acidosis; Lead; Link; Lipodystrophy; Liver diseases; Measures; Methods; Mitochondria; Mitochondrial DNA; Mitochondrial Diseases; Modification; Molecular; Mutation; Nucleosides; Nucleotides; Onset of illness; Oxidative Phosphorylation; Patients; Pharmaceutical Preparations; Phenotype; Point Mutation; Polymerase; Proline; Ptosis; Reporting; Resistance; Reverse Transcriptase Inhibitors; Safety; Seizures; Severities; Severity of illness; Structural Models; Structure; Symptoms; Testing; Therapeutic; Toxic effect; Violence; early childhood; human DNA; human disease; mitochondrial genome; mutant; novel; nucleoside analog; oxidative damage; preclinical study; premature; repaired; stem

DESCRIPTION (provided by applicant): Mitochondrial toxicity can result from altered activity of human mitochondrial DNA polymerase 3 (DNA pol 3), the enzyme responsible for replication of the mitochondrial genome. Mitochondrial DNA deletion or depletion resulting from deficient DNA pol 3 activity has been observed in many mitochondrial diseases which affect about 60,000 Americans (1), and in HIV which currently affects an estimated one million Americans. Several known point mutations in DNA pol 3 have been linked to mitochondrial diseases such as Alpers Syndrome, which is characterized by violent seizures and liver disease and causes death in early childhod, and progressive external ophthalmoplegia, an adult onset disorder which causes ptosis and ataxia and is usualy non-fatal. Effort has been made to determine the molecular mechanism of the alteration of DNA pol 3 activity by mutation, which can stem from the modification of any of the steps in the catalytic cycle including the nucleotide incorporation efficiency, the affinity for incoming nucleotides and the DNA template, and the overall fidelity. However, kinetic characterization has not been undertaken for most mutations. For patients infected with HIV, nucleoside reverse transcriptase inhibitors (NRTIs) are a critical component of treatment, but unfortunately DNA pol 3 also can use these nucleoside analogs as substrates, resulting in chain termination during mitochondrial DNA replication. Symptoms similar to those seen in progressive external ophthalmoplegia can result, as well as lactic acidosis and lipodystrophy, and toxicity can be severe enough to require the halt of this type of treatment. Problems of toxicity and resistance highlight the demand for new NRTIs, and it is critical to test these novel drugs for DNA pol 3 activity to assess safety. This proposal seeks to characterize the kinetic mechanism of toxicity for four DNA pol 3 mutants, A957P, A957S, R1096H, and R1096C, which are associated with disease ranging in severity from mild progressive external ophthalmoplegia to Alpers Syndrome. First, biophysical methods will be used to characterize changes in the overall structure of the mutant enzymes, and steady-state and pre-steady-state kinetics will be utilized to determine the molecular mechanism of toxicity. Second, elucidation of the pre-steady-state kinetics of DNA pol 3 will be used measure the efficiency of incorporation and the rate of excision of the novel anti-HIV drug, FLT, which is known to exhibit some toxicity. Determining the kinetic mechanism of how mutation and NRTI treatment alters DNA pol 3 is a critical step in understanding the cause and progression of mitochondrial diseases, most of which currently have no cure, and for assessing the safety of NRTI drugs under development.

描述（由申请人提供）：线粒体毒性可能是由人线粒体DNA聚合酶3（DNA POL 3）的活性改变的，该酶是负责复制线粒体基因组的酶。在许多线粒体疾病中，已经观察到影响约60,000名美国人（1）的线粒体DNA POL 3活性导致的线粒体DNA缺失或耗尽，目前影响了100万美国人。 DNA POL 3中的几个已知点突变与线粒体疾病（如Alpers综合征）有关，Alpers综合征的特征是剧烈癫痫发作和肝病，并导致早期儿童的死亡，以及进行性外科眼质疾病，一种成人的发病障碍，是一种导致Ptosis and ataxia and Ataxia且ataxia且exaxia and Ataxia and as astaxia and as Praneaual is as Pranual nobaly非fatal。已经努力通过突变来确定DNA POL 3活性改变的分子机制，这可能源于催化循环中任何步骤的修改，包括核苷酸掺入效率，对传入核苷酸和DNA模板的亲和力以及整体保真度。但是，对于大多数突变，动力学表征尚未进行。对于感染HIV的患者，核苷逆转录酶抑制剂（NRTIS）是治疗的关键组成部分，但不幸的是，DNA Pol 3也可以将这些核苷类似物用作底物，从而导致线粒体DNA复制过程中的链终止。与渐进性外科治疗中所见的症状相似，可能会导致乳酸性酸中毒和脂肪营养不良，并且毒性可能足够严重，需要停止这种类型的治疗。毒性和抵抗力的问题突出了对新NRTI的需求，至关重要的是，测试这些新型药物以评估安全性的DNA POL 3活性。该提案旨在表征四个DNA Pol 3突变体的毒性动力机制，即A957p，A957S，R1096H和R1096C，它们与疾病的疾病范围从轻度进行性渐进外部外部眼科乳腺癌的隔离范围相关，到Alpers综合征。首先，将使用生物物理方法来表征突变酶整体结构的变化，并将利用稳态和稳态的稳态动力学来确定毒性的分子机制。其次，将使用DNA POL 3的稳态态动力学的阐明，以测量掺入的效率和新型抗HIV药物FLT的切除率，该抗HIV药物FLT已知表现出一些毒性。确定突变和NRTI治疗如何改变DNA POL 3的动力学机制是理解线粒体疾病的原因和进展的关键步骤，线粒体疾病目前尚无治愈方法以及评估正在开发的NRTI药物的安全性。