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

基本信息

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
项目类别：
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&apos 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

项目摘要

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于人线粒体 DNA 聚合酶 3 (DNA pol 3) 活性改变造成的，该酶负责线粒体基因组的复制。在影响约 60,000 名美国人的许多线粒体疾病中以及目前影响约 100 万美国人的 HIV 中，已观察到因 DNA pol 3 活性缺陷而导致的线粒体 DNA 缺失或耗竭。 DNA pol 3 中的几个已知点突变与线粒体疾病有关，例如阿尔珀斯综合征（其特征是剧烈癫痫发作和肝病，导致儿童早期死亡）和进行性眼外肌麻痹（一种成人发病的疾病，导致上睑下垂和共济失调）通常是非致命的。人们已努力确定通过突变改变 DNA pol 3 活性的分子机制，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、对引入核苷酸的亲和力和 DNA 模板，以及整体保真度。然而，大多数突变尚未进行动力学表征。对于感染 HIV 的患者，核苷逆转录酶抑制剂 (NRTI) 是治疗的关键组成部分，但不幸的是 DNA pol 3 也可以使用这些核苷类似物作为底物，导致线粒体 DNA 复制过程中的链终止。可能会导致类似于进行性外眼肌麻痹的症状，以及乳酸性酸中毒和脂肪营养不良，并且毒性可能严重到需要停止此类治疗。毒性和耐药性问题凸显了对新型 NRTI 的需求，测试这些新型药物的 DNA pol 3 活性以评估安全性至关重要。该提案旨在描述四种 DNA pol 3 突变体 A957P、A957S、R1096H 和 R1096C 的毒性动力学机制，这些突变体与从轻度进行性外眼肌麻痹到阿尔珀斯综合征等严重程度的疾病相关。首先，将使用生物物理方法来表征突变酶整体结构的变化，并利用稳态和前稳态动力学来确定毒性的分子机制。其次，对 DNA pol 3 前稳态动力学的阐明将用于测量新型抗 HIV 药物 FLT 的掺入效率和切除率，已知 FLT 具有一定的毒性。确定突变和 NRTI 治疗如何改变 DNA pol 3 的动力学机制是了解线粒体疾病（其中大多数疾病目前无法治愈）的原因和进展以及评估正在开发的 NRTI 药物安全性的关键一步。