MOLECULAR CHARACTERIZATION OF THE MITOCHONDRIAL DNA POLYMERASE

线粒体 DNA 聚合酶的分子表征

• 批准号: 6290046
• 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/6290046
• 关键词: DNA directed DNA polymerase; enzyme activity; gene mutation; human genetic material tag; isozymes; mitochondria; mitochondrial DNA; tissue /cell culture

Summary of Work: Human mitochondrial DNA evolves faster and is more prone to oxidative damage than is nuclear DNA. Mutations in mitochondrial DNA causes a wide range of mitochondrial dysfunctional diseases affecting some 50 million people in the US. These mutations may occur during replication by the DNA polymerase gamma. AZT and dideoxynucleotides produce mitochondrial toxicity by inhibition of the DNA polymerase gamma. How the mitochondrial DNA polymerase makes mutations and what structural properties set this polymerase apart from the nuclear DNA polymerases to give rise to its inhibition patterns are poorly understood. To address these questions we previously cloned the human DNA polymerase gamma cDNA. Using our overproduced human DNA polymerase gamma we have discovered that the polymerase can perform translesion synthesis past cisplatinin adducts. We purified the native human DNA polymerase complex and identified the smaller subunit to be 55 kDa. The cDNA for the Hp55 was isolated and overexpressed in E. coli and protein purified to homogeneity. The Hp55 accessory protein stimulates DNA polymerase activity, confers highly processive DNA synthesis to DNA polymerase gamma by providing greater affinity to DNA, binds DNA with tight affinity in the absence of catalytic polymerase subunit, and protects DNA polymerase gamma from N-ethylmaleimide inhibition. The fidelity of DNA synthesis of the wild type and exonuclease deficient DNA polymerase was determined and we find that the catalytic subunit has a large propensity to make errors in homopolymeric runs. We have isolated a new gene in yeast that regulates mitochondrial copper levels. Deletion of this gene results in slow growth, increased sensitivity to copper, and a dramatic increase in the rate of loss of mitochondrial function. This gene is highly conserved, and a human homolog has been identified. Mutation of the human gene could result in a phenotype similar to other diseases with defective mitochondrial copper regulation, e.g. Friedreichs ataxia or Wilsons disease. - Catalysis, Gene Library, Hominidae, Nucleotide Mapping, Point Mutation, Saccharomyces Cerevisiae

工作摘要：与核DNA相比，人线粒体DNA的演变更快，更容易受到氧化损害。线粒体DNA中的突变会导致各种线粒体功能障碍疾病影响美国约5000万人。这些突变可能在通过DNA聚合酶γ复制过程中发生。 AZT和二乙氧基核苷酸通过抑制DNA聚合酶伽马产生线粒体毒性。线粒体DNA聚合酶如何产生突变，以及哪些结构特性使该聚合酶与核DNA聚合酶不同以产生其抑制模式。为了解决这些问题，我们以前克隆了人DNA聚合酶γcDNA。使用我们生产过多的人DNA聚合酶伽玛，我们发现聚合酶可以在顺铂加合物上进行跨性质合成。我们纯化了人类DNA聚合酶复合物，并确定较小的亚基为55 kDa。将HP55的cDNA分离并过表达大肠杆菌，并纯化为均匀性。 HP55辅助蛋白刺激DNA聚合酶活性，通过提供对DNA的更高亲和力，在没有催化性聚合酶亚基的情况下与DNA相关的DNA含量更高，并在没有催化性聚合酶亚基中结合DNA，并保护DNA聚合酶GAMMA，并保护DNA聚合酶Gamma从N-乙基甲基甲米底氨基倍氨基倍率中进行保护。确定了野生型和核酸外切酶缺陷DNA聚合酶的DNA合成的保真度，我们发现催化亚基具有很大的倾向是在均聚物运行中犯错。我们已经在酵母中分离了一个新基因，该基因调节线粒体铜水平。该基因的缺失会导致生长缓慢，对铜的敏感性增加以及线粒体功能损失率的显着增加。该基因是高度保守的，并且已经鉴定出人类同源物。人类基因的突变可能导致与其他有缺陷的线粒体铜调节的疾病相似的表型，例如弗里德里希共济失调或威尔逊氏病。 - 催化，基因文库，hominidae，核苷酸映射，点突变，酿酒酵母