MITOCHONDRIAL DNA POLYMERASE--MECHANISM AND STRUCTURE

线粒体 DNA 聚合酶——机制和结构

• 批准号: 2838575
• 负责人: LAURIE SIMON KAGUNI
• 金额: $ 18.78万
• 依托单位: MICHIGAN STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-07-01 至 2000-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2838575
• 关键词: DNA binding protein; DNA directed DNA polymerase; DNA footprinting; DNA replication; Drosophilidae; enzyme mechanism; enzyme reconstitution; enzyme structure; enzyme substrate; gel mobility shift assay; genetically modified animals; immunoprecipitation; laboratory rabbit; mitochondria; mitochondrial DNA; mitochondrial membrane; nucleic acid sequence; polymerase chain reaction; polymerization; protein sequence; protein structure function; site directed mutagenesis; western blottings

DESCRIPTION: The long term objective is the elucidation of mechanisms of DNA replication in animal mitochondria. This may be accomplished, among other ways, when purified enzymes are reconstituted in vitro to yield replication complexes capable of initiation and elongation of DNA synthesis. The studies will provide the framework for research to determine the regulation of these processes, as they occur in vivo. The proposal focuses on the major replicative enzyme in Drosophila mitochondria, DNA Polymerase gamma (Pol gamma). A combined approach of biochemistry and molecular biology is being employed, to pursue mechanistic and structure-function analyses of this p125:p35 heterodimeric DNA polymerase, to overexpress and purify the two subunits both together and individually, and to elucidate the role of the small subunit in enzyme function. Additional goals are to generate altered enzyme forms for comparative studies, and to examine the role of mitochondrial single-stranded DNA-binding protein (SSB) in Pol gamma function. The applicant will also use a transgenic fly model to examine possible relationships between Pol gamma function, mitochondrial DNA replication fidelity, aging, and disease. The control of animal cell reproduction during normal development, and the loss of control during cancerous development, is of central importance in the processes of human growth, aging and disease. Mitochondrial biogenesis proceeds in parallel with cell proliferation, but it is neither tightly coupled to mitochondrial DNA replication nor to the cell cycle. Nevertheless, because both the DNA content of the mitochondrion and the number of mitochondria in cell remain relatively constant, specific regulatory mechanisms are likely required to couple mitochondrial DNA replication and biogenesis, to nuclear DNA replication and cell division. A detailed analysis of the key enzyme involved in mitochondrial DNA replication will represent a major contribution toward an eventual understanding of mitochondrial biogenesis and function in normal and diseased tissues. A variety of mitochondrial DNA diseases have recently been documented. This and an increased recognition that antiviral and antitumor drugs frequently affect mitochondrial DNA function, and in particular, the activity of Pol gamma demonstrate a critical need for an in-depth understanding of this DNA polymerase. The Specific Aims as follows: 1. To continue mechanistic analysis of Pol gamma to examine the mechanisms and fidelity of nucleotide polymerization, and the role of mitochondrial (mtSSB) in Pol gamma function; 2. To overexpress and purify recombinant forms of the two subunits of Pol gamma in E. coli and in bacculovirus-infected insect cells; 3. To continue structure-function studies of Pol gamma involving native, recombinant and altered forms, to elucidate the role of the small subunit in enzyme function, to dissect functional domains in the catalytic subunit, and begin to elucidate the molecular architecture of the enzyme; and 4. To initiate the development of a transgenic fly model to examine the relationship between Pol gamma function, mitochondrial DNA replication fidelity, aging and disease.

描述：长期目标是阐明机制 动物线粒体中的DNA复制。 这可能是在 其他方式，当纯化的酶在体外重组以产生 能够启动和伸长DNA合成的复制复合物。 研究将为研究框架提供确定的框架 这些过程的调节，因为它们在体内发生。 该提案重点 在果蝇线粒体中的主要复制酶上，DNA聚合酶 伽玛（Pol Gamma）。 生物化学和分子的组合方法 正在采用生物学，追求机械和结构功能 该p125的分析：p35异二聚体DNA聚合酶，过表达和 将两个亚基一起纯化在一起，并单独净化，并阐明 小亚基在酶功能中的作用。 其他目标是 产生改变的酶形式进行比较研究，并检查 线粒体单链DNA结合蛋白（SSB）在POL GAMMA中的作用 功能。 申请人还将使用转基因飞行模型检查 POL GAMMA功能，线粒体DNA之间的可能关系 复制保真度，衰老和疾病。 在正常发育过程中控制动物细胞繁殖， 癌性发育过程中失去控制的丧失是至关重要的 人类生长，衰老和疾病的过程。 线粒体生物发生 与细胞增殖并行进行，但既不紧密 与线粒体DNA复制或细胞周期耦合。 然而，因为线粒体的DNA含量和 细胞中线粒体的数量保持相对恒定，特异性 可能需要采用线粒体DNA的调节机制 复制和生物发生，核DNA复制和细胞分裂。 一个 对线粒体DNA涉及的关键酶的详细分析 复制将代表最终的重大贡献 了解线粒体生物发生和正常和功能 患病的组织。 最近已经记录了各种线粒体DNA疾病。 这 并越来越多地认识到抗病毒和抗肿瘤药物经常 影响线粒体DNA功能，尤其是POL的活性 伽玛表明对该DNA的深入了解的迫切需要 聚合酶。 具体目的如下：1。继续机械 POL伽马的分析以检查核苷酸的机制和保真度 聚合和线粒体（MTSSB）在POL GAMMA功能中的作用； 2。过表达和净化POL的两个亚基的重组形式 大肠杆菌中的伽马和收culovirus感染的昆虫细胞中的伽马； 3。继续 涉及天然，重组和重组的POL伽马的结构功能研究 改变形式，以阐明小亚基在酶中的作用 功能，剖析催化亚基中的功能域，然后开始 阐明酶的分子结构；和4。发起 转基因飞蝇模型的开发以检查关系 在Pol Gamma功能，线粒体DNA复制保真度，老化之间 和疾病。