MITOCHONDRIAL DNA POLYMERASE--MECHANISM AND STRUCTURE

描述：长期目标是阐明 动物线粒体中的DNA复制。这是可以实现的，其中 其他方法，当纯化的酶在体外重组以产生 能够启动和延长DNA合成的复制复合体。 这些研究将提供研究框架，以确定 对这些过程的调节，因为它们发生在体内。该提案将重点放在 果蝇线粒体主要复制酶DNA聚合酶的研究 伽马(波尔伽马)。生物化学与分子生物学相结合的方法 生物学正在被用来追求机械和结构功能 P125：p35异源二聚体DNA聚合酶的分析 同时和单独纯化这两个亚基，并阐明 小亚基在酶功能中的作用。其他目标是 产生改变的酶形式用于比较研究，并检查 线粒体单链DNA结合蛋白(SSB)在Pol-γ中的作用 功能。申请者还将使用转基因苍蝇模型来检查 波尔伽马功能与线粒体DNA的可能关系 复制保真度、衰老和疾病。 在正常发育期间对动物细胞繁殖的控制，以及 在癌症发展过程中失去控制，在 人类成长、衰老和疾病的过程。线粒体生物发生 与细胞增殖同步进行，但两者都不是紧密的 与线粒体DNA复制或细胞周期有关。 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 可能需要调节机制来连接线粒体DNA 复制和生物发生，到核DNA复制和细胞分裂。一个 线粒体DNA中关键酶的详细分析 复制将是对最终实现 对正常人和正常人线粒体生物发生和功能的认识 有病的组织。 最近有多种线粒体DNA疾病被记录在案。这 人们越来越多地认识到，抗病毒和抗肿瘤药物经常 影响线粒体DNA功能，特别是Pol的活性 伽马证明了深入了解这种DNA的迫切需要 聚合酶。具体目标如下：1.继续机械化 分析波尔伽玛来考察核苷酸的作用机制和保真度 聚合，以及线粒体(MtSSB)在波尔伽马功能中的作用； 2.高效表达和纯化Pol两个亚基的重组形式 在大肠杆菌和杆状病毒感染的昆虫细胞中的伽马；3.继续 波尔伽马天然、重组和重组蛋白的结构与功能研究 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，并开始 阐明该酶的分子结构；以及4.启动 建立转基因果蝇模型以检验两者之间的关系 在波尔伽马功能、线粒体DNA复制保真度、衰老之间 和疾病。

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.