MITOCHONDRIAL DNA POLYMERASE--MECHANISM AND STRUCTURE

线粒体 DNA 聚合酶——机制和结构

• 批准号: 3304711
• 负责人: LAURIE SIMON KAGUNI
• 金额: $ 14.15万
• 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 1995-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3304711
• 关键词: DNA directed DNA polymerase; DNA directed RNA polymerase; DNA primase; DNA replication; Drosophilidae; SDS polyacrylamide gel electrophoresis; chemical kinetics; deoxyribonucleoside triphosphate; enzyme linked immunosorbent assay; enzyme mechanism; enzyme reconstitution; enzyme structure; enzyme substrate; epitope mapping; immunoprecipitation; ionic strengths; laboratory mouse; laboratory rabbit; mitochondria; mitochondrial DNA; mitochondrial membrane; molecular cloning; monoclonal antibody; nucleic acid sequence; polymerization; protein purification; thioredoxin; western blottings

The long term objective of the proposed work is the elucidation of the mechanism of DNA replication in animal mitochondria. This may be accomplished 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 current proposal focuses on the major replicative enzyme in mitochondria, DNA polymerase gamma. A combined approach involving biochemical, immunological and molecular biological methods will be employed to undertake mechanistic and structure-function analysis of the mitochonchial DNA polymerase and to done and overproduce its two subunits. Secondary goals involve isolation of a mitochondrial RNA polymerase / DNA primase and examination of the co-ordination of RNA priming and DNA synthesis on mitochondrial DNA templates. 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. Prior to every cell division, the DNA complement of each cell is duplicated. Because DNA replication and cell division are tightly coupled, an understanding of the mechanisms of DNA replication functioning in animal cells is essential to our understanding of these basic processes. 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 cells 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, gamma polymerase, will represent a major contribution toward an eventual understanding of mitochondrial biogenesis.

拟议工作的长期目标是阐明 动物线粒体中DNA复制的机制。 这可能是 当纯化的酶在体外重组以产生时完成 能够启动和伸长DNA的复制络合物 合成。 研究将为研究框架 确定这些过程在体内发生的调节。 当前的提议着重于主要的复制酶 线粒体，DNA聚合酶γ。涉及的组合方法 生化，免疫学和分子生物学方法将是 用于进行机械和结构功能分析 线性DNA聚合酶并完成并生产其两个 亚基。 次要目标涉及隔离线粒体RNA 聚合酶 / DNA初步酶和RNA协调的检查 线粒体DNA模板上的启动和DNA合成。 在正常情况下控制动物细胞繁殖 发育，癌性发育过程中失去控制的丧失是 人类生长，衰老和疾病的过程中的核心重要性。 在每个细胞分裂之前，每个细胞的DNA补体为 重复。 因为DNA复制和细胞分裂紧密 耦合，了解DNA复制机制 在动物细胞中的功能对于我们对这些的理解至关重要 基本过程。 线粒体生物发生与细胞并行进行 扩散，但既不紧密耦合到线粒体DNA 复制或对细胞周期。 然而，因为两个DNA 线粒体的含量和细胞中线粒体的数量 保持相对恒定，可能是特定的调节机制 需要将线粒体DNA复制和生物发生与 核DNA复制和细胞分裂。 对 与线粒体DNA复制有关的关键酶，伽马聚合酶， 将代表最终理解的主要贡献 线粒体生物发生。