MITOCHONDRIAL DNA POLYMERASE--MECHANISM AND STRUCTURE

拟议工作的长期目标是澄清 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 能够启动和延长DNA的复制复合体 综合。这些研究将为研究提供框架，以 确定这些过程在体内发生时的调节。 目前的提案集中在体内的主要复制酶上 线粒体，DNA聚合酶，伽马。一种涉及以下内容的组合方法 生化、免疫学和分子生物学方法将是 用来进行机械结构和结构功能分析 线粒体DNA聚合酶及其两个多聚酶的制备和过度生产 亚单位。次要目标包括分离线粒体rna。 聚合酶/DNA底物酶与RNA配位的检测 线粒体DNA模板的启动和DNA合成。 正常状态下动物细胞繁殖的控制 发展，以及在癌症发展过程中失去控制是 在人类成长、衰老和疾病的过程中起着核心作用。 在每次细胞分裂之前，每个细胞的DNA补体是 复制的。因为DNA复制和细胞分裂是紧密相连的 再加上对DNA复制机制的理解 在动物细胞中的功能对于我们理解这些 基本流程。 线粒体生物发生与细胞平行进行 增殖，但既不与线粒体DNA紧密结合 复制也不会影响细胞周期。然而，因为DNA和 细胞内线粒体的含量和数量 保持相对不变，可能会有特定的监管机制 需要将线粒体DNA复制和生物发生耦合到 核DNA复制和细胞分裂。详细分析了 参与线粒体DNA复制的关键酶，伽玛聚合酶， 将是对最终理解 线粒体生物发生。

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.