MITOCHONDRIAL DNA POLYMERASE: MECHANISM AND STRUCTURE

描述（由申请人提供）：拟议工作的长期目标是阐明动物线粒体中DNA复制的机制，及其与线粒体突变和人类疾病的关系。结合生物化学和分子遗传学的现有方法，研究线粒体中主要的复制酶，DNA聚合酶y （pol y）的机制、结构和生理，以及它与线粒体复制叉蛋白的功能相互作用。poly的结构-功能研究将包括对天然形式和改变形式的比较动力学和物理分析，继续阐明小亚基在酶功能中的作用，并剖析催化亚基和辅助亚基的功能域。突变研究将集中于阐明催化核心的间隔区域的作用，物理分析将绘制亚基接触图。生理学研究将集中于扩大转基因果蝇模型的发展，以研究poly功能、线粒体DNA复制保真度、衰老和疾病之间的关系。在线粒体生物发生的背景下，一种新的重组敲入方法将被用来介绍和研究几种与人类疾病相关的polyy等位基因。

DESCRIPTION (provided by applicant): The long-term objective of the proposed work is the elucidation of the mechanism of DNA replication in animal mitochondria, and its relationship to mitochondrial mutagenesis and human disease. A combined approach of current methods in biochemistry and molecular genetics is being pursued to study the mechanism, structure and physiology of the major replicative enzyme in mitochondria, DNA polymerase y (pol y), and its functional interactions with proteins at the mitochondrial replication fork. Structure-function studies of pol y will involve comparative kinetic and physical analyses of native and altered forms, to continue to elucidate the role of the small subunit in enzyme function, and to dissect functional domains in both the catalytic and accessory subunits. Mutational studies will focus on elucidating the roles of the spacer region of the catalytic core, and physical analyses will map subunit contacts. Physiological studies will focus on expanding the development of a transgenic fly model to examine the relationship between pol y function, mitochondrial DNA replication fidelity, aging and disease. A novel recombinational knock-in approach will be taken to introduce and study several human-disease-related alleles of pol y in the context of mitochondrial biogenesis. 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 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, DNA polymerase y, 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 anti-tumor drugs frequently affect mitochondrial DNA function, and in particular, the activity of pol y, demonstrate a critical need for an in-depth understanding of this essential cellular DNA polymerase, and other proteins that function at the replication fork.