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。目前生物化学和分子遗传学的方法相结合的方法正在寻求研究线粒体中的主要复制酶，DNA聚合酶y（poly）的机制，结构和生理学，及其与蛋白质在线粒体复制叉的功能相互作用。多聚体的结构-功能研究将涉及对天然和改变形式的动力学和物理分析的比较，以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中的功能结构域。突变研究将侧重于阐明催化核心的间隔区的作用，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开发，以检查多聚体功能，线粒体DNA复制保真度，衰老和疾病之间的关系。将采用一种新的重组敲入方法，在线粒体生物发生的背景下引入和研究多聚体y的几个人类疾病相关等位基因。 在正常发育过程中对动物细胞繁殖的控制以及在癌症发展过程中的失控，在人类生长、衰老和疾病的过程中至关重要。线粒体生物合成与细胞增殖平行进行，但它既不与线粒体DNA复制紧密耦合，也不与细胞周期紧密耦合。然而，由于线粒体的DNA含量和细胞中线粒体的数量保持相对恒定，因此可能需要特定的调节机制将线粒体DNA复制和生物发生与核DNA复制和细胞分裂偶联。对线粒体DNA复制中的关键酶DNA聚合酶γ的详细分析将为最终理解正常和患病组织中线粒体的生物发生和功能做出重大贡献。! 最近已经记录了各种线粒体DNA疾病。这一点，以及越来越多的认识，即抗病毒和抗肿瘤药物经常影响线粒体DNA功能，特别是多聚酶的活性，表明迫切需要深入了解这种必需的细胞DNA聚合酶，以及其他在复制叉发挥作用的蛋白质。

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.