Ribonucleotide Reductase and DNA Precursor Pool Control

在致力于DNA合成的第一反应中，核苷酸还原酶将四种核苷酸底物还原为相应的2‘-脱氧核糖核苷酸。该酶受核苷三磷酸效应物的调控，与两类变构位点相互作用，控制酶的专一性，从而合成与基因组碱基组成成比例的脱氧核糖核苷三磷酸(DNTPs)。使用一种检测程序，在与体内相似的底物和效应器浓度下，监测一个混合物中的四种反应，该项目将研究哺乳动物细胞中dNTP池大小控制的几个相互关联的方面。因为平衡的dNTP池对于准确的DNA复制是必不可少的，这些研究应该阐明细胞如何决定DNA复制的保真度和速率1。ADP已被发现特别抑制GDP和ADP本身的减少。使用四底物分析、竞争结合实验和光亲和标记，该项目将询问该效应是否涉及ADP在催化部位、变构部位或先前未描述的部位的结合。羟基脲是一种自由基清除剂，可以抑制RNDP还原酶的所有四种活性。然而，用羟基脲处理细胞会导致dATP池的优先耗尽。这个项目将询问这种效应是否源于ADP在体内的积累，从而抑制ADP的还原。另一种模型涉及ADP还原的首要变构激活剂dGTP的选择性耗竭。已经发现，在低氧条件下培养细胞可以选择性地缩小dCTP池，而其他三种dNTP会积累。由于RNDP还原酶的激活需要氧气，因此在低pO2时可能对CDP还原的激活有特定的抑制作用。这一假说将通过上述四种底物试验进行检验。线粒体内的dNTP组成的池与服务于核基因组的较大池不同。此外，线粒体还含有一种独特的RNDP还原酶。这项研究旨在帮助我们了解酶在其自然环境--活细胞中是如何发挥作用的，核糖核苷酸还原酶是催化致力于DNA合成的第一反应的主要酶。有可能在能量代谢和基因组复制之间的关系、DNA复制的控制、细胞对氧胁迫的反应以及线粒体DNA复制方面获得重要的见解。

Ribonucleotide reductase reduces four ribonucleotide substrates to the corresponding 2'-deoxyribo-nucleotides in the first reaction committed to DNA synthesis. The enzyme is regulated by nucleoside triphosphate effectors that interact with two classes of allosteric sites to control the specificity of the enzyme, so that deoxyribonucleoside triphosphates (dNTPs) are synthesized in proportion to the base composition of the genome. Using an assay procedure that monitors the four reactions in one mixture, at substrate and effector concentrations similar to those in vivo, the project will investigate several interrelated aspects of the control of dNTP pool sizes in mammalian cells. Because balanced dNTP pools are essential for accurate DNA replication, these studies should illuminate how cells determine the fidelity as well as the rate of DNA replication.1. ADP has been found to specifically inhibit the reduction of GDP and of ADP itself. Using the four-substrate assay, competitive binding experiments, and photoaffinity labeling, this project will ask whether the effect involves ADP binding at the catalytic site, an allosteric site, or a previously undescribed site.2. Hydroxyurea, a free radical scavenger, inhibits all four activities of rNDP reductase. However, treating cells with hydroxyurea causes preferential depletion of the dATP pool. This project will ask whether this effect results from accumulation of ADP, with consequent inhibition of ADP reduction in vivo. An alternative model relates to the selective depletion of dGTP, the prime allosteric activator of ADP reduction.3. Culturing cells under hypoxic conditions has been found to selectively shrink the dCTP pool, while the other three dNTPs accumulate. Since oxygen is required for rNDP reductase activation, there may be a specific inhibition of the activation of CDP reduction at low pO2. This hypothesis will be tested by use of the four-substrate assay mentioned above.4. dNTPs within mitochondria comprise pools distinct from the larger pools that serve the nuclear genome. Also, mitochondria contain a distinctive rNDP reductase. The mitochondrial enzyme will be purified and characterized with regard to regulation, genetic origin, and role in synthesizing mtDNA precursors.This research is designed to help us understand how enzymes function in their natural environment, the living cell, using as the principal enzyme ribonucleotide reductase, which catalyzes the first reaction committed to DNA synthesis. Possibly, significant insights will be gained into relationships between energy metabolism and genome replication, into the control of DNA replication, into cellular responses to oxygen stress, and into mitochondrial DNA replication.