Ribonucleotide Reductase and DNA Precursor Pool Control

核糖核苷酸还原酶在DNA合成的第一个反应中将四种核糖核苷酸底物还原为相应的2'-脱氧核糖核苷酸。该酶受三磷酸核苷效应物的调控，该效应物与两类变构位点相互作用以控制酶的特异性，因此脱氧核糖核苷三磷酸（dNTPs）的合成与基因组的碱基组成成比例。使用一种检测程序，在底物和效应物浓度与体内相似的情况下，监测一种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关方面。由于平衡的dNTP池对于精确的DNA复制至关重要，这些研究应该阐明细胞如何决定DNA复制的保真度和速率。研究发现，ADP能特异性抑制GDP和ADP本身的降低。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ADP的作用是否涉及在催化位点、变弹性位点或先前描述的位点上的结合。羟基脲是一种自由基清除剂，能抑制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池优先耗尽。该项目将探讨这种效应是否源于ADP的积累，从而抑制体内ADP的减少。另一种模型与dGTP的选择性消耗有关，dGTP是ADP还原的主要变构激活剂。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则会累积。由于rNDP还原酶的激活需要氧气，因此在低pO2条件下，CDP还原的激活可能存在特异性抑制。这个假设将通过使用上面提到的四底物试验来检验。线粒体内的dNTPs组成的池与服务于核基因组的较大池不同。此外，线粒体含有一种独特的rNDP还原酶。线粒体酶将被纯化和表征关于调控，遗传起源，并在合成mtDNA前体中的作用。这项研究的目的是帮助我们了解酶在其自然环境中是如何发挥作用的，活细胞使用作为主要酶的核糖核苷酸还原酶，它催化了DNA合成的第一个反应。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、细胞对氧应激的反应以及线粒体DNA复制方面，将获得重要的见解。

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.