Ribonucleotide Reductases: Structure and Function

描述(申请人提供)：核糖核苷酸还原酶(RNRs)在所有生物体中催化核苷酸转化为脱氧核苷酸，提供DNA复制和修复所需的单体前体。RNRs作为一个范例，用来理解大自然如何通过精确的控制来利用蛋白质和核苷酸自由基的增强反应性来产生脱氧核苷酸。RNRs在核酸代谢中的中心作用使其成为临床上使用的两种药物的成功靶点：2‘，2“-二氟-2’-脱氧胞苷(Gemzar)和羟基脲。这两种化合物都干扰RNRs的自由基化学，其细节正在本提案中进行调查。I类RNRs需要二铁酪氨酸基(Y()辅因子)来催化。它的功能是在35A的距离内启动前所未有的远程质子耦合电子转移(PCET)，即自由基传播步骤。这一过程发生在RNR的亚基之间：R1和R2。介绍了用定点附着式探头、PELDOR和DQC方法测量这一距离的方法。方法通过内含素介导的蛋白质连接或同源tRNA/tRNA合成酶结合体内的翻译机制，将非天然氨基酸(FnY，x=1-4)插入到每个亚基中，研究PCET。为了检测瞬时氨基酸自由基中间体，提出了用光触发PCET的方法。研究正常的还原过程、基于机理的抑制剂、自由基的传播以及核苷酸还原的特异性和速率的调节，都需要了解R1的四级结构及其与R2的相互作用。为了解决这一问题，提出了在R1和R2中使用定点放置的荧光和交联剂的生物物理方法(超速离心法、动态光散射法、停流荧光法)。这些研究对于实现我们的长期目标至关重要：了解细胞如何生物合成并维持RNR活性所必需的diferric-Y(辅因子)，以及定量了解体内控制dNTP池的复杂调控层。大肠埃希氏菌RNR、酿酒葡萄球菌和人类RNRs是我们努力的重点。

DESCRIPTION (provided by applicant): Ribonucleotide reductases (RNRs) catalyze the conversion of nucleotides to deoxynucleotides in all organisms providing the monomeric precursors required for DNA replication and repair. The RNRs serve as a paradigm for understanding how Nature has harnessed the enhanced reactivity of protein and nucleotide free radicals with exquisite control to produce deoxynucleotides. The central role of RNRs in nucleic acid metabolism has made them the successful target of two drugs used clinically: 2', 2"-difluoro-2'-deoxycytidine (gemzar) and hydroxyurea. Both compounds interfere with the radical chemistry of the RNRs, the details of which are being investigated in the present proposal. The class I RNRs, require a diferric-tyrosyl radical (Y() cofactor for catalysis. Its function is to initiate the unprecedented long range proton coupled electron transfer (PCET), the radical propagation step, over a 35 A distance. This process occurs between the subunits of the RNR: R1 and R2. Methods to measure this distance with site-specifically attached probes and PELDOR and DQC methods are presented. Methods to study PCET using unnatural amino acids (FnYs with x = 1-4) placed into each subunit by intein mediated protein ligations or orthologous tRNA/tRNA synthetase pairs in conjunction with the translational machinery in vivo are described. Methods to trigger the PCET with light are presented in an effort to detect transient amino acid radical intermediates. Studies of the normal reduction process, mechanism based inhibitors, radical propagation and regulation of the specificity and rate of nucleotide reduction, all require an understanding of the quaternary structure of R1 and its interaction with R2. Biophysical methods (ultracentrifugation, dynamic light scattering, stopped flow fluorescence) using site- specifically placed fluorescent and cross-linking agents within R1 and R2 are presented to address this issue. These studies are essential for achieving our long range goals: to understand how cells biosynthesize and maintain the diferric-Y( cofactor essential for RNR activity and to understand quantitatively the complex layers of regulation that control dNTP pools in vivo. The E. coli RNR and the S. cerevisiae and human RNRs are the focus of our efforts.