Ribonucleotide Reductase Regulation: Diferric Y* assembly/maintenance and Sml1

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，并提供DNA复制和DNA修复所需的单体前体。I类RNRs由两个亚基组成：N亚基结合四种NDP底物，变构效应(NTPs和dATP)控制底物专一性和周转率。那是什么？亚基中含有重要的二铁-酪氨酸基(Y)辅因子，在？N上启动化学困难的还原过程。活性的RNR复合体是？N？2。RNR的调节在很大程度上负责控制dNTP池的相对比例和数量，这对DNA复制和修复的保真度至关重要。失去这种控制可能会导致细胞死亡、遗传不稳定，并在人类中易患癌症。RnR在核酸代谢中的核心作用使其成为治疗多种恶性肿瘤的成功靶点。RNR活性的调节在多个水平上发生：在转录水平上，通过控制？N和？2的亚细胞定位，通过变构效应(ATP，dNTPs与？N)的结合，通过控制蛋白质的降解，通过控制双铁金属辅因子产生的Y的浓度，以及通过小的蛋白抑制剂。这项提案的重点是对来自大肠杆菌和酿酒酵母的I类RNRs进行调控。将综合使用生化和遗传方法来研究两种调控机制。第一和第二个特定的目的是阐明在大肠杆菌和酿酒酵母中重要的差异铁-Y辅因子？2的生物合成和维持(修复)途径。提出了确定铁和减少当量输送所需的装配系数的实验。辅因子的Y是羟基脲用于治疗血液系统恶性肿瘤的靶点，也是二期临床试验中三平的靶点。因此，了解集群是否可以修复可以在临床上产生戏剧性的结果。酿酒酵母的第三个特定目的是了解小蛋白Sml1和新发现的Spd1在RNR抑制中的作用机制。这一认识可以确定一个新的治疗靶点。长期目标是定量地了解如何在不同的生长条件下整合所有的调控机制来控制细胞dNTPs池。与公共卫生相关：在所有生物体中，核苷酸还原酶催化核苷酸转化为脱氧核苷酸。它们的调控对于控制dNTP池至关重要，而dNTP池对于DNA复制和修复的保真度至关重要。在这项提案中检查了两种调节机制；了解这些机制可能会导致新的治疗靶点。

DESCRIPTION (provided by applicant): Ribonucleotide reductases (RNRs) catalyze the conversion of nucleotides to deoxynucleotides in all organisms and provide the monomeric precursors required for DNA replication and DNA repair. The class I RNRs are composed of two subunits: the ?n subunit binds the four NDP substrates and the allosteric effectors (NTPs and dATP) that govern substrate specificity and turnover rate. The ?? subunit houses the essential diferric-tyrosyl radical (Y) cofactor required to initiate the chemically difficult reduction process on ?n. The active RNR complex is ?n?2. Regulation of RNRs is largely responsible for controlling the relative ratios and amounts of the dNTP pools, which is critical to the fidelity of DNA replication and repair. Loss of this control can lead to cell death, genetic instability, and in humans, a predisposition to cancer. RNR's central role in nucleic acid metabolism has made them the successful target in the treatment of a number of malignancies. Regulation of RNR activity occurs at multiple levels: transcriptionally, by control of the subcellular localization of ?n and ?2, by the binding of allosteric effectors (ATP, dNTPs to ?n), by control of protein degradation, by control of the concentration of the Y generated by the di-iron metallo-cofactor, and by small protein inhibitors. This proposal focuses on the regulation of the class I RNRs from E. coli and S. cerevisiae. Two regulatory mechanisms will be examined using an integration of biochemical and genetic approaches. The first and second specific aims are to elucidate the biosynthetic and maintenance (repair) pathways of the essential diferric-Y cofactor of ?2 in E. coli and S. cerevisiae. Experiments are presented to identify the assembly factors required for iron and reducing equivalent delivery. The Y of the cofactor is the target of hydroxyurea used in the treatment of hematologic malignancies and of triapine in phase II clinical trials. Thus understanding whether the clusters can be repaired can have dramatic outcomes clinically. The third specific aim in S. cerevisiae is to understand mechanism of the small proteins: Sml1 and the newly discovered Spd1, in RNR inhibition. This understanding could identify a new therapeutic target. The long-range goal is to understand quantitatively how all of the regulatory mechanisms are integrated to control cellular dNTPs pools under different growth conditions. PUBLIC HEALTH RELEVANCE: Ribonucleotide reductases catalyze the conversion of nucleotides to deoxynucleotides in all organisms. Their regulation is essential for controlling dNTP pools, critical for the fidelity of DNA replication and repair. Two regulatory mechanisms are examined in this proposal; understanding these mechanisms could lead to new therapeutic targets.