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

描述（由申请人提供）：核糖核苷酸还原酶（RNR）在所有生物体中催化核苷酸向脱氧核苷酸的转化，并提供DNA复制和DNA修复所需的单体前体。I类RNR由两个亚基组成：n亚基结合四种NDP底物和控制底物特异性和周转率的变构效应物（NTP和dATP）。那？？亚基容纳了必需的二铁酪氨酰自由基（Y）辅因子，该辅因子是启动对？n.活性RNR复合体是？你好吗？2. RNRs的调节主要负责控制dNTP库的相对比率和量，这对DNA复制和修复的保真度至关重要。失去这种控制会导致细胞死亡、基因不稳定，在人类中，还会导致癌症。RNR在核酸代谢中的核心作用使其成为许多恶性肿瘤治疗的成功靶点。RNR活性的调节发生在多个水平：转录水平，通过控制亚细胞定位？n和？2、通过变构效应物（ATP、dNTPs与？n）、通过控制蛋白质降解、通过控制由二铁金属辅因子产生的Y的浓度、以及通过小蛋白抑制剂。该建议集中于来自E. coli和革兰氏阳性菌S.啤酒。将使用生物化学和遗传学方法的整合来检查两种调节机制。第一个和第二个具体目标是阐明必需的异铁-Y辅因子的生物合成和维持（修复）途径？2在E. coli和革兰氏阳性菌S.啤酒。通过实验确定了铁和还原当量输送所需的装配系数。辅因子的Y是羟基脲用于治疗恶性血液病和曲氮平在II期临床试验中的靶点。因此，了解是否可以修复集群可以有戏剧性的临床结果。S.目的：研究小分子蛋白Sml 1和新发现的Spd 1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。长期目标是定量了解在不同生长条件下，如何整合所有调控机制以控制细胞dNTP库。公共卫生相关性：核糖核苷酸还原酶在所有生物体中催化核苷酸向脱氧核苷酸转化。它们的调节对于控制dNTP池至关重要，dNTP池对于DNA复制和修复的保真度至关重要。在本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.