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（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（NTPs和dATP）。的? ?亚基包含必需的异酪氨酸自由基(Y)辅因子，以启动化学上困难的还原过程。活性RNR复合物为？n?2。rrna的调控主要负责控制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。失去这种控制会导致细胞死亡、遗传不稳定，对人类来说，更容易患癌症。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，通过控制亚细胞定位？N和？2、由变构效应物（ATP、dNTPs）结合到？n)，通过控制蛋白质降解，通过控制二铁金属辅助因子和小蛋白质抑制剂产生的Y的浓度。本文主要研究了大肠杆菌和酿酒葡萄球菌I类RNRs的调控。两种调节机制将使用生物化学和遗传方法的整合进行审查。第一个和第二个具体目标是阐明？的基本差分- y辅因子的生物合成和维持（修复）途径。2在大肠杆菌和酿酒杆菌中。提出了确定铁所需的组装因子和减少等效交货的实验。辅助因子的Y是用于治疗血液恶性肿瘤的羟基脲和II期临床试验中的曲平的靶标。因此，了解是否可以修复群集可以有显著的临床结果。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。这种认识可以确定一个新的治疗靶点。长期目标是定量地了解在不同生长条件下如何整合所有的调节机制来控制细胞dNTPs池。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的转化。它们的调节对于控制dNTP池至关重要，对DNA复制和修复的保真度至关重要。本提案审查了两种监管机制；了解这些机制可以找到新的治疗靶点。

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.