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

核糖核苷酸还原酶调节：Diferric Y* 组装/维护和 Sml1

• 批准号: 7527517
• 负责人: JOANNE STUBBE
• 金额: $ 42.81万
• 依托单位: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-07-02 至 2012-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7527517
• 关键词: Affinity Chromatography; Amaze; Anabolism; Arabinose; Binding; Biochemical Genetics; C-terminal; Candidate Disease Gene; Cell Death; Cells; Chemicals; Chemistry; Class; Complex; Condition; Crystallography; Cytochromes b5; DNA; DNA Repair; DNA biosynthesis; Drug usage; Electrons; Ensure; Escherichia coli; Escherichia coli Proteins; Ferredoxin; Fission Yeast; Flavodoxin; Free Radicals; Genetic; Goals; Growth; Hematologic Neoplasms; Homologous Gene; Housing; Human; In Vitro; Iron; Kinetics; Label; Lead; Link; Maintenance; Malignant Neoplasms; Modeling; Monitor; Mossbauer Spectroscopy; Mutagenesis; Nucleotides; Numbers; Organism; Outcome; Pathway interactions; Phase II Clinical Trials; Phosphorylation; Phosphorylation Site; Phosphotransferases; Play; Predisposition; Process; Proteins; Public Health; Range; Rate; Reaction; Recycling; Reducing Agents; Regulation; Relative (related person); Ribonucleotide Reductase; Role; Saccharomyces cerevisiae; Site; Source; Substrate Specificity; Testing; Triapine; Viral; Yeasts; cofactor; crosslink; drug development; hydroxyurea; in vivo; inhibitor/antagonist; interest; mutant; novel therapeutics; nucleic acid metabolism; protein degradation; repaired; research study; therapeutic target; tumor

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.

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：fin 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子（NTP 和 dATP）。这 ？？亚基含有启动 ?n 上化学上困难的还原过程所需的必需二铁酪氨酰自由基 (Y) 辅因子。活性RNR复合物是πnπ2。 RNR 的调节主要负责控制 dNTP 库的相对比例和数量，这对于 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、遗传不稳定以及人类患癌症的倾向。 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 活性的调节发生在多个水平：转录，通过控制 ?n 和 ?2 的亚细胞定位，通过变构效应子（ATP、dNTP 到 ?n）的结合，通过控制蛋白质降解，通过控制浓度由二铁金属辅因子和小蛋白抑制剂产生的 Y。该提案重点关注大肠杆菌和酿酒酵母 I 类 RNR 的监管。将使用生化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大肠杆菌和酿酒酵母中必需的二铁-Y 辅因子 α2 的生物合成和维持（修复）途径。通过实验来确定铁和减少当量输送所需的装配系数。辅因子的 Y 是用于治疗血液恶性肿瘤的羟基脲和 II 期临床试验中的三甲平的靶标。因此，了解这些簇是否可以修复可以在临床上产生显着的结果。酿酒酵母的第三个具体目标是了解小蛋白：Sml1 和新发现的 Spd1 在 RNR 抑制中的机制。这种理解可以确定新的治疗靶点。长期目标是定量了解所有调控机制如何整合以控制不同生长条件下的细胞 dNTP 库。公共卫生相关性：核糖核苷酸还原酶催化所有生物体中核苷酸向脱氧核苷酸的转化。它们的调节对于控制 dNTP 库至关重要，对于 DNA 复制和修复的保真度至关重要。该提案审查了两种监管机制；了解这些机制可能会带来新的治疗靶点。