DEOXYRIBONUCLEOTIDE METABOLISM IN ESCHERICHIA COLI

核糖核苷酸还原酶催化 核糖核苷酸到脱氧核糖核苷酸。 分离的酶 已经被广泛表征， 作为其他核糖核苷酸还原酶的模型， 源 高等真核生物中的酶似乎非常 与E.大肠杆菌酶。 一种酶催化 所有核糖核苷酸二磷酸和底物的还原 特异性和整体活性由变构控制 调控 该酶合成的调节是 不寻常. 核糖核苷酸编码基因的表达 还原酶（nrd）似乎与DNA复制的控制平行。 因此，了解控制的分子机制， NRD表达将是理解 DNA复制的调节。 这反过来将是一个重大的 了解细胞生长控制的一步。 由于这种酶的水平与细胞生长成正比， 真核生物和E.大肠杆菌，这种酶似乎是一种 抗肿瘤化疗药物理想靶点 细胞 由于这种酶是由疱疹病毒和爱泼斯坦-酒吧编码 病毒，它可能是化疗药物的一个可能的目标 对抗这些病毒。 为了了解nrd调节的分子细节， 迄今为止的实验已经利用胸腺嘧啶剥夺来改变NRD 表情 本提案的一个目标是调查NRD 在指数生长细胞中的表达作为细胞的函数 周期 初步实验表明，nrd表达 在胸腺嘧啶剥夺期间观察到的调节结果 通常发生在细胞周期中。 为了进一步研究 NRD调控的细节，含有NRD的质粒 与lacZ融合的调节区将用于分离和 表征反式作用突变体。 这些变种人将被用来 克隆并表征“野生型”等位基因。 A DNA蛋白 结合聚丙烯酰胺凝胶分析将用于鉴定和 纯化调节蛋白。 DNA酶足迹法将用于 显示这些蛋白质结合到被鉴定为操纵基因的位点上， 网站. 在体外产生的点突变体的调控区5' 对NRD的结构基因进行表征和测序 为了进一步确定涉及阳性和阴性的位点， 调控

Ribonucleotide reductases catalyze the reduction of ribonucleotides to deoxyribonucleotides. The enzyme isolated from Escherichia coli has been extensively characterized and serves as the model for ribonucleotide reductases from other sources. The enzyme in higher eucaryotes appears to be very similar to the E. coli enzyme. A single enzyme catalyzes the reduction of all ribonucleotide diphosphates and the substrate specificity and overall activity is controlled by allosteric regulation. The regulation of the synthesis of the enzyme is unusual. The expression of the genes encoding ribonucleotide reductase (nrd) appear to parallel the control of DNA replication. Thus, understanding the molecular mechanism of the control of nrd expression will be an important step in the understanding of the regulation of DNA replication. This in turn will be a major step in understanding the control of cell growth. Since the level of this enzyme is proportional to cell growth in both eucaryotes and in E. coli, this enzyme would appear to be an ideal target for chemotherapeutic agents active against tumor cells. Since this enzyme is encoded by Herpes and Epstein-Bar virus, it could be a possible target for chemotherapeutic agents against these viruses. To understand the molecular details of nrd regulation, experiments to date have utilized thymine deprivation to alter nrd expression. One objective of this proposal is to investigate nrd expression in exponentially growing cells as a function of the cell cycle. Preliminary experiments suggest that the nrd expression observed during thymine deprivation results from regulation normally occurring during the cell cycle. To further investigate the details of nrd regulation, plasmids containing the nrd regulatory region fused to lacZ will be utilized to isolate and characterize transacting mutants. These mutants will be used to clone and characterize the "wild type" allele. A DNA-protein binding polyacrylamide gel assay will be used to identify and purify the regulatory proteins. DNase footprinting will be used to show that these proteins bind to the sites identified as operator sites. In vitro generated point mutants in the regulatory region 5' to the structural genes of nrd will be characterized and sequenced to further define sites involved in both positive and negative regulation.