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.
