Regulation of ribonucleotide reductase M2 expression by the upstream AUGs

Ribonucleotide reductase catalyzes a rate-limiting reaction in DNA synthesis by converting ribonucleotides to deoxyribonucleotides. It consists of two subunits and the small one, M2 (or R2), plays an essential role in regulating the enzyme activity and its expression is finely controlled. Changes in the M2 level influence the dNTP pool and, thus, DNA synthesis and cell proliferation. M2 gene has two promoters which produce two major mRNAs with 5′-untranslated regions (5′-UTRs) of different lengths. In this study, we found that the M2 mRNAs with the short (63 nt) 5′-UTR can be translated with high efficiency whereas the mRNAs with the long (222 nt) one cannot. Examination of the long 5′-UTR revealed four upstream AUGs, which are in the same reading frame as the unique physiological translation initiation codon. Further analysis demonstrated that these upstream AUGs act as negative cis elements for initiation at the downstream translation initiation codon and their inhibitory effect on M2 translation is eIF4G dependent. Based on the findings of this study, we conclude that the expression of M2 is likely regulated by fine tuning the translation from the mRNA with a long 5′-UTR during viral infection and during the DNA replication phase of cell proliferation.     

Mutations at several loci cause increased expression of ribonucleotide reductase in Escherichia coli

Production of deoxyribonucleotides for DNA synthesis is an essential and tightly regulated process. The class Ia ribonucleotide reductase (RNR), the product of the nrdAB genes, is required for aerobic growth of Escherichia coli. In catalyzing the reduction of ribonucleotides, two of the cysteines of RNR become oxidized, forming a disulfide bond. To regenerate active RNR, the cell uses thioredoxins and glutaredoxins to reduce the disulfide bond. Strains that lack thioredoxins 1 and 2 and glutaredoxin 1 do not grow because RNR remains in its oxidized, inactive form. However, suppressor mutations that lead to RNR overproduction allow glutaredoxin 3 to reduce sufficient RNR for growth of these mutant strains. We previously described suppressor mutations in the dnaA and dnaN genes that had such effects. Here we report the isolation of new mutations that lead to increased levels of RNR. These include mutations that were not known to influence production of RNR previously, such as a mutation in the hda gene and insertions in the nrdAB promoter region of insertion elements IS1 and IS5. Bioinformatic analysis raises the possibility that IS element insertion in this region represents an adaptive mechanism in nrdAB regulation in E. coli and closely related species. We also characterize mutations altering different amino acids in DnaA and DnaN from those isolated before.     

Involvement of ribonucleotide reductase in cellular differentiation

L₆ and L₈ rat myoblast cell lines have been selected for resistance to hydroxyurea, an antineoplastic agent whose intracellular target is the rate-limiting enzyme activity of DNA synthesis, ribonucleotide reductase. In contrast to the differentiation-competent parental lines from which they were selected, the drug-resistant lines exhibit a grossly altered or absent myogenic capacity. Independent selections have revealed a strong correlation between changes in ribonucleotide reductase, as determined by velocity levels and product pool analyses, and altered myogenic potential. These results provide the first indication that alterations in this key enzyme activity and its accompanying deoxyribonucleoside triphosphate pools can affect cellular differentiation.     

Cell cycle-dependent expression of mammalian ribonucleotide reductase. Differential regulation of the two subunits

Consistent with its specialized role in DNA synthesis, the activity of ribonucleotide reductase is cell cycle-dependent, reaching its maximum during S-phase. This paper demonstrates, however, the levels of the two protein subunits, M1 and M2, of this enzyme vary independently of one another. The level of protein M1 was determined by use of a two-site monoclonal antibody-enzyme immunoassay and found to be constant throughout the cell cycle in bovine kidney MDBK cells. Pulse-chase experiments showed that the half-life of protein M1 was 15 h. This contrasts with our previous results demonstrating an S-phase-correlated increase in the concentration of protein M2 and a half-life of this subunit of 3 h. Therefore, ribonucleotide reductase is controlled during the cell cycle by the level of protein M2.     

Thionucleotides as inhibitors of ribonucleotide reductase

Ribonucleosides and xylonucleosides bearing a disulfide function on the sugar ring were synthesized. Ribonucleosides belonging to the cytidine series were found to efficiently reduce dNTP pools in the human lymphoblastoid CEM/SS cell line.     

Micro-array analysis of resistance for gemcitabine results in increased expression of ribonucleotide reductase subunits

To study in detail the relation between gene expression and resistance against gemcitabine, a cell line was isolated from a tumor for which gemcitabine resistance was induced in vivo. Similar to the in vivo tumor, resistance in this cell line, C 26-G, was not related to deficiency of deoxycytidine kinase (dCK). Micro-array analysis showed increased expression of ribonucleotide reductase (RR) subunits M1 and M2 as confirmed by real time PCR analysis (28- and 2.7-fold, respectively). In cell culture, moderate cross-resistance (about 2-fold) was observed to 1-ss-D-arabinofuranosylcytosine (ara-C), 2-chloro-2'deoxyadenosine (CdA), LY231514 (ALIMTA), and cisplatin (CDDP), and pronounced cross-resistance (>23-fold) to 2',2'-difluorodeoxyuridine (dFdU) and 2',2'-difluorodeoxyguanosine (dFdG). Culture in the absence of gemcitabine reduced resistance as well as RRM1 RNA expression, demonstrating a direct relationship of RRM1 RNA expression with acquired resistance to gemcitabine.     

8-Azidoadenosine and ribonucleotide reductase.

Inhibitors of ribonucleotide reductase are potential antiproliferative agents, since they deplete cells from DNA precursors. Substrate nucleoside analogues, carrying azido groups at the base moiety, are shown to have strong cytostatic properties, as measured by the inhibition of the incorporation of thymidine into DNA. One compound, 8-azidoadenosine, inhibits CDP reduction in cytosolic extracts from cancer cells. The corresponding diphosphate behaves as a substrate for ribonucleotide reductase while the triphosphate is an allosteric effector.     

Localization of ribonucleotide reductase in mammalian cells.

The results of immunocytochemical studies using two different monoclonal antibodies against the M1 subunit of ribonucleotide reductase show an exclusively cytoplasmic localization of this subunit both in cultured MDBK and mouse 3T6 cells, and in cells from various rat tissues. By fluorescent light microscopy, there is a diffuse staining of the cytoplasm, while by electron microscopy the immunoreactive material appears to be associated with ribosomes. In the rat tissues, only actively dividing cells show M1-specific immunofluorescence revealing a strong correlation between the presence of protein M1 and DNA synthesis. Therefore M1 immunofluorescence could be used to study cell proliferation in normal, inflammatory or neoplastic tissue. A lesser variation in M1 staining is observed between individual cells in tissue culture, where most cells are positive, but neither here nor in the tissues examined are any cells with nuclear staining detected. We interpret our results to mean that in mammalian cells ribonucleotide reduction takes place in the cytoplasm and from there the deoxyribonucleotides are transported into the nucleus to serve in DNA synthesis.     

Presence of oxygen-consuming ribonucleotide reductase in corrinoid-deficientPropionibacterium freudenreichii

Corrinoid-deficient Propionibacterium freuden- reichii subsp. shermanii showed adenosylcobalamin-(AdoCbl)-independent ribonucleotide reductase (RNR) activity in the presence of air. Increasing the incubation time with free access of O₂ led to an increase in RNR activity. As polarographic estimations of O₂ uptake demonstrated, AdoCbl-independent RNR activity (with ADP as substrate) in a cell-free system of corrinoid-deficient P. freudenreichii was accompanied by specific molecular oxygen consumption. The activity was not inhibited by carbonyl cyanide m-chlorophenylhydrazone (CCCP) or carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCCP). The activity was present in the cytoplasmic membrane-free soluble fraction of the cell extract, and it was inhibited by hydroxyurea. Manganese ions were important for the cell division of corrinoid-deficient P. freudenreichii and stimulated RNR activity after 8-hydroxyquinoline or EDTA treatment of the cell extract. We therefore concluded that P. freudenreichii is able to form DNA (deoxyribosylic precursors) using AdoCbl-dependent ribonucleotide reductase and also with an alternative AdoCbl-independent molecular-oxygen-consuming RNR system. Received: 29 May 1995 / Accepted: 14 August 1995     

Paramagnetic form of ribonucleotide reductase in human tissues

The paramagnetic form of ribonucleotide reductase was detected by ESR method in human cervix tissues, especially in tumor ones. The magnetic relaxation rate was proved to be slower for this form than for that in normal animal tissues having a high level of proliferative activity or in Ehrlich tumor cells studied before.     

Aerobic-type ribonucleotide reductase in the anaerobe Bacteroides fragilis.

Bacteroides fragilis, a component of the normal intestinal flora, is an obligate anaerobe capable of long-term survival in the presence of air. Survival is attributed to an elaborate oxidative stress response that controls the induction of more than 28 peptides, but there is limited knowledge concerning the identities of these peptides. In this report, RNA fingerprinting by arbitrarily primed PCR identified five new genes whose expression increased following exposure to O2. Nucleotide sequence analysis of the cloned genes indicated that they encoded an outer membrane protein, an aspartate decarboxylase, an efflux pump, heat shock protein HtpG, and an NrdA ortholog constituting the large subunit of a class Ia ribonucleotide reductase (RRase). Attention was focused on the nrdA gene since class I RRases are obligate aerobic enzymes catalyzing the reduction of ribonucleoside 5'-diphosphates by a mechanism that requires molecular oxygen for activity. Sequence analysis of the nrd locus showed that two genes, nrdA and nrdB, are located in the same orientation in a 4.5-kb region. Northern hybridization and primer extension experiments confirmed induction of the genes by O2 and suggested they are an operon. The B. fragilis nrdA and nrdB genes were overexpressed in Escherichia coli, and CDP reductase assays confirmed that they encoded an active enzyme. The enzyme activity was inhibited by hydroxyurea, and ATP was shown to be a positive effector of CDP reductase activity, while dATP was an inhibitor, indicating that the enzyme was a class Ia RRase. A nrdA mutant was viable under anaerobic conditions but had decreased survival following exposure to O2, and it could not rapidly resume growth after O2 treatment. The results presented indicate that during aerobic conditions B. fragilis NrdAB may have a role in maintaining deoxyribonucleotide pools for DNA repair and growth recovery.