Cell cycle regulation of the Escherichia coli nrd operon: requirement for a cis-acting upstream AT-rich sequence.

The expression of the nrd operon encoding ribonucleotide reductase in Escherichia coli has been shown to be cell cycle regulated. To identify the cis-acting elements required for the cell cycle regulation of the nrd promoter, different 5' deletions as well as site-directed mutations were translationally fused to a lacZ reporter gene. The expression of beta-galactosidase from these nrd-lacZ fusions in single-copy plasmids was determined with synchronously growing cultures obtained by repeated phosphate starvation as well as with exponentially growing cultures by flow cytometry analysis. Although Fis and DnaA, two regulatory proteins that bind at multiple sites on the E. coli chromosome, have been found to regulate the nrd promoter, the results in this study demonstrated that neither Fis nor DnaA was required for nrd cell cycle regulation. A cis-acting upstream AT-rich sequence was found to be required for the cell cycle regulation. This sequence could be replaced by a different sequence that maintained the AT richness. A flow cytometry analysis that combined specific immunofluorescent staining of beta-galactosidase with a DNA-specific stain was developed and employed to study the nrd promoter activity in cells at specific cell cycle positions. The results of the flow cytometry analysis confirmed the results obtained from studies with synchronized cells.     

Identification of the stable free radical tyrosine residue in ribonucleotide reductase.

The small subunit of iron-dependent ribonucleotide reductases contains a stable organic free radical, which is essential for enzyme activity and which is localized to a tyrosine residue. Tyrosine-122 in the B2 subunit of Escherichia coli ribonucleotide reductase has been changed into a phenylalanine. The mutation was introduced with oligonucleotide-directed mutagenesis in an M13 recombinant and verified by DNA sequencing. Purified native and mutant B2 protein were found to have the same size, iron content and iron-related absorption spectrum. The sole difference observed is that the mutant protein lacks tyrosyl radical and enzymatic activity. These results identify Tyr122 of E. coli protein B2 as the tyrosyl radical residue. An expression vector was constructed for manipulation and expression of ribonucleotide reductase subunits. It contains the entire nrd operon with its own promoter in a 2.3-kb fragment from pBR322. Both the B1 and the B2 subunits were expressed at a 25-35 times higher level as compared to the host strain.     

A 45 bp inverted repeat is required for cell cycle regulation of the Escherichia coli nrd operon

Expression of β-galactosidase from a nrd–lacZ fusion was used to determine the role in nrd regulation of an inverted sequence upstream of the promoter. Removal or replacement of a 45 bp inverted repeat with an altered sequence including a 48 bp perfect inverted repeat resulted in a mutant phenotype that was low in nrd expression in an exponentially growing culture and that did not increase during DNA synthesis inhibition. Changing the 22 bp in the upstream half of the inverted repeat resulted in the same phenotype, whereas changing the 22 bp in the downstream half of the inverted repeat decreased nrd expression to a lesser extent in an exponentially growing culture and had only a smaller effect on nrd expression during DNA synthesis inhibition. As other mutants with the phenotype of the upstream inverted repeat mutant were found to lack cell cycle regulation, expression of nrd – lac mRNA produced from a plasmid with this mutation in the nrd–lacZ fusion gene was compared with nrd mRNA produced from the chromosomal nrd gene in a synchronized culture. The results indicated that the upstream half of the nrd inverted repeat contains a cis -acting element essential for nrd cell cycle regulation.     

Cell cycle regulation of ribonucleoside diphosphate reductase activity in permeable mouse L cells and in extracts

Ribonucleoside diphosphate reductase (EC1.17.4.1) was previously characterized in exponentially growing mouse L cells selectively permeabilized to small molecules by treatment with dextran sulfate (Kucera and Paulus, 1982b). This characterization has now been extended to cells in specific phases of the cell cycle and in transition between cell cycle phases, with activity studied both in situ (permeabilized cells) and in cell extracts. Cells at various stages in the cell cycle were obtained by unit-gravity sedimentation employing a commercially available reorienting chamber device, by G1 arrest induced by isoleucine limitation, and by metaphase arrest induced by Colcemid. G1 cells from both cycling and noncycling populations had negligible levels of ribonucleotide reductase activity as measured by CDP reduction both in situ and in extracts. When G1 arrested cells were allowed to progress to S phase, ribonucleotide reductase activity increased in parallel with [3H]thymidine incorporation into DNA. Ribonucleotide reductase activity in extracts increased at a somewhat greater rate than in situ activity. S phase ribonucleotide reductase activity measured in situ resembled the previously characterized activity in exponentially growing cells with respect to an absolute dependence on ATP or its analogs as positive allosteric effector, sensitivity to the negative allosteric effector dATP, and low susceptibility to stimulation by NADPH, dithiothreitol, and FeCl3. Disruption of permeabilized cells caused reductase activity to become highly dependent on the presence of both dithiothreitol and FeCl3. As synchronized cultures progressed from S into G2/M phase, no significant change in ribonucleotide reductase activity was seen. On the other hand, when cells that had been arrested in metaphase by Colcemid were allowed to resume cell cycle traversal by removing the drug, in situ ribonucleotide reductase activity decreased by 75% within 2.5 h. This decrease seemed to be a late mitotic event, since it was not correlated with the percentage of cells entering G1 phase. The cause of a subsequent slight increase of in situ ribonucleotide reductase activity is not clear. Parallel measurements of ribonucleotide reductase activity in cell extracts indicated also an initial decline accompanied by increasing dependence on added dithiols and FeCl3, followed by complete activity loss. Our results suggest a cell cycle pattern of ribonucleotide reductase activity that involves negligible levels in G1 phase, a progressive increase of activity upon entry into S phase paralleling overall DNA synthesis, continued retention of significant ribonucleotide reductase activity well into the metaphase period of mitosis, and a very rapid decline in activity during the later phases of mitosis. The periods of increase and decrease of ribonucleotide reductase activity were accompanied by modulation of the properties of the enzyme as indicated by differential changes in enzyme activity measured in situ and in extracts.     

Mutationally altered ribonucleotide reductase from Escherichia coli: characterization of mutations isolated on multicopy plasmids.

The Escherichia coli ribonucleotide reductase genes (nrd genes) were mutagenized at random. Point mutations were introduced in vitro into a recombinant nrd plasmid. Transformants were initially screened for altered tolerance toward the drug hydroxyurea and further characterized by enzymatic and immunological methods. The screening procedure could pick out defects in either of the two subunits of ribonucleotide reductase. Cells carrying the nrd plasmid pPS2 were earlier shown to have levels of ribonucleotide reductase molecules that were 10 to 20 times higher than those in wild-type cells. We now demonstrate that the enzymatic activity in gently lysed pPS2-containing cells on cellophane disks is six times higher than in wild-type cells. Supplementation of the pPS2-containing lysates with a purified thioredoxin system results in a further 4.5-fold stimulation of the enzymatic activity, which implies a functional shortage of the electron donor system(s) for ribonucleotide reduction in pPS2-containing cells.     

Regulation of ribonucleoside diphosphate reductase synthesis in Escherichia coli: increased enzyme synthesis as a result of inhibition of deoxyribonucleic acid synthesis.

Inhibition of deoxyribonucleic acid (DNA) synthesis in Escherichia coli by chemical inhibitors or by shifting cultures of temperature-sensitive elongation (dnaE and dnaB) or initiation (dnaA) mutants to nonpermissive conditions led to greatly increased synthesis of the enzyme ribonucleoside diphosphate reductase, which catalyzes the first reaction unique to the pathway leading to DNA replication. In contrast to the Gudas and Pardee proposed model for control of the synthesis of DNA repair enzymes, in which both DNA inhibition and DNA degradation are involved, DNA synthesis inhibition in recA, recB, recC, or lex strains results in increased synthesis of ribonucleotide reductase, which suggests that DNA degradation is not required. We propose that inhibition of DNA synthesis causes a cell to accumulate an unknown compound that stimulates the initiation of a new round of DNA replication, and that this same signal is used to induce ribonucleotide reductase synthesis.     

The small subunit of ribonucleotide reductase is encoded by one of the most abundant translationally regulated maternal RNAs in clam and sea urchin eggs

In both clam oocytes and sea urchin eggs, fertilization triggers the synthesis of a set of proteins specified by stored maternal mRNAs. One of the most abundant of these (p41) has a molecular weight of 41,000. This paper describes the identification of p41 as the small subunit of ribonucleotide reductase, the enzyme that provides the precursors necessary for DNA synthesis. This identification is based mainly on the amino acid sequence deduced from cDNA clones corresponding to p41, which shows homology with a gene in Herpes Simplex virus that is thought to encode the small subunit of viral ribonucleotide reductase. Comparison with the B2 (small) subunit of Escherichia coli ribonucleotide reductase also shows striking homology in certain conserved regions of the molecule. However, our attention was originally drawn to protein p41 because it was specifically retained by an affinity column bearing the monoclonal antibody YL 1/2, which reacts with alpha-tubulin (Kilmartin, J. V., B. Wright, and C. Milstein, 1982, J. Cell Biol., 93:576-582). The finding that this antibody inhibits the activity of sea urchin embryo ribonucleotide reductase confirmed the identity of p41 as the small subunit. The unexpected binding of the small subunit of ribonucleotide reductase can be accounted for by its carboxy-terminal sequence, which matches the specificity requirements of YL 1/2 as determined by Wehland et al. (Wehland, J., H. C. Schroeder, and K. Weber, 1984, EMBO [Eur. Mol. Biol. Organ.] J., 3:1295-1300). Unlike the small subunit, there is no sign of synthesis of a corresponding large subunit of ribonucleotide reductase after fertilization. Since most enzymes of this type require two subunits for activity, we suspect that the unfertilized oocytes contain a stockpile of large subunits ready for combination with newly made small subunits. Thus, synthesis of the small subunit of ribonucleotide reductase represents a very clear example of the developmental regulation of enzyme activity by control of gene expression at the level of translation.     

Deoxyribonucleotide metabolism and cyclic AMP resistance in hydroxyurea-resistant S49 T-lymphoma cells

We investigated the cell cycle regulation of deoxyribonucleoside triphosphate (dNTP) metabolism in hydroxyurea-resistant (HYUR) murine S49 T-lymphoma cell lines. Cell lines 10- to 40-fold more hydroxyurea-resistant were selected in a stepwise manner. These HYUR cells exhibited increased CDP reductase activity (5- to 8-fold) and increased dNTP pools (up to 5-fold) that appeared to result from increased activity of the M2 subunit (binding site of hydroxyurea) of ribonucleotide reductase. These characteristics remained stable when the cells were grown in the absence of hydroxyurea for up to 2 years. In both wild type and hydroxyurea-resistant cell populations synchronized by elutriation, dCTP and dTTP pools increased in S phase, whereas dATP and dGTP pools generally remained the same or decreased, suggesting that allosteric effector mechanisms were operating to regulate pool sizes. Additionally, CDP reductase activity measured in permeabilized cells increased in S phase in both wild type and hydroxyurea-resistant cells, suggesting a nonallosteric mechanism of increased ribonucleotide reductase activity during periods of active DNA synthesis. While wild type S49 cells could be arrested in the G1 phase of the cell cycle by dibutyryl cyclic AMP, hydroxyurea-resistant cell lines could not be arrested in the G1 phase by exogenous cyclic AMP or agents that elevate the concentration of endogenous cyclic AMP. These data suggest that cyclic AMP-generated G1 arrest in S49 cells might be mediated by the M2 subunit of ribonucleotide reductase.     

Iron deprivation decreases ribonucleotide reductase activity and DNA synthesis

The effects of the iron-chelator, desferrioxamine, and monoclonal antibodies against transferrin receptors of DNA synthesis and ribonucleotide reductase activity were examined in human leukemia K562 cells. Treatment of the cells with desferrioxamine resulted in decreases of ribonucleotide reductase activity, DNA synthesis, and cell growth. Exposure of the cells to anti-transferrin receptor antibody, 42/6, which blocks iron supplement into cells caused decreases of ribonucleotide reductase activity and DNA synthesis, in a parallel fashion. Decreases of ribonucleotide reductase activity and DNA synthesis by 42/6 were restored by the addition of ferric nitriloacetate. These results indicate that ribonucleotide reductase activity is dependent on the iron-supply and also regulates cell proliferation.     

Ribonucleotide reductase activity and deoxyribonucleoside triphosphate metabolism during the cell cycle of S49 wild-type and mutant mouse T-lymphoma cells

We investigated deoxyribonucleoside triphosphate metabolism in S49 mouse T-lymphoma cells synchronized in different phases of the cell cycle. S49 wild-type cultures enriched for G1 phase cells by exposure to dibutyryl cyclic AMP (Bt2cAMP) for 24 h had lower dCTP and dTTP pools but equivalent or increased pools of dATP and dGTP when compared with exponentially growing wild-type cells. Release from Bt2cAMP arrest resulted in a maximum enrichment of S phase occurring 24 h after removal of the Bt2cAMP, and was accompanied by an increase in dCTP and dTTP levels that persisted in colcemid-treated (G2/M phase enriched) cultures. Ribonucleotide reductase activity in permeabilized cells was low in G1 arrested cells, increased in S phase enriched cultures and further increased in G2/M enriched cultures. In cell lines heterozygous for mutations in the allosteric binding sites on the M1 subunit of ribonucleotide reductase, the deoxyribonucleotide pools in S phase enriched cultures were larger than in wild-type S49 cells, suggesting that feedback inhibition of ribonucleotide reductase is an important mechanism limiting the size of deoxyribonucleoside triphosphate pools. The M1 and M2 subunits of ribonucleotide reductase from wild-type S49 cells were identified on two-dimensional polyacrylamide gels, but showed no significant change in intensity during the cell cycle. These data are consistent with allosteric inhibition of ribonucleotide reductase during the G1 phase of the cycle and release of this inhibition during S phase. They suggest that the increase in ribonucleotide reductase activity observed in permeabilized S phase-enriched cultures may not be the result of increased synthesis of either the M1 or M2 subunit of the enzyme.     

Ribonucleotide reductase and the regulation of DNA replication: an old story and an ancient heritage

All organisms that synthesize their own DNA have evolved mechanisms for maintaining a constant DNA/cell mass ratio independent of growth rate. The DNA/cell mass ratio is a central parameter in the processes controlling the cell cycle. The co-ordination of DNA replication with cell growth involves multiple levels of regulation. DNA synthesis is initiated at specific sites on the chromosome termed origins of replication, and proceeds bidirectionally to elongate and duplicate the chromosome. These two processes, initiation and elongation, therefore determine the total rate of DNA synthesis in the cell. In Escherichia coli, initiation depends on the DnaA protein while elongation depends on a multiprotein replication factory that incorporates deoxyribonucleotides (dNTPs) into the growing DNA chain. The enzyme ribonucleotide reductase (RNR) is universally responsible for synthesizing the necessary dNTPs. In this review we examine the role RNR plays in regulating the total rate of DNA synthesis in E. coli and, hence, in maintaining constant DNA/cell mass ratios during normal growth and under conditions of DNA stress.     

Half-site reactivity of the tyrosyl radical of ribonucleotide reductase from Escherichia coli

A C-terminally truncated form of protein B2, the homodimeric small subunit of ribonucleotide reductase from Escherichia coli, was found as the result of an apparently specific proteolysis. Truncated homodimers contain an intact binuclear iron center and a normal tyrosyl radical but have no binding capacity for the other ribonucleotide reductase subunit, protein B1, and are consequently enzymatically inactive. Heterodimers, consisting of one full-length and one truncated polypeptide, formed spontaneously during a chelation-reconstitution cycle and were easily separated from the two homodimeric variants. The heterodimeric form of B2 shows a weak interaction with the B1 subunit resulting in low enzyme activity. Using heterodimers containing deuterated tyrosine on the full-length side and protonated tyrosine on the truncated side, we could demonstrate that the tyrosyl radical was randomly generated in one or the other of the two polypeptide chains of the heterodimeric B2 subunit. The small subunit of ribonucleotide reductase thus conforms to a half-site reactivity.     

Multiple cis-acting sites positively regulate Escherichia coli nrd expression

Regulation of nrd expression in Escherichia coli by cis -acting elements was found to be more complex than previously reported. At least five upstream sites appear to positively regulate nrd expression including a Fis binding site, a DnaA binding site, an AT-rich region, an inverted repeat and a 10 bp site between the AT-rich region and the inverted repeat. Double mutants defective in these sites indicate that all sites tested act independently when regulating nrd expression. As the decrease in nrd expression in exponentially growing cultures paralleled the decrease observed in DNA synthesis-inhibited cultures for all single and double mutants, we concluded that nrd is regulated by the same mechanism in these physiological states. As mutants unable to induce nrd expression during inhibition of DNA synthesis also fail to exhibit cell cycle-regulated nrd expression, we conclude that cell cycle nrd regulation is controlled by these same sites. Site-directed mutagenesis was used to show that the absence of an increase in nrd expression during DNA inhibition previously observed for deletion of the AT-rich region results from deletion of both the Fis binding site and the AT-rich region.     

Selective suppression of growth factor-induced cell cycle gene expression by Na+/H+ antiport inhibitors.

Activation of Na+/H+ exchange activity is a ubiquitous response to growth factors and has been implicated in the mitogenic response. Little is known of how the antiport influences events in the nucleus which ultimately control the cell cycle. Using potent Na+/H+ exchange inhibitors we show for normal mouse bone marrow-derived macrophages that this activity is required for the colony-stimulating factor-1-induced gene expression of the M1 and M2 subunits of ribonucleotide reductase, an enzyme critical for DNA synthesis. Suppression of M1 and M2 mRNA levels occurred when the inhibitors were added up to 8 h after the growth factor, mirroring their ability to prevent entry into S phase at similar times. Antiport activity was not required for the induction of other genes associated with cell cycle progression including proliferating cell nuclear antigen and the G1 cyclin, CYL1. These results highlight the differential expression of various cell cycle-associated genes and demonstrates that non-coordinate regulation of CYL1 cyclin and DNA synthesis gene expression can occur. The selective dependence of ribonucleotide reductase subunit gene expression on Na+/H+ exchange activity may provide a biochemical basis for the requirement of persistent antiporter activity during G1 for subsequent entry into S phase.     

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.     

Modulation of transferrin receptor expression by inhibitors of nucleic acid synthesis

We investigated the effects of the iron chelator desferrioxamine on the expression of transferrin receptors (TfR) by CCRF-CEM human T-cell leukaemia and B16 mouse melanoma cells growing in tissue culture. Desferrioxamine (DFOA) enhanced TfR expression when added in the dose range of 10(-5)-10(-4) to CCRF-CEM cells, but was toxic to these cells, the lower concentrations producing a slowing of cell growth with a build up in S-phase, while higher concentrations caused cell death with a block at the G1/S-phase interface. These toxic effects are compatible with its previously reported inhibition of the non-haem iron containing (M2) subunit of ribonucleotide reductase. In marked contrast, DFOA caused the growth of B16 melanoma cells to arrest in G1, without loss of cloning efficiency, and resulted in a fall in TfR expression to approximately 50% of control values. These results suggested that the effects of DFOA on TfR expression were linked to DNA synthesis rather than to a more generalised inhibition of iron-dependent cellular processes. It was subsequently found that inhibition of the M2 subunit of ribonucleotide reductase in CCRF-CEM cells with 5 X 10(-5) M hydroxyurea, which is not an iron chelator, also enhanced TfR expression, as did thymidine and cytosine arabinoside, which have different enzyme targets. By measuring cellular DNA and RNA content simultaneously it was shown that all of these agents caused unbalanced growth, i.e., inhibited DNA synthesis more than RNA synthesis. In contrast, 6-thioguanine was more inhibitory to RNA synthesis, and treatment with this drug caused a fall in TfR expression. Thus, although CCRF-CEM cells treated with DFOA show enhanced TfR expression, similar effects are also seen with other inhibitors of DNA synthesis, provided that RNA synthesis is allowed to continue. These results provide further evidence that the regulation of TfR expression by proliferating cells is specifically linked to DNA synthesis rather than to the iron requirements of other cellular processes.     

Amplification of the genes for both components of ribonucleotide reductase in hydroxyurea resistant mammalian cells

Ribonucleotide reductase catalyzes the formation of deoxyribonucleotides from ribonucleoside diphosphate precursors, and is a rate-limiting step in the synthesis of DNA. The enzyme consists of two dissimilar subunits usually called M1 and M2. The antitumor agent, hydroxyurea, is a specific inhibitor of DNA synthesis and acts by destroying the tyrosyl free radical of the M2 subunit of ribonucleotide reductase. Two highly drug resistant cell lines designated HR-15 and HR-30 were isolated by exposing a population of mouse L cells to increasing concentrations of hydroxyurea. HR-15 and HR-30 cells contained elevated levels of ribonucleotide reductase activity, and were 68 and 103 times, respectively, more resistant than wild type to the cytotoxic effects of hydroxyurea. Northern and Southern blot analysis indicated that the two drug resistant lines contained elevated levels of M2 mRNA and M2 gene copy numbers. Similar studies with M1 specific cDNA demonstrated that HR-15 and HR-30 cell lines also contained increased M1 message levels, and showed M1 gene amplification. Mutant cell lines altered in expression and copy numbers for both the M1 and M2 genes are useful for obtaining information relevant to the regulation of ribonucleotide reductase, and its role in DNA synthesis and cell proliferation.