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Query: UNIPROT:P06889 (Mol)
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A multicopy suppressor gene which rescues the temperature-sensitive growth defect of Saccharomyces cerevisiae mutants in the mitochondrial DNA (mtDNA) polymerase-encoding MIP1 gene has been isolated and identified as the RNR1 gene. This gene, whose transcript is cell cycle-regulated and mainly expressed at the G1 to S phase transition, encodes the large subunit of ribonucleotide reductase. This enzyme catalyses a limiting step in the production of deoxynucleotides needed for DNA synthesis. The presence of a high copy number of the RNR1 gene also decreases the accumulation of rho- mutants observed in diploids that harbour a single copy of the MIP1 gene. In cell cycle-synchronised cells, the presence of a high copy number of RNR1 does not modify its cell cycle transcription regulation and increases its transcript level by a factor of 10 throughout the cell cycle. Our results show that an increased supply of dNTPs in mitochondria can stimulate the mtDNA polymerase activity and indicate that the dNTP concentration may be rate limiting for the replication of mtDNA.
Mol Gen Genet 1995 Nov 01
PMID:Overexpression of the RNR1 gene rescues Saccharomyces cerevisiae mutants in the mitochondrial DNA polymerase-encoding MIP1 gene. 855 25

Salmonella typhimurium and Escherichia coli cells have two different class I ribonucleotide reductases encoded by the nrdEF and nrdAB operons. Despite the presence of one additional ribonucleotide reductase, the nrdAB-encoded enzyme is essential to the aerobic growth of the cell because nrdAB-defective mutants of both species are not viable in the presence of oxygen. Several factors controlling nrdAB gene transcription have been analysed intensively. Nothing is known about the expression of the nrdEF genes. To study this subject, and after cloning of E. coli nrdEF genes and sequencing of their 5' ends, the promoter of this operon has been identified by primer extension in both bacterial species. The +1 position was 691 bp and 692 bp upstream of the translational start points of the nrdE genes of S. typhimurium and E. coli, respectively. Downstream of the +1 position, and before the nrdE gene, two open reading frames (ORFs) of 81 and 136 amino acid residues are present in both bacteria. The synthesis of a polypeptide with a molecular mass of 9 kDa, corresponding to the first of these two ORFs, was observed by using the T7 RNA polymerase expression system. Comparison of the amino acid predicted sequence of this ORF reveals a significant similarity with glutaredoxin proteins. Competitive, reverse-transcription polymerase chain reaction experiments indicate that transcription from the nrdEF promoter normally takes place in wild-type cells. nrdEF transcription is increased by hydroxyurea, which inhibits class I ribonucleotide reductase activity, in both RecA+ and RecA- cells. nrdA(ts) mutants show a higher level of nrdEF transcription than wild-type cells at either the permissive or the restrictive temperature. nrdEF expression was unaffected by changes in DNA supercoiling whether caused by the introduction of either topA::Tn10 and hns::Tn10 mutations or by the inhibition of DNA gyrase with the antibiotic novobiocin. In contrast to the nrdAB genes, the nrdEF operon is not essential to the cells because nrdEF-defective mutants are viable under both aerobic and anaerobic conditions.
Mol Microbiol 1996 Feb
PMID:Promoter identification and expression analysis of Salmonella typhimurium and Escherichia coli nrdEF operons encoding one of two class I ribonucleotide reductases present in both bacteria. 882 Jun 48

Ribonucleotide reductase (EC 1. 17. 4. 1.) is an essential enzyme providing 2'-deoxy-ribonucleotides for DNA replication. Ribonucleotide reductase from Streptomyces aureofaciens was purified 3365-fold with a yield of 6.5%. After homogenization of cells by ultrasonic homogenizer and DNA removing by 7% (w/v) solution of streptomycin sulphate, the sample was chromatographed on a DEAE-Sepharose CL 6 B, Phenyl-Sepharose CL 4 B, Heparin-Sepharose CL 6 B and a Sephacryl S-200. The specific activity of the purified protein was 1740 pmol per s per mg. Sephacryl S-200 chromatography and sodium dodecyl sulphate-polyacrylamide gel electrophoresis revealed that in the presence of calcium ions the enzyme appears to be a dimer with an apparent molecular weight of 125.9 kDa. In the absence of calcium dimer dissociates into a monomer with the apparent molecular weight of 64.3 kDa. On the basis of these results, we suggest that calcium plays a role in the formation of the dimer, which is the biologically active form of ribonucleotide reductase.
Biochem Mol Biol Int 1996 Mar
PMID:Purification and partial characterization of Ca(2+)-dependent ribonucleotide reductase from Streptomyces aureofaciens. 882 8

The suc22+ gene of Schizosaccharomyces pombe encodes the small subunit of ribonucleotide reductase. Two transcripts that hybridise to suc22+ have previously been described: a constitutive transcript of 1.5 kb, and a transcript of approximately 1.9 kb that is induced when DNA replication is blocked by hydroxyurea. In this paper we show that both transcripts derive from the suc22+ gene, are polyadenylated, and have transcription initiation sites separated by approximately 550 nucleotides. The absence of translation initiation codons and predicted intron splice sites within this 550 nucleotide region suggests strongly that both transcripts encode the same protein. Under normal growth conditions, the larger suc22+ transcript is present at a very low level. This low level expression is periodic during the cell cycle, showing a pattern similar to that of other genes under regulation by MCB elements with a maximum in G1/S phase. Consistent with this, there are MCB elements upstream of the initiation site of the transcript. This pattern of expression contrasts with the continuous expression, at a much higher level, of the smaller suc22+ transcript. The larger suc22+ transcript is induced by exposure of cells to 4-nitroquinoline oxide (4-NQO),a UV-mimetic agent that causes DNA damage. The transcriptional response to 4-NQO is observed in cells previously arrested in G2 by a cdc2ts mutation, demonstrating that induction can occur outside S phase. We show that the rad1+ gene, part of the mitotic checkpoint, is required for induction of the large transcript. Exposure of cells to heat shock also induces the suc22+ large transcript: a consensus heat shock element has been identified upstream of the large transcript start site.
Mol Gen Genet 1996 Sep 13
PMID:Cell cycle, DNA damage and heat shock regulate suc22+ expression in fission yeast. 884 48

The large subunit of ribonucleotide reductase (RNR) contains a ten-stranded beta/alpha barrel of a new type consisting of two antiparallel halves. The two halves of the barrel are pseudo 2-fold-related, have similar folds but different additional intervening secondary structure elements and loops. The inner diameter of the RNR barrel, 15 A to 20 A, is significantly larger than for the (beta alpha)3 barrels. The larger barrel forms a stable framework which holds an inserted hairpin loop rigidly and exposes active site residues at its tip. The barrel organization allows three cysteine residues to be positioned close to each other without forming unfavorable disulfide bridges between Cys439 on the tip of the inserted loop and the redox-active cysteine residues on the barrel strands. Redox-active cysteine residues separated by more than 200 residues are held in close proximity to each other on adjacent barrel strands.
J Mol Biol 1996 Sep 27
PMID:The ten-stranded beta/alpha barrel in ribonucleotide reductase protein R1. 884 1

The three-dimensional structure of mouse ribonucleotide reductase R2 has been determined at 2.3 A resolution using molecular replacement and refined to an R-value of 19.1% (Rfree = 25%) with good stereo-chemistry. The overall tertiary structure architecture of mouse R2 is similar to that from Escherichia coli R2. However, several important structural differences are observed. Unlike the E. coli protein, the mouse dimer is completely devoid of beta-strands. The sequences differ significantly between the mouse and E. coli R2s, but there is high sequence identity among the eukaryotic R2 proteins, and the identities are localized over the whole sequence. Therefore, the three-dimensional structures of other mammalian ribonucleotide reductase R2 proteins are expected to be very similar to that of the mouse enzyme. In mouse R2 a narrow hydrophobic channel leads to the proposed binding site for molecular oxygen near to the iron-radical site in the interior of the protein. In E. coli R2 this channel is blocked by the phenyl ring of a tyrosine residue, which in mouse R2 is a serine. These structural variations may explain the observed differences in sensitivity to radical scavengers. The structure determination is based on diffraction data from crystals grown at pH 4.7. Unexpectedly, the protein is not iron-free, but contains one iron ion bound at one of the dinuclear iron sites. This ferric ion is bound with partial occupancy and is coordinated by three glutamic acids (one bidentate) and one histidine in a bipyramidal coordination that has a free apical coordination position. Soaking of crystals in a solution of ferrous salt at pH 4.7 increased the occupancy on the already occupied site, but without any detectable binding at the second site.
J Mol Biol 1996 Oct 11
PMID:The three-dimensional structure of mammalian ribonucleotide reductase protein R2 reveals a more-accessible iron-radical site than Escherichia coli R2. 887 48

Control of cell proliferation involves a finely interwoven network of positive and negative cell cycle regulators. Signal transduction pathways linking c-fms (CSF-1R) to cellular proliferation and differentiation are being explored. Part of the strategy is to use a series of G1 inhibitors to help pinpoint relevant targets. Several inhibitors-8Br-cAMP, interferon gamma (IFN gamma), INF alpha/beta, lipopolysaccharide (LPS), tumor necrosis factor-alpha (TNF alpha), and dimethylamiloride-suppress CSF-1-stimulated proliferation in murine bone marrow-derived macrophages (BMM) even when added in the mid- to late-G1 phase of the cell cycle. The down-modulating effects of the inhibitors on the expression of the following cell cycle regulators have been examined: c-myc, cyclin D1 and D2, cdk4, Rb phosphorylation, E2F binding activity, ribonucleotide reductase subunits, and PCNA. Some differences in the negative control of such regulators were found, for example, in the manner in which IFN gamma and cAMP down-regulate c-myc expression. Using blocking antibodies and BMM from type I IFN receptor knockout mice, it appears that one of these inhibitors, IFN alpha/beta, acts as an endogenous inhibitor in CSF-1-treated BMM and is also responsible, at least in part, for the inhibition of cell cycle progression by LPS and TNF alpha. Another strategy has been to attempt to relate early biochemical changes induced by CSF-1 to later changes in the G1 phase, partly by studying cycling versus noncycling macrophages and partly by using cells expressing c-fms with tyrosine mutations in the intracytoplasmic region. CSF-1-mediated effects on the following signal transduction molecules in these systems will be described: PI3-kinase, myelin basic protein kinases, Erks, and STAT transcription factors.
Mol Reprod Dev 1997 Jan
PMID:CSF-1 and cell cycle control in macrophages. 898 59

We have cloned and sequenced cDNAs coding for the R1 and R2 proteins of ribonucleotide reductase from zebrafish (Danio rerio). This ribonucleotide reductase shows high amino acid sequence identity to those of other vertebrates. The R1 cDNA has a coding sequence of 2382 bp, yielding a 794 amino acid protein, and the R2 cDNA has a coding sequence of 1158 bp, yielding a 386 amino acid protein. The zebrafish R1 shows 94% similarity and R2 shows 91% similarity to the human R1 and R2, respectively. The similarity extends to intron positions, of which the equivalent of mouse R2 intron 3 has been studied.
Mol Mar Biol Biotechnol 1996 Dec
PMID:Cloning and sequencing of cDNAs encoding ribonucleotide reductase from zebrafish Danio rerio. 898 96

The synthesis of DNA in CCl 39 cells is inhibited by the presence of the Fe2+ chelator bathophenanthroline disulfonate (BPS) when growth is stimulated by thrombin EGF plus insulin, but not by fetal calf serum. The presence of transferrin and Fe3+ in fetal calf serum can be the basis for lack of BPS effect with serum. The impermeable Fe3+ chelator Tiron does not, by itself, inhibit growth factor induced DNA synthesis, but it induces together with BPS inhibition on fetal calf serum induced DNA synthesis. The combined effect of BPS and Tiron is similar to inhibition of DNA synthesis by impermeable polyvalent DTPA which can chelate both Fe2+ and Fe3+ but does not inhibit ribonucleotide reductase in intact cells. Ferrous iron that bind BPS can relieve the inhibition at stoichiometric concentration. Ferric iron also prevents the inhibition even though it does not bind BPS. BPS does not inhibit DNA synthesis in HeLa cells. BPS reacts with iron from CCl 39 cells but not from HeLa cells. Data show that iron available for impermeable external chelators is in the ferrous state, and that exogenous iron should be reduced before it reverses the inhibition.
Biochem Mol Biol Int 1997 Feb
PMID:Inhibition of DNA synthesis in CCL 39 cells by impermeable iron chelators. 906 70

Flavodoxin reductase from Escherichia coli is an FAD-containing oxidoreductase that transports electrons between flavodoxin or ferredoxin and NADPH. Together with flavodoxin, the enzyme is involved in the reductive activation of three E. coli enzymes: cobalamin-dependent methionine synthase, pyruvate formate lyase and anaerobic ribonucleotide reductase. An additional function for the oxidoreductase appears to be to protect the bacteria against oxygen radicals. The three-dimensional structure of flavodoxin reductase has been solved by multiple isomorphous replacement, and has been refined at 1.7 A to an R-value of 18.4% and Rfree 24.8%. The monomeric molecule contains one beta-sandwich FAD domain and an alpha/beta NADP domain. The overall structure is similar to other reductases of the NADP-ferredoxin reductase family in spite of the low sequence similarities within the family. Flavodoxin reductase lacks the loop which is involved in the binding of the adenosine moiety of FAD in other FAD binding enzymes of the superfamily but is missing in the FMN binding phthalate dioxygenase reductase. Instead of this loop, the adenine interacts with an extra tryptophan at the C terminus. The FAD in flavodoxin reductase has an unusual bent conformation with a hydrogen bond between the adenine and the isoalloxazine. This is probably the cause of the unusual spectrum of the enzyme. There is a pronounced cleft close to the isoalloxazine that appears to be well suited for binding of flavodoxin/ferredoxin. Two extra short strands of the NADP-binding domain probably act as an anchor point for the binding of flavodoxin.
J Mol Biol 1997 Apr 25
PMID:The three-dimensional structure of flavodoxin reductase from Escherichia coli at 1.7 A resolution. 914 48


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