| 1. |
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| 2. |
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| 3. |
- Berner, Boel, 1945-, et al.
(författare)
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Inledning
- 2008
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Ingår i: Samhällsforskning 2008. Betydelsen för människorna, hembygden och regionen av ett slutförvar för använt kärnbränsle. - Stockholm : SKB.
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Bokkapitel (populärvet., debatt m.m.)
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| 4. |
- Berner, Boel, 1945-, et al.
(författare)
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Inledning
- 2007
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Ingår i: Samhällsforskning 2007. Betydelsen för människorna, hembygden och regionen av ett slutförvar för använt kärnbränsle. - Stockholm : SKB.
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Bokkapitel (populärvet., debatt m.m.)abstract
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| 5. |
- Berner, Boel, 1945-, et al.
(författare)
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Inledning
- 2006
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Ingår i: Samhällsforskning 2006. Betydelsen för människorna, hembygden och regionen av ett slutförvar för använt kärnbränsle. - Stockholm : SKB.
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Bokkapitel (populärvet., debatt m.m.)abstract
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| 6. |
- Berner, Boel, 1945-, et al.
(författare)
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Inledning
- 2005
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Ingår i: Samhällsforskning 2005. Betydelsen för människorna, hembygden och regionen av ett slutförvar för använt kärnbränsle. - Stockholm : SKB. - 9197560634
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Bokkapitel (populärvet., debatt m.m.)
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| 7. |
- Berner, Boel, et al.
(författare)
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Introduktion
- 2009
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Ingår i: Samhällsforskning 2009. - Stockholm : SKB. - 9789197786287 ; , s. 4-17
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Bokkapitel (populärvet., debatt m.m.)
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| 8. |
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| 9. |
- Due, Karin, 1953-
(författare)
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Fysik, lärande samtal och genus. : En studie av gymnasieelevers gruppdiskussioner i fysik.
- 2009
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- This thesis investigates how students in upper secondary school solve problems and discuss physics in small groups. The study examines how gender, knowledge in physics and the image of the subject physics are constructed in the students’ conversation and how these processes are related to each other. The theoretical framework includes a sociocultural perspective on learning and a gender perspective that views gender as both process and discourse and focus on how femininity and masculinity are constructed in social relations. 28 students in two classes at the science program participated in the study. 8 videotaped group discussions and 15 audiotaped interviews where analysed through thematic analysis and discourse analysis according to different research questions. The results show the complexity of a learning conversation. The character of the dialogue in the groups, the difficulties the student encounter when dealing with the tasks, and the social interaction in the groups, has a deep impact on the possibilities to develop the discussion about physics phenomena and concepts. The images of the subject physics and the images of a student skilled in physic that are constructed in the interviews are reconstructed in the students’ discussions and in their acting in the groups. But there are also inconsistencies and counter discourses. The possibilities for learning are related to the construction of gender and to equality issues within the groups. Traditional gendered positions are to a large extent reconstructed in the students’ interaction. Boys are for instance positioned as more competent in physics than girls. But traditional gendered positions are also resisted and challenged.
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| 10. |
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| 11. |
- Kolberg, Matthias, et al.
(författare)
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A new tyrosyl radical on Phe208 as ligand to the diiron center in Escherichia coli ribonucleotide reductase, mutant R2-Y122H. Combined x-ray diffraction and EPR/ENDOR studies.
- 2005
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Ingår i: J Biol Chem. - 0021-9258. ; 280:12, s. 11233-11246
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- The R2 protein subunit of class I ribonucleotide reductase (RNR) belongs to a structurally related family of oxygen bridged diiron proteins. In wild-type R2 of Escherichia coli, reductive cleavage of molecular oxygen by the diferrous iron center generates a radical on a nearby tyrosine residue (Tyr122), which is essential for the enzymatic activity of RNR, converting ribonucleotides into deoxyribonucleotides. In this work, we characterize the mutant E. coli protein R2-Y122H, where the radical site is substituted with a histidine residue. The x-ray structure verifies the mutation. R2-Y122H contains a novel stable paramagnetic center which we name H, and which we have previously proposed to be a diferric iron center with a strongly coupled radical, Fe(III)Fe(III)R.. Here we report a detailed characterization of center H, using 1H/2H -14N/15N- and 57Fe-ENDOR in comparison with the Fe(III)Fe(IV) intermediate X observed in the iron reconstitution reaction of R2. Specific deuterium labeling of phenylalanine residues reveals that the radical results from a phenylalanine. As Phe208 is the only phenylalanine in the ligand sphere of the iron site, and generation of a phenyl radical requires a very high oxidation potential, we propose that in Y122H residue Phe208 is hydroxylated, as observed earlier in another mutant (R2-Y122F/E238A), and further oxidized to a phenoxyl radical, which is coordinated to Fe1. This work demonstrates that small structural changes can redirect the reactivity of the diiron site, leading to oxygenation of a hydrocarbon, as observed in the structurally similar methane monoxygenase, and beyond, to formation of a stable iron-coordinated radical.
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| 12. |
- Larsson Birgander, Pernilla, et al.
(författare)
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Nucleotide-dependent formation of catalytically competent dimers from engineered monomeric ribonucleotide reductase protein R1
- 2005
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 280:15, s. 14997-15003
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Tidskriftsartikel (refereegranskat)abstract
- Each catalytic turnover by aerobic ribonucleotide reductase requires the assembly of the two proteins, R1 (α2) and R2 (β2), to produce deoxyribonucleotides for DNA synthesis. The R2 protein forms a tight dimer, whereas the strength of the R1 dimer differs between organisms, being monomeric in mouse R1 and dimeric in Escherichia coli. We have used the known E. coli R1 structure as a framework for design of eight different mutations that affect the helices and proximal loops that comprise the dimer interaction area. Mutations in loop residues did not affect dimerization, whereas mutations in the helices had very drastic effects on the interaction resulting in monomeric proteins with very low or no activity. The monomeric N238A protein formed an interesting exception, because it unexpectedly was able to reduce ribonucleotides with a comparatively high capacity. Gel filtration studies revealed that N238A was able to dimerize when bound by both substrate and effector, a result in accordance with the monomeric R1 protein from mouse. The effects of the N238A mutation, fit well with the notion that E. coli protein R1 has a comparatively small dimer interaction surface in relation to its size, and the results illustrate the stabilization effects of substrates and effectors in the dimerization process. The identification of key residues in the dimerization process and the fact that there is little sequence identity between the interaction areas of the mammalian and the prokaryotic enzymes may be of importance in drug design, similar to the strategy used in treatment of HSV infection.
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| 13. |
- Larsson Birgander, Pernilla, 1974-
(författare)
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The influence of nucleotides on ribonucleotide reductase assambly in class I ribonucleotide reductase from Escherichia coli
- 2005
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The components of DNA, the deoxyribonucleotides, are produced from the components of RNA, the ribonucleotides. One single substitution is needed to convert a ribonucleotide into a deoxyribonucleotide i.e. a replacement of a hydroxyl group with a hydrogen atom. The reaction is catalysed by ribonucleotide reductase, an enzyme that is present in all living organisms. At first this conversion may seem trivial but in reality it is a difficult chemical reaction requiring much energy. In ribonucleotide reductase this energy is provided by an amino acid radical that upon each catalytic turnover is transferred from its stable position in the R2 protein to the active site of protein R1. The need for deoxyribonucleotides in the cell varies, therefore the activity of ribonucleotide reductase must be regulated. A complex allosteric regulation controls both the level of enzymatic activity and the substrate specificity to make sure that the deoxyribonucleotides are produced in correct amounts. In this work, we have shown that the reason why enzymatic activity is turned off when dATP binds is due to formation of a constrained R1: R2 interaction and we have proposed that a conserved hydrogen bond is important in this mechanism. We evaluated the effect of nucleotides on the R1: R2 interaction further using the surface plasmon resonance technique and found that allosteric effectors and substrates as well as the presence of thioredoxin considerably enhances the interaction.The second allosteric site that controls substrate specificity is located at the interaction area of the two polypeptides constituting protein R1. We have identified key residues in the dimerisation process of the two polypeptides and we have established a stabilisation effect of allosteric effectors and substrates on the dimer interaction using a mutant protein. The radical is believed to be transferred between the two proteins that constitute ribonucleotide reductase by a chain of hydrogen bonded amino acid residues. We have developed a new in vivo activity assay in which we have shown the physiological importance of these residues. Another methodological approach in this work was an attempt to turn protein R1 of ribonucleotide reductase into a selenoprotein by genetically substituting one of the active site cysteines for a selenocysteine. In a selenocysteine-substituted protein, this particular residue can be distinguished from the other cysteines which would be advantageous in subsequent biophysical characterisations of thiyl radicals.
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| 14. |
- Lundin, Daniel, 1965-, et al.
(författare)
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RNRdb, a curated database of the universal enzyme family ribonucleotide reductase, reveals a high level of misannotation in sequences deposited to Genbank
- 2009
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Ingår i: BMC Genomics. - : Springer Science and Business Media LLC. - 1471-2164. ; 10:1
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Tidskriftsartikel (refereegranskat)abstract
- Ribonucleotide reductases (RNRs) catalyse the only known de novo pathway for deoxyribonucleotide synthesis, and are therefore essential to DNA-based life. While ribonucleotide reduction has a single evolutionary origin, significant differences between RNRs nevertheless exist, notably in cofactor requirements, subunit composition and allosteric regulation. These differences result in distinct operational constraints (anaerobicity, iron/oxygen dependence and cobalamin dependence), and form the basis for the classification of RNRs into three classes. PMID: 19995434
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| 15. |
- Nord, David, et al.
(författare)
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A functional homing endonuclease in the Bacillus anthracis nrdE group I intron
- 2007
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Ingår i: Journal of Bacteriology. - 0021-9193 .- 1098-5530. ; 189:14, s. 5293-5301
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Tidskriftsartikel (refereegranskat)abstract
- The essential Bacillus anthracis nrdE gene carries a self-splicing group I intron with a putative homing endonuclease belonging to the GIY-YIG family. Here, we show that the nrdE pre-mRNA is spliced and that the homing endonuclease cleaves an intronless nrdE gene 5 nucleotides (nt) upstream of the intron insertion site, producing 2-nt 3' extensions. We also show that the sequence required for efficient cleavage spans at least 4 bp upstream and 31 bp downstream of the cleaved coding strand. The position of the recognition sequence in relation to the cleavage position is as expected for a GIY-YIG homing endonuclease. Interestingly, nrdE genes from several other Bacillaceae were also susceptible to cleavage, with those of Bacillus cereus, Staphylococcus epidermidis (nrdE1), B. anthracis, and Bacillus thuringiensis serovar konkukian being better substrates than those of Bacillus subtilis, Bacillus lichenformis, and S. epidermidis (nrdE2). On the other hand, nrdE genes from Lactococcus lactis, Escherichia coli, Salmonella enterica serovar Typhimurium, and Corynebacterium ammoniagenes were not cleaved. Intervening sequences (IVSs) residing in protein-coding genes are often found in enzymes involved in DNA metabolism, and the ribonucleotide reductase nrdE gene is a frequent target for self-splicing IVSs. A comparison of nrdE genes from seven gram-positive low-G + C bacteria, two bacteriophages, and Nocardia farcinica showed five different insertion sites for self-splicing IVSs within the coding region of the nrdE gene.
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| 16. |
- Nord, David, 1975-
(författare)
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Homing Endonucleases and Horizontal Gene Transfer in Bacteria and Bacteriophages
- 2007
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Homing endonuclease genes (HEGs) are selfish genetic elements that mediate their own super-Mendelian inheritance. This is mediated by the homing endonuclease cleavage of a HEG- allele followed by recombination-repair with a HEG+ allele.The majority of the HEGs are encoded in intervening sequences (IVSs). The insertion of the IVS interrupts the endonuclease recognition site, making the genome with the IVS resistant to further cleavage by homing endonucleases with specificity for that particular sequence, but susceptible for homing endonucleases with a target not affected by the IVS insert. In 39 studied strains of the Bacillus cereus group, 28 IVSs were found in the nrdIEF operon. Phylogenetic studies of these sequences showed a scattered distribution of the IVSs, indicating a frequent horizontal gene transfer and that there might be competition between the different IVSs in the nrdIEF operon in the Bacillaceae family. One novel group I intron was shown to encode a functional homing endonuclease with a GIY-(X)8-YIG motif, expanding the family motif to GIY-(X)8-11-YIG. Interestingly, by studying the known insertion sites for IVSs in the ribonuclotide reductase genes, we show that the majority of the insertions are at conserved motifs, indicating that conservation is important for IVS survival.Most freestanding HEGs in bacteriophage T4 cleave both HEG+ and HEG- alleles, possibly providing a competitive advantage for the host organism when two phages infect the same bacterium. Two novel freestanding HEGs replace two putative HEGs in T4 in some T-even-like phages. The characterisation of these HEGs showed that both cleave double stranded DNA. SegH was shown to promote homing of its gene. Hef showed no homing, possibly due to general exclusion of other phages. The mobE putative HEG was shown to be homing proficient and showed strong general DNA degradation when expressed in Escherichia coli.
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| 17. |
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| 18. |
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| 19. |
- Rofougaran, Reza, et al.
(författare)
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Oligomerization status directs overall activity regulation of the Escherichia coli class Ia ribonucleotide reductase
- 2008
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Ingår i: Journal of Biological Chemistry. - : American Society for Biochemistry and Molecular Biology. - 0021-9258 .- 1083-351X. ; 283:51, s. 35310-35318
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Tidskriftsartikel (refereegranskat)abstract
- Ribonucleotide reductase (RNR) is a key enzyme for the synthesis of the four DNA building blocks. Class Ia RNRs contain two subunits, denoted R1 (α) and R2 (β). These enzymes are regulated via two nucleotide-binding allosteric sites on the R1 subunit, termed the specificity and overall activity sites. The specificity site binds ATP, dATP, dTTP, or dGTP and determines the substrate to be reduced, whereas the overall activity site binds dATP (inhibitor) or ATP. By using gas-phase electrophoretic mobility macromolecule analysis and enzyme assays, we found that the Escherichia coli class Ia RNR formed an inhibited α4β4 complex in the presence of dATP and an active α2β2 complex in the presence of ATP (main substrate: CDP), dTTP (substrate: GDP) or dGTP (substrate: ADP). The R1-R2 interaction was 30–50 times stronger in the α4β4 complex than in the α2β2complex, which was in equilibrium with free α2 and β2 subunits. Studies of a known E. coli R1 mutant (H59A) showed that deficient dATP inhibition correlated with reduced ability to form α4β4 complexes. ATP could also induce the formation of a generally inhibited α4β4 complex in the E. coli RNR but only when used in combination with high concentrations of the specificity site effectors, dTTP/dGTP. Both allosteric sites are therefore important for α4β4 formation and overall activity regulation. The E. coli RNR differs from the mammalian enzyme, which is stimulated by ATP also in combination with dGTP/dTTP and forms active and inactive α6β2 complexes.
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| 20. |
- Sandegren, Linus, et al.
(författare)
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SegH and Hef: two novel homing endonucleases whose genes replace the mobC and mobE genes in several T4-related phages.
- 2005
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Ingår i: Nucleic Acids Res. - 1362-4962. ; 33:19, s. 6203-13
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- T4 contains two groups of genes with similarity to homing endonucleases, the seg-genes (similarity to endonucleases encoded by group I introns) containing GIY-YIG motifs and the mob-genes (similarity to mobile endonucleases) containing H-N-H motifs. The four seg-genes characterized to date encode homing endonucleases with cleavage sites close to their respective gene loci while none of the mob-genes have been shown to cleave DNA. Of 18 phages screened, only T4 was found to have mobC while mobE genes were found in five additional phages. Interestingly, three phages encoded a seg-like gene (hereby called segH) with a GIY-YIG motif in place of mobC. An additional phage has an unrelated gene called hef (homing endonuclease-like function) in place of the mobE gene. The gene products of both novel genes displayed homing endonuclease activity with cleavage site specificity close to their respective genes. In contrast to intron encoded homing endonucleases, both SegH and Hef can cleave their own DNA as well as DNA from phages without the genes. Both segH and mobE (and most likely hef) can home between phages in mixed infections. We discuss why it might be a selective advantage for phage freestanding homing endonucleases to cleave both HEG-containing and HEG-less genomes.
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| 21. |
- Sandegren, Linus, et al.
(författare)
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Self-splicing of the bacteriophage T4 group I introns requires efficient translation of the pre-mRNA in vivo and correlates with the growth state of the infected bacterium.
- 2007
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Ingår i: J Bacteriol. - 0021-9193. ; 189:3, s. 980-90
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- Bacteriophage T4 contains three self-splicing group I introns in genes in de novo deoxyribonucleotide biosynthesis (in td, coding for thymidylate synthase and in nrdB and nrdD, coding for ribonucleotide reductase). Their presence in these genes has fueled speculations that the introns are retained within the phage genome due to a possible regulatory role in the control of de novo deoxyribonucleotide synthesis. To study whether sequences in the upstream exon interfere with proper intron folding and splicing, we inhibited translation in T4-infected bacteria as well as in bacteria containing recombinant plasmids carrying the nrdB intron. Splicing was strongly reduced for all three T4 introns after the addition of chloramphenicol during phage infection, suggesting that the need for translating ribosomes is a general trait for unperturbed splicing. The splicing of the cloned nrdB intron was markedly reduced in the presence of chloramphenicol or when translation was hindered by stop codons inserted in the upstream exon. Several exon regions capable of forming putative interactions with nrdB intron sequences were identified, and the removal or mutation of these exon regions restored splicing efficiency in the absence of translation. Interestingly, splicing of the cloned nrdB intron was also reduced as cells entered stationary phase and splicing of all three introns was reduced upon the T4 infection of stationary-phase bacteria. Our results imply that conditions likely to be frequently encountered by natural phage populations may limit the self-splicing efficiency of group I introns. This is the first time that environmental effects on bacterial growth have been linked to the regulation of splicing of phage introns.
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| 22. |
- Torrents, Eduard, et al.
(författare)
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Efficient growth inhibition of Bacillus anthracis by knocking out the ribonucleotide reductase tyrosyl radical.
- 2005
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Ingår i: Proc Natl Acad Sci U S A. - 0027-8424. ; 102:50, s. 17946-51
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- Bacillus anthracis, the causative agent of anthrax, is a worldwide problem because of the need for effective treatment of respiratory infections shortly after exposure. One potential key enzyme of B. anthracis to be targeted by antiproliferative drugs is ribonucleotide reductase. It provides deoxyribonucleotides for DNA synthesis needed for spore germination and growth of the pathogen. We have cloned, purified, and characterized the tyrosyl radical-carrying NrdF component of B. anthracis class Ib ribonucleotide reductase. Its EPR spectrum points to a hitherto unknown three-dimensional geometry of the radical side chain with a 60 degrees rotational angle of C(alpha)-(C(beta)-C(1))-plane of the aromatic ring. The unusual relaxation behavior of the radical signal and its apparent lack of line broadening at room temperature suggest a weak interaction with the nearby diiron site and the presence of a water molecule plausibly bridging the phenolic oxygen of the radical to a ligand of the diiron site. We show that B. anthracis cells are surprisingly resistant to the radical scavenger hydroxyurea in current use as an antiproliferative drug, even though its NrdF radical is efficiently scavenged in vitro. Importantly, the antioxidants hydroxylamine and N-methyl hydroxylamine scavenge the radical several orders of magnitude faster and prevent B. anthracis growth at several hundred-fold lower concentrations compared with hydroxyurea. Phylogenetically, the B. anthracis NrdF protein clusters together with NrdFs from the pathogens Bacillus cereus, Bacillus thuringiensis, Staphylococcus aureus, and Staphylococcus epidermidis. We suggest the potential use of N-hydroxylamines in combination therapies against infections by B. anthracis and closely related pathogens.
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| 23. |
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| 24. |
- Torrents, Eduard, et al.
(författare)
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Ribonucleotide reductase modularity : Atypical duplication of the ATP-cone domain in Pseudomonas aeruginosa.
- 2006
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Ingår i: J Biol Chem. - 0021-9258. ; 281:35, s. 25287-96
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- The opportunistic pathogen Pseudomonas aeruginosa, which causes serious nosocomial infections, is a gamma-proteobacterium that can live in many different environments. Interestingly P. aeruginosa encodes three ribonucleotide reductases (RNRs) that all differ from other well known RNRs. The RNR enzymes are central for de novo synthesis of deoxyribonucleotides and essential to all living cells. The RNR of this study (class Ia) is a complex of the NrdA protein harboring the active site and the allosteric sites and the NrdB protein harboring a tyrosyl radical necessary to initiate catalysis. P. aeruginosa NrdA contains an atypical duplication of the N-terminal ATP-cone, an allosteric domain that can bind either ATP or dATP and regulates the overall enzyme activity. Here we characterized the wild type NrdA and two truncated NrdA variants with precise N-terminal deletions. The N-terminal ATP-cone (ATP-c1) is allosterically functional, whereas the internal ATP-cone lacks allosteric activity. The P. aeruginosa NrdB is also atypical with an unusually short lived tyrosyl radical, which is efficiently regenerated in presence of oxygen as the iron ions remain tightly bound to the protein. The P. aeruginosa wild type NrdA and NrdB proteins form an extraordinarily tight complex with a suggested alpha4beta4 composition. An alpha2beta2 composition is suggested for the complex of truncated NrdA (lacking ATP-c1) and wild type NrdB. Duplication or triplication of the ATP-cone is found in some other bacterial class Ia RNRs. We suggest that protein modularity built on the common catalytic core of all RNRs plays an important role in class diversification within the RNR family.
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| 25. |
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| 26. |
- Torrents, Eduard, et al.
(författare)
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Two proteins mediate class II ribonucleotide reductase activity in Pseudomonas aeruginosa : expression and transcriptional analysis of the aerobic enzymes.
- 2005
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Ingår i: J Biol Chem. - 0021-9258. ; 280:17, s. 16571-8
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- The opportunistic human pathogen Pseudomonas aeruginosa is one of a few microorganisms that code for three different classes (I, II, and III) of the enzyme ribonucleotide reductase (RNR). Class II RNR of P. aeruginosa differs from all hitherto known class II enzymes by being encoded by two consecutive open reading frames denoted nrdJa and nrdJb and separated by 16 bp. Split nrdJ genes were also found in the few other gamma-proteobacteria that code for a class II RNR. Interestingly, the two genes encoding the split nrdJ in P. aeruginosa were co-transcribed, and both proteins were expressed. Exponentially growing aerobic cultures were predominantly expressing the class I RNR (encoded by the nrdAB operon) compared with the class II RNR (encoded by the nrdJab operon). Upon entry to stationary phase, the relative amount of nrdJa transcript increased about 6-7-fold concomitant with a 6-fold decrease in the relative amount of nrdA transcript. Hydroxyurea treatment known to knock out the activity of class I RNR caused strict growth inhibition of P. aeruginosa unless 5'-deoxyadenosylcobalamin, a cofactor specifically required for activity of class II RNRs, was added to the rich medium. Rescue of the hydroxyurea-treated cells in the presence of the vitamin B12 cofactor strongly implies that P. aeruginosa produces a functionally active NrdJ protein. Biochemical studies showed for the first time that presence of both NrdJa and NrdJb subunits were absolutely essential for enzyme activity. Based on combined genetic and biochemical results, we suggest that the two-component class II RNR in P. aeruginosa is primarily used for DNA repair and/or possibly DNA replication at low oxygen tension.
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