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  • 1.
    Björklund, Stefan
    et al.
    Umeå universitet, Medicinsk fakultet, Medicinsk kemi och biofysik.
    Hjortsberg, K
    Johansson, Erik
    Umeå universitet, Medicinsk fakultet, Medicinsk kemi och biofysik.
    Thelander, Lars
    Umeå universitet, Medicinsk fakultet, Medicinsk kemi och biofysik.
    Structure and promoter characterization of the gene encoding the large subunit (R1 protein) of mouse ribonucleotide reductase.1993Ingår i: Proceedings of the National Academy of Science U S A, ISSN 0027-8424, Vol. 90, nr 23, s. 11322-6Artikel i tidskrift (Refereegranskat)
  • 2.
    Björklund, Stefan
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Skog, Sven
    Tribukait, Bernard
    Thelander, Lars
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    S-phase-specific expression of mammalian ribonucleotide reductase R1 and R2 subunit mRNAs1990Ingår i: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 29, nr 23, s. 5452-5458Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Ribonucleotide reductase in mammalian cells is composed of two nonidentical subunits, proteins R1 and R2, each inactive alone. The R1 protein is present in excess in proliferating cells, and its levels are constant during the cell cycle. Expression of the R2 protein, which is limiting for enzyme activity, is strictly S-phase-correlated. In this paper, we have used antisense RNA probes in a solution hybridization assay to measure the levels of R1 and R2 mRNA during the cell cycle in centrifugally elutriated cells and in cells synchronized by isoleucine or serum starvation. The levels of both transcripts were very low or undetectable in G0/G1-phase cells, showed a pronounced increase as cells progressed into S phase, and then declined when cells progressed into G2 + M phase. The R1 and R2 transcripts increased in parallel, starting slightly before the rise in S-phase cells, and reached the same levels. The relative lack of cell cycle dependent variation in R1 protein levels, obtained previously, may therefore simply be a consequence of the long half-life of the R1 protein. Hydroxyurea-resistant, R2-overproducing mouse TA3 cells showed the same regulation of the R1 and R2 transcripts as the parental cells, but with R2 mRNA at a 40-fold higher level.

  • 3.
    Björklund, Stefan
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Skogman, E
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Thelander, Lars
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    An S-phase specific release from a transcriptional block regulates the expression of mouse ribonucleotide reductase R2 subunit1992Ingår i: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 11, nr 13, s. 4953-4959Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Ribonucleotide reductase (RR) activity in mammalian cells is closely linked to DNA synthesis. The RR enzyme is composed of two non-identical subunits, proteins R1 and R2. Both proteins are required for holoenzyme activity, which is regulated by S-phase specific de novo synthesis and breakdown of the R2 subunit. In quiescent cells stimulated to proliferate and in elutriated cell populations enriched in the various cell cycle phases the R2 protein levels are correlated to R2 mRNA levels that are low in G0/G1-phase cells but increase dramatically at the G1/S border. Using an R2 promoter-luciferase reporter gene construct we demonstrate an unexpected early activation of the R2 promoter as cells pass from quiescence to proliferation. However, due to a transcriptional block, this promoter activation only results in very short R2 transcripts until cells enter the S-phase, when full-length R2 transcripts start to appear. The position for the transcriptional block was localized to a nucleotide sequence approximately 87 bp downstream from the first exon/intron boundary by S1 nuclease mapping of R2 transcripts from modified in vitro nuclear run-on experiments. These results identify blocking of transcription as a mechanism to control cell cycle regulated gene expression.

  • 4.
    Chabes, Andrei
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Domkin, Vladimir
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Larsson, Göran
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Liu, Aimin
    Gräslund, Astrid
    Wijmenga, Sybren S
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Thelander, Lars
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Yeast ribonucleotide reductase has a heterodimeric iron-radical-containing subunit2000Ingår i: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 97, nr 6, s. 2474-2479Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Ribonucleotide reductase (RNR) catalyzes the de novo synthesis of deoxyribonucleotides. Eukaryotes have an alpha(2)beta(2) form of RNR consisting of two homodimeric subunits, proteins R1 (alpha(2)) and R2 (beta(2)). The R1 protein is the business end of the enzyme containing the active site and the binding sites for allosteric effectors. The R2 protein is a radical storage device containing an iron center-generated tyrosyl free radical. Previous work has identified an RNR protein in yeast, Rnr4p, which is homologous to other R2 proteins but lacks a number of conserved amino acid residues involved in iron binding. Using highly purified recombinant yeast RNR proteins, we demonstrate that the crucial role of Rnr4p (beta') is to fold correctly and stabilize the radical-storing Rnr2p by forming a stable 1:1 Rnr2p/Rnr4p complex. This complex sediments at 5.6 S as a betabeta' heterodimer in a sucrose gradient. In the presence of Rnr1p, both polypeptides of the Rnr2p/Rnr4p heterodimer cosediment at 9.7 S as expected for an alpha(2)betabeta' heterotetramer, where Rnr4p plays an important role in the interaction between the alpha(2) and the betabeta ' subunits. The specific activity of the Rnr2p complexed with Rnr4p is 2,250 nmol deoxycytidine 5'-diphosphate formed per min per mg, whereas the homodimer of Rnr2p shows no activity. This difference in activity may be a consequence of the different conformations of the inactive homodimeric Rnr2p and the active Rnr4p-bound form, as shown by CD spectroscopy. Taken together, our results show that the Rnr2p/Rnr4p heterodimer is the active form of the yeast RNR small subunit.

  • 5.
    Chabes, Andrei
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Georgieva, Bilyana
    Domkin, Vladimir
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Zhao, Xiaolan
    Rothstein, Rodney
    Thelander, Lars
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Survival of DNA damage in yeast directly depends on increased dNTP levels allowed by relaxed feedback inhibition of ribonucleotide reductase.2003Ingår i: Cell, ISSN 0092-8674, E-ISSN 1097-4172, Vol. 112, nr 3, s. 391-401Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    In eukaryotes, DNA damage elicits a multifaceted response that includes cell cycle arrest, transcriptional activation of DNA repair genes, and, in multicellular organisms, apoptosis. We demonstrate that in Saccharomyces cerevisiae, DNA damage leads to a 6- to 8-fold increase in dNTP levels. This increase is conferred by an unusual, relaxed dATP feedback inhibition of ribonucleotide reductase (RNR). Complete elimination of dATP feedback inhibition by mutation of the allosteric activity site in RNR results in 1.6-2 times higher dNTP pools under normal growth conditions, and the pools increase an additional 11- to 17-fold during DNA damage. The increase in dNTP pools dramatically improves survival following DNA damage, but at the same time leads to higher mutation rates. We propose that increased survival and mutation rates result from more efficient translesion DNA synthesis at elevated dNTP concentrations.

  • 6.
    Chabes, Andrei
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Thelander, Lars
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    DNA building blocks at the foundation of better survival.2003Ingår i: Cell Cycle, ISSN 1538-4101, E-ISSN 1551-4005, Vol. 2, nr 3, s. 171-3Artikel i tidskrift (Refereegranskat)
  • 7.
    Chabes, Anna Lena
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Björklund, Stefan
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Thelander, Lars
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    S phase-specific transcription of the mouse ribonucleotide reductase R2 gene is dependent on an upstream promoter activating region and a proximal repressive E2F binding site.Manuskript (Övrigt vetenskapligt)
  • 8.
    Chabes, Anna Lena
    et al.
    Umeå universitet, Medicinsk fakultet, Medicinsk kemi och biofysik.
    Björklund, Stefan
    Umeå universitet, Medicinsk fakultet, Medicinsk kemi och biofysik.
    Thelander, Lars
    Umeå universitet, Medicinsk fakultet, Medicinsk kemi och biofysik.
    S Phase-specific transcription of the mouse ribonucleotide reductase R2 gene requires both a proximal repressive E2F-binding site and an upstream promoter activating region.2004Ingår i: Journal of biological chemistry, ISSN 0021-9258, Vol. 279, nr 11, s. 10796-807Artikel i tidskrift (Refereegranskat)
  • 9.
    Chabes, Anna Lena
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Pfleger, C.M.
    Kirschner, M.W.
    Thelander, Lars
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Mouse ribonucleotide reductase R2 protein: A new target for anaphase-promoting complex-Cdh1-mediated proteolysis.2003Ingår i: Proc. Natl. Acad. Sci. U S A., Vol. 100, nr 7, s. 3925-3929Artikel i tidskrift (Refereegranskat)
  • 10.
    Domkin, Vladimir
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Thelander, Lars
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Chabes, Andrei
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Yeast DNA damage-inducible Rnr3 has a very low catalytic activity strongly stimulated after the formation of a cross-talking Rnr1/Rnr3 complex.2002Ingår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 277, nr 21, s. 18574-8Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The ribonucleotide reductase system in Saccharomyces cerevisiae includes four genes (RNR1 and RNR3 encoding the large subunit and RNR2 and RNR4 encoding the small subunit). RNR3 expression, nearly undetectable during normal growth, is strongly induced by DNA damage. Yet an rnr3 null mutant has no obvious phenotype even under DNA damaging conditions, and the contribution of RNR3 to ribonucleotide reduction is not clear. To investigate the role of RNR3 we expressed and characterized the Rnr3 protein. The in vitro activity of Rnr3 was less than 1% of the Rnr1 activity. However, a strong synergism between Rnr3 and Rnr1 was observed, most clearly demonstrated in experiments with the catalytically inactive Rnr1-C428A mutant, which increased the endogenous activity of Rnr3 by at least 10-fold. In vivo, the levels of Rnr3 after DNA damage never reached more than one-tenth of the Rnr1 levels. We propose that heterodimerization of Rnr3 with Rnr1 facilitates the recruitment of Rnr3 to the ribonucleotide reductase holoenzyme, which may be important when Rnr1 is limiting for dNTP production. In complex with inactive Rnr1-C428A, the activity of Rnr3 is controlled by effector binding to Rnr1-C428A. This result indicates cross-talk between the Rnr1 and Rnr3 polypeptides of the large subunit.

  • 11.
    Elfving, Anna
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Thelander, Lars
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Characterization of the promoters of the mouse ribonucleotide reductase R1, R2 and p53R2-genesManuskript (preprint) (Övrigt vetenskapligt)
    Abstract [en]

    Reduction of ribonucleotides into deoxyribonucleotides is catalyzed by the enzyme ribonucleotide reductase. The mouse enzyme is composed of two protein subunits, the R1 protein and the R2/p53R2 protein, and its subunit constellation differs during the cell cycle. We report here how the promoters of each of these subunits are regulated during the cell cycle. Previous DNase footprinting experiments of the R1 and the R2 promoter gave us an idea of how these promoters are structured. The R1 promoter contains four elements; Inr, α (binding YY1), β (binding YY1) and γ, while the R2 promoter contains four different elements; TATA-box (binding TBP), CCAAT-box (binding NFY), E2F element (binding E2F4) and an upstream activating region. The p53R2 promoter is uncharacterized; only the transcription start has been suggested in Genebank.

    We found that activation of both subunits needed for S phase specific activity (R1 and R2) is dependent of release of the repressor E2F4 from each promoter. Previous results showed that the mouse R2 promoter harbors an E2F4 binding element and our result, using transient transfections, indicates that this is also the case for the mouse R1 promoter. Using primer extension experiments on the mouse p53R2 promoter we show that the transcription start colocalizes with an earlier unidentified Inr element similar to the Inr element in the mouse R1 promoter. Our transcription start site is localized 126 bp downstream from the start site indicated in Genebank. We also show that it is possible to partially purify the transcription factor(s) binding to the upstream activating region in the mouse R2 promoter by using phosphocellulose chromatography and oligonucleotides immobilized on magnetic beads.

  • 12.
    Fijolek, Artur
    et al.
    Umeå universitet, Medicinsk fakultet, Medicinsk kemi och biofysik.
    Hofer, Anders
    Umeå universitet, Medicinsk fakultet, Medicinsk kemi och biofysik.
    Thelander, Lars
    Umeå universitet, Medicinsk fakultet, Medicinsk kemi och biofysik.
    Expression, purification, characterization, and in vivo targeting of trypanosome CTP synthetase for treatment of African sleeping sickness.2007Ingår i: Journal of biological chemistry, ISSN 0021-9258, Vol. 282, nr 16, s. 11858-11865Artikel i tidskrift (Refereegranskat)
  • 13. Filatov, D
    et al.
    Björklund, Stefan
    Umeå universitet, Medicinsk fakultet, Medicinsk kemi och biofysik.
    Johansson, Erik
    Umeå universitet, Medicinsk fakultet, Medicinsk kemi och biofysik.
    Thelander, Lars
    Umeå universitet, Medicinsk fakultet, Medicinsk kemi och biofysik.
    Induction of the mouse ribonucleotide reductase R1 and R2 genes in response to DNA damage by UV light.1996Ingår i: Journal of Biological Chemistry, ISSN 0021-9258, Vol. 271, nr 39, s. 23698-704Artikel i tidskrift (Refereegranskat)
  • 14.
    Guittet, Olivier
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Håkansson, Pelle
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Voevodskaya, Nina
    Fridd, Susan
    Gräslund, Astrid
    Arakawa, Hirofumi
    Nakamura, Yusuke
    Thelander, Lars
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Mammalian p53R2 protein forms an active ribonucleotide reductase in vitro with the R1 protein, which is expressed both in resting cells in response to DNA damage and in proliferating cells2001Ingår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 276, nr 44, s. 40647-40651Artikel i tidskrift (Refereegranskat)
  • 15. Hayakawa, H
    et al.
    Hofer, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Thelander, Lars
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Kitajima, S
    Cai, Y
    Oshiro, S
    Yakushiji, H
    Nakabeppu, Y
    Kuwano, M
    Sekiguchi, M
    Metabolic fate of oxidized guanine ribonucleotides in mammalian cells.1999Ingår i: Biochemistry, ISSN 0006-2960, Vol. 38, nr 12, s. 3610-4Artikel i tidskrift (Refereegranskat)
  • 16.
    Hofer, Anders
    et al.
    Umeå universitet, Medicinsk fakultet, Medicinsk kemi och biofysik.
    Ekanem, J T
    Thelander, Lars
    Umeå universitet, Medicinsk fakultet, Medicinsk kemi och biofysik.
    Allosteric regulation of Trypanosoma brucei ribonucleotide reductase studied in vitro and in vivo.1998Ingår i: Journal of Biological Chemistry, ISSN 0021-9258, Vol. 273, nr 51, s. 34098-104Artikel i tidskrift (Refereegranskat)
  • 17.
    Hofer, Anders
    et al.
    Umeå universitet, Medicinsk fakultet, Medicinsk kemi och biofysik.
    Schmidt, P P
    Gräslund, Astrid
    Thelander, Lars
    Umeå universitet, Medicinsk fakultet, Medicinsk kemi och biofysik.
    Cloning and characterization of the R1 and R2 subunits of ribonucleotide reductase from Trypanosoma brucei.1997Ingår i: Proceedings of the National Academy of Sciences of the U S A, ISSN 0027-8424, Vol. 94, nr 13, s. 6959-64Artikel i tidskrift (Refereegranskat)
  • 18.
    Hofer, Anders
    et al.
    Umeå universitet, Medicinsk fakultet, Medicinsk kemi och biofysik.
    Steverding, D
    Chabes, Andrei
    Umeå universitet, Medicinsk fakultet, Medicinsk kemi och biofysik.
    Brun, R
    Thelander, Lars
    Umeå universitet, Medicinsk fakultet, Medicinsk kemi och biofysik.
    Trypanosoma brucei CTP synthetase: a target for the treatment of African sleeping sickness.2001Ingår i: Proceedings of the National Academy of Sciences of the U S A, ISSN 0027-8424, Vol. 98, nr 11, s. 6412-6Artikel i tidskrift (Refereegranskat)
  • 19.
    Håkansson, Pelle
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Dahl, Lina
    Chilkova, Olga
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Domkin, Vladimir
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Thelander, Lars
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    The Schizosaccharomyces pombe replication inhibitor Spd1 regulates ribonucleotide reductase activity and dNTPs by binding to the large Cdc22 subunit.2006Ingår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 281, nr 3, s. 1778-1783Artikel i tidskrift (Refereegranskat)
  • 20.
    Håkansson, Pelle
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Hofer, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Thelander, Lars
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Regulation of mammalian ribonucleotide reduction and dNTP pools after DNA damage and in resting cells.2006Ingår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 281, nr 12, s. 7834-7841Artikel i tidskrift (Refereegranskat)
  • 21.
    Johansson, Erik
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Björklund, Stefan
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Thelander, Lars
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Gene structure and regulation of the expression of the R1 and R2 subunits of mouse ribonucleotide reductase.1994Ingår i: Advances in Experimental Medicine and Biology, ISSN 0065-2598, E-ISSN 2214-8019, Vol. 370, s. 721-4Artikel i tidskrift (Refereegranskat)
  • 22.
    Johansson, Erik
    et al.
    Umeå universitet, Medicinsk fakultet, Medicinsk kemi och biofysik.
    Hjortsberg, K
    Thelander, Lars
    Umeå universitet, Medicinsk fakultet, Medicinsk kemi och biofysik.
    Two YY-1-binding proximal elements regulate the promoter strength of the TATA-less mouse ribonucleotide reductase R1 gene.1998Ingår i: Journal of Biological Chemistry, ISSN 0021-9258, Vol. 273, nr 45, s. 29816-21Artikel i tidskrift (Refereegranskat)
  • 23.
    Johansson, Erik
    et al.
    Umeå universitet, Medicinsk fakultet, Medicinsk kemi och biofysik.
    Skogman, E
    Thelander, Lars
    Umeå universitet, Medicinsk fakultet, Medicinsk kemi och biofysik.
    The TATA-less promoter of mouse ribonucleotide reductase R1 gene contains a TFII-I binding initiator element essential for cell cycle-regulated transcription.1995Ingår i: Journal of Biological Chemistry, ISSN 0021-9258, Vol. 270, nr 50, s. 30162-7Artikel i tidskrift (Övrigt vetenskapligt)
  • 24.
    Kotova, Irina
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Chabes, Anna Lena
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Lobov, Sergei
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Thelander, Lars
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Björklund, Stefan
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Sequences downstream of the transcription initiation site are important for proper initiation and regulation of mouse ribonucleotide reductase R2 gene transcription2003Ingår i: European Journal of Biochemistry, ISSN 0014-2956, E-ISSN 1432-1033, Vol. 270, nr 8, s. 1791-1801Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Ribonucleotide reductase is essential for the synthesis of all four dNTPs required for DNA replication. The enzyme is composed of two proteins, R1 and R2, which are both needed for activity. Expression of the R1 and R2 mRNAs is restricted to the S-phase of the cell cycle, but the R1 and R2 promoters show no obvious sequence homologies that could indicate coordination of transcription. Here we study initiation of transcription at the natural mouse R2 promoter, which contains an atypical TATA-box with the sequence TTTAAA, using a combination of in vivo reporter gene assays and in vitro transcription. Our results indicate that in constructs where sequences from the R2 5'-UTR are present, the mouse R2 TATA-box is dispensable both for unregulated, basal transcription from the R2 promoter and for S-phase specific activity. Instead, initiation of R2 transcription is directed by sequences downstream from the transcription start. We report that this region contains a conserved palindrome sequence that interacts with TAF(II)s. This interaction down-regulates basal transcription from the R2 promoter, both in the absence and in the presence of the TATA-box.

  • 25.
    Kotova, Irina
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Chabes, Anna Lena
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Segerman, Bo
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Flodell, Sara
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Thelander, Lars
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Björklund, Stefan
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    A mouse in vitro transcription system reconstituted from highly purified RNA polymerase II, TFIIH, and recombinant TBP, TFIIB, TFIIE and TFIIF.2001Ingår i: European Journal of Biochemistry, ISSN 0014-2956, E-ISSN 1432-1033, Vol. 268, nr 16, s. 4527-4536Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Unregulated transcription of protein-encoding genes in vitro is dependent on 12-subunit core RNA polymerase II and five general transcription factors; TATA binding protein (TBP), transcription factor (TF)IIB, TFIIE, TFIIF, and TFIIH. Here we describe cloning of the mouse cDNAs encoding TFIIB and the small and large TFIIE and TFIIF subunits. The cDNAs have been used to express the corresponding proteins in recombinant form in Escherichia coli and in Sf21 insect cells, and all proteins have been purified to > 90% homogeneity. We have also purified a recombinant His6-tagged mouse TBP to near homogeneity and show that it is active in both a reconstituted mouse in vitro transcription system and a TBP-dependent in vitro transcription system from Saccharomyces cerevisiae. The more complex general transcription factors, TFIIH and RNA polymerase II, were purified more than 1000-fold and to near homogeneity, respectively, from tissue cultured mouse cells. When combined, the purified factors were sufficient to initiate transcription from different promoters in vitro. Functional studies of the S-phase-specific mouse ribonucleotide reductase R2 promoter using both the highly purified system described here (a mouse cell nuclear extract in vitro transcription system) and in vivo R2-promoter reporter gene assays together identify an NF-Y interacting promoter proximal CCAAT-box as being essential for high-level expression from the R2 promoter.

  • 26. Lembo, D
    et al.
    Gribaudo, G
    Hofer, Anders
    Umeå universitet, Medicinsk fakultet, Medicinsk kemi och biofysik.
    Riera, L
    Cornaglia, M
    Mondo, A
    Angeretti, A
    Gariglio, M
    Thelander, Lars
    Umeå universitet, Medicinsk fakultet, Medicinsk kemi och biofysik.
    Landolfo, S
    Expression of an altered ribonucleotide reductase activity associated with the replication of murine cytomegalovirus in quiescent fibroblasts.2000Ingår i: Journal of Virology, ISSN 0022-538X, Vol. 74, nr 24, s. 11557-65Artikel i tidskrift (Refereegranskat)
  • 27. Lembo, David
    et al.
    Donalisio, Manuela
    Hofer, Anders
    Umeå universitet, Medicinsk fakultet, Medicinsk kemi och biofysik.
    Cornaglia, Maura
    Brune, Wolfram
    Koszinowski, Ulrich
    Thelander, Lars
    Umeå universitet, Medicinsk fakultet, Medicinsk kemi och biofysik.
    Landolfo, Santo
    The ribonucleotide reductase R1 homolog of murine cytomegalovirus is not a functional enzyme subunit but is required for pathogenesis.2004Ingår i: Journal of virology, ISSN 0022-538X, Vol. 78, nr 8, s. 4278-88Artikel i tidskrift (Refereegranskat)
  • 28. Narváez, Ana J
    et al.
    Voevodskaya, Nina
    Thelander, Lars
    Umeå universitet, Medicinsk fakultet, Medicinsk kemi och biofysik.
    Gräslund, Astrid
    The involvement of Arg265 of mouse ribonucleotide reductase R2 protein in proton transfer and catalysis.2006Ingår i: Journal of biological chemistry, ISSN 0021-9258, Vol. 281, nr 36, s. 26022-8Artikel i tidskrift (Refereegranskat)
  • 29. Pontarin, Giovanna
    et al.
    Ferraro, Paola
    Håkansson, Pelle
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Thelander, Lars
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Reichard, Peter
    Bianchi, Vera
    p53R2-dependent ribonucleotide reduction provides deoxyribonucleotides in quiescent human fibroblasts in the absence of induced DNA damage2007Ingår i: Journal of Biological Chemistry, ISSN 0021-9258, Vol. 282, nr 23, s. 16820-8Artikel i tidskrift (Refereegranskat)
  • 30. Pontarin, Giovanna
    et al.
    Fijolek, Artur
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Pizzo, Paola
    Ferraro, Paola
    Rampazzo, Chiara
    Pozzan, Tullio
    Thelander, Lars
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Reichard, Peter A
    Bianchi, Vera
    Ribonucleotide reduction is a cytosolic process in mammalian cells independently of DNA damage.2008Ingår i: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 105, nr 46, s. 17801-17806Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Ribonucleotide reductase provides deoxynucleotides for nuclear and mitochondrial (mt) DNA replication and repair. The mammalian enzyme consists of a catalytic (R1) and a radical-generating (R2 or p53R2) subunit. During S-phase, a R1/R2 complex is the major provider of deoxynucleotides. p53R2 is induced by p53 after DNA damage and was proposed to supply deoxynucleotides for DNA repair after translocating from the cytosol to the cell nucleus. Similarly R1 and R2 were claimed to move to the nucleus during S-phase to provide deoxynucleotides for DNA replication. These models suggest translocation of ribonucleotide reductase subunits as a regulatory mechanism. In quiescent cells that are devoid of R2, R1/p53R2 synthesizes deoxynucleotides also in the absence of DNA damage. Mutations in human p53R2 cause severe mitochondrial DNA depletion demonstrating a vital function for p53R2 different from DNA repair and cast doubt on a nuclear localization of the protein. Here we use three independent methods to localize R1, R2, and p53R2 in fibroblasts during cell proliferation and after DNA damage: Western blotting after separation of cytosol and nuclei; immunofluorescence in intact cells; and transfection with proteins carrying fluorescent tags. We thoroughly validate each method, especially the specificity of antibodies. We find in all cases that ribonucleotide reductase resides in the cytosol suggesting that the deoxynucleotides produced by the enzyme diffuse into the nucleus or are transported into mitochondria and supporting a primary function of p53R2 for mitochondrial DNA replication.

  • 31. Popović-Bijelić, Ana
    et al.
    Voevodskaya, Nina
    Domkin, Vladimir
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Thelander, Lars
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Gräslund, Astrid
    Metal binding and activity of ribonucleotide reductase protein R2 mutants: conditions for formation of the mixed manganese-iron cofactor2009Ingår i: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 48, nr 27, s. 6532-6539Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Class Ic ribonucleotide reductase (RNR) from Chlamydia trachomatis (C. tm.) lacks the tyrosyl radical and uses a Mn(IV)-Fe(III) cluster for cysteinyl radical initiation in the large subunit. Here we investigated and compared the metal content and specific activity of the C. tm. wild-type R2 protein and its F127Y mutant, as well as the native mouse R2 protein and its Y177F mutant, all produced as recombinant proteins in Escherichia coli. Our results indicate that the affinity of the RNR R2 proteins for binding metals is determined by the nature of one specific residue in the vicinity of the dimetal site, namely the one that carries the tyrosyl radical in class Ia and Ib R2 proteins. In mouse R2, this tyrosyl residue is crucial for the activity of the enzyme, but in C. tm., the corresponding phenylalanine plays no obvious role in activation or catalysis. However, for the C. tm. wild-type R2 protein to bind Mn and gain high specific activity, there seems to be a strong preference for F over Y at this position. In studies of mouse RNR, we find that the native R2 protein does not bind Mn whereas its Y177F mutant incorporates a significant amount of Mn and exhibits 1.4% of native mouse RNR activity. The observation suggests that a manganese-iron cofactor is associated with the weak activity in this protein.

  • 32.
    Ranjbarian, Farahnaz
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Vodnala, Munender
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Vodnala, Sharvani Munender
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Rofougaran, Reza
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik. Department of Biochemistry, Institute of Biochemistry and Biophysics, Tehran University, Tehran, Iran.
    Thelander, Lars
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Hofer, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Trypanosoma brucei thymidine kinase is tandem protein consisting of two homologous parts, which together enable efficient substrate binding2012Ingår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 287, nr 21, s. 17628-17636Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Trypanosoma brucei causes African sleeping sickness, a disease for which existing chemotherapies are limited by their toxicity or lack of efficacy. We have found that four parasites, including T. brucei, contain genes where two or four thymidine kinase (TK) sequences are fused into a single open reading frame. The T. brucei full-length enzyme as well as its two constituent parts, domain 1 and domain 2, were separately expressed and characterized. Of potential interest for nucleoside analog development, T. brucei TK was less discriminative against purines than human TK1 with the following order of catalytic efficiencies: thymidine > deoxyuridine ≫ deoxyinosine > deoxyguanosine. Proteins from the TK1 family are generally dimers or tetramers, and the quaternary structure is linked to substrate affinity. T. brucei TK was primarily monomeric but can be considered a two-domain pseudodimer. Independent kinetic analysis of the two domains showed that only domain 2 was active. It had a similar turnover number (k(cat)) as the full-length enzyme but could not self-dimerize efficiently and had a 5-fold reduced thymidine/deoxyuridine affinity. Domain 1, which lacks three conserved active site residues, can therefore be considered a covalently attached structural partner that enhances substrate binding to domain 2. A consequence of the non-catalytic role of domain 1 is that its active site residues are released from evolutionary pressure, which can be advantageous for developing new catalytic functions. In addition, nearly identical 89-bp sequences present in both domains suggest that the exchange of genetic material between them can further promote evolution.

  • 33. Saitoh, Shigeaki
    et al.
    Chabes, Andrei
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    McDonald, W Hayes
    Thelander, Lars
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
    Yates, John R
    Russell, Paul
    Cid13 is a cytoplasmic poly(A) polymerase that regulates ribonucleotide reductase mRNA.2002Ingår i: Cell, ISSN 0092-8674, E-ISSN 1097-4172, Vol. 109, nr 5, s. 563-73Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Fission yeast Cid13 and budding yeast Trf4/5 are members of a newly identified nucleotidyltransferase family conserved from yeast to man. Trf4/5 are thought to be essential DNA polymerases. We report that Cid13 is a poly(A) polymerase. Unlike conventional poly(A) polymerases, which act in the nucleus and indiscriminately polyadenylate all mRNA, Cid13 is a cytoplasmic enzyme that specifically targets suc22 mRNA that encodes a subunit of ribonucleotide reductase (RNR). cid13 mutants have reduced dNTP pools and are sensitive to hydroxyurea, an RNR inhibitor. We propose that Cid13 defines a cytoplasmic form of poly(A) polymerase important for DNA replication and genome maintenance.

  • 34.
    Thelander, Lars
    Umeå universitet, Medicinsk fakultet, Medicinsk kemi och biofysik.
    Ribonucleotide reductase and mitochondrial DNA synthesis.2007Ingår i: Nature. Genetics, ISSN 1061-4036, Vol. 39, nr 6, s. 703-4Artikel i tidskrift (Refereegranskat)
  • 35. Voevodskaya, N
    et al.
    Narvaez, A-J
    Domkin, Vladimir
    Umeå universitet, Medicinsk fakultet, Medicinsk kemi och biofysik.
    Torrents, E
    Thelander, Lars
    Umeå universitet, Medicinsk fakultet, Medicinsk kemi och biofysik.
    Gräslund, Astrid
    Chlamydial ribonucleotide reductase: tyrosyl radical function in catalysis replaced by the FeIII-FeIV cluster.2006Ingår i: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, Vol. 103, nr 26, s. 9850-4Artikel i tidskrift (Refereegranskat)
  • 36. Zhao, X
    et al.
    Georgieva, B
    Chabes, Andrei
    Umeå universitet, Medicinsk fakultet, Medicinsk kemi och biofysik.
    Domkin, Vladimir
    Umeå universitet, Medicinsk fakultet, Medicinsk kemi och biofysik.
    Ippel, J H
    Schleucher, Jurgen
    Umeå universitet, Medicinsk fakultet, Medicinsk kemi och biofysik.
    Wijmenga, S
    Thelander, Lars
    Umeå universitet, Medicinsk fakultet, Medicinsk kemi och biofysik.
    Rothstein, R
    Mutational and structural analyses of the ribonucleotide reductase inhibitor Sml1 define its Rnr1 interaction domain whose inactivation allows suppression of mec1 and rad53 lethality.2000Ingår i: Mol Cell Biol, ISSN 0270-7306, Vol. 20, nr 23, s. 9076-83Artikel i tidskrift (Refereegranskat)
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