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Domkin, Vladimir
Publications (9 of 9) Show all publications
Domkin, V. & Chabes, A. (2014). Phosphines are ribonucleotide reductase reductants that act via C-terminal cysteines similar to thioredoxins and glutaredoxins. Scientific Reports, 4, 5539
Open this publication in new window or tab >>Phosphines are ribonucleotide reductase reductants that act via C-terminal cysteines similar to thioredoxins and glutaredoxins
2014 (English)In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 4, p. 5539-Article in journal (Refereed) Published
Abstract [en]

Ribonucleotide reductases (RNRs) catalyze the formation of 2'-deoxyribonucleotides. Each polypeptide of the large subunit of eukaryotic RNRs contains two redox-active cysteine pairs, one in the active site and the other at the C-terminus. In each catalytic cycle, the active-site disulfide is reduced by the C-terminal cysteine pair, which in turn is reduced by thioredoxins or glutaredoxins. Dithiols such as DTT are used in RNR studies instead of the thioredoxin or glutaredoxin systems. DTT can directly reduce the disulfide in the active site and does not require the C-terminal cysteines for RNR activity. Here we demonstrate that the phosphines tris(2-carboxyethyl)phosphine (TCEP) and tris(3-hydroxypropyl)phosphine (THP) are efficient non-thiol RNR reductants, but in contrast to the dithiols DTT, bis(2-mercaptoethyl)sulfone (BMS), and (S)-(1,4-dithiobutyl)-2-amine (DTBA) they act specifically via the C-terminal disulfide in a manner similar to thioredoxin and glutaredoxin. The simultaneous use of phosphines and dithiols results in ~3-fold higher activity compared to what is achieved when either type of reductant is used alone. This surprising effect can be explained by the concerted action of dithiols on the active-site cysteines and phosphines on the C-terminal cysteines. As non-thiol and non-protein reductants, phosphines can be used to differentiate between the redox-active cysteine pairs in RNRs.

Place, publisher, year, edition, pages
Macmillan Publishers Ltd., 2014
National Category
Other Basic Medicine
Identifiers
urn:nbn:se:umu:diva-91043 (URN)10.1038/srep05539 (DOI)000338421000012 ()24986213 (PubMedID)
Available from: 2014-07-09 Created: 2014-07-09 Last updated: 2018-06-07Bibliographically approved
Björnberg, O., Vodnala, M., Domkin, V., Hofer, A., Rasmussen, A., Andersen, G. & Piskur, J. (2010). Ribosylurea accumulates in yeast urc4 mutants. Nucleosides, Nucleotides & Nucleic Acids, 29(4-6), 433-437
Open this publication in new window or tab >>Ribosylurea accumulates in yeast urc4 mutants
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2010 (English)In: Nucleosides, Nucleotides & Nucleic Acids, ISSN 1525-7770, E-ISSN 1532-2335, Vol. 29, no 4-6, p. 433-437Article in journal (Refereed) Published
Abstract [en]

Yeast Saccharomyces (Lachancea) kluyveri urc4 mutants, unable to grow on uracil, biotransformed (14)C(2)-uracil into two labeled compounds, as detected by high performance liquid chromatography (HPLC). These two compounds could also be obtained following organic synthesis of ribosylurea. This finding demonstrates that in the URC pyrimidine degradation pathway, the opening of the uracil ring takes place when uracil is attached to the ribose moiety. Ribosylurea has not been reported in the cell metabolism before and the two observed compounds likely represent an equilibrium mixture of the pyranosyl and furanosyl forms.

Keywords
Uracil degradation, ribosylurea, URC pathway, Saccharomyces (Lachancea) kluyveri
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-42558 (URN)10.1080/15257771003741265 (DOI)000278713200029 ()20544532 (PubMedID)
Available from: 2011-04-11 Created: 2011-04-11 Last updated: 2018-06-08Bibliographically approved
Popović-Bijelić, A., Voevodskaya, N., Domkin, V., Thelander, L. & Gräslund, A. (2009). Metal binding and activity of ribonucleotide reductase protein R2 mutants: conditions for formation of the mixed manganese-iron cofactor. Biochemistry, 48(27), 6532-6539
Open this publication in new window or tab >>Metal binding and activity of ribonucleotide reductase protein R2 mutants: conditions for formation of the mixed manganese-iron cofactor
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2009 (English)In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 48, no 27, p. 6532-6539Article in journal (Refereed) Published
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.

Identifiers
urn:nbn:se:umu:diva-42882 (URN)10.1021/bi900693s (DOI)19492792 (PubMedID)
Available from: 2011-04-14 Created: 2011-04-14 Last updated: 2018-06-08Bibliographically approved
Voevodskaya, N., Narvaez, A.-J., Domkin, V., Torrents, E., Thelander, L. & Gräslund, A. (2006). Chlamydial ribonucleotide reductase: tyrosyl radical function in catalysis replaced by the FeIII-FeIV cluster.. Proceedings of the National Academy of Sciences of the United States of America, 103(26), 9850-4
Open this publication in new window or tab >>Chlamydial ribonucleotide reductase: tyrosyl radical function in catalysis replaced by the FeIII-FeIV cluster.
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2006 (English)In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, Vol. 103, no 26, p. 9850-4Article in journal (Refereed) Published
Keywords
Bacterial Proteins/*chemistry, Catalysis, Chlamydia trachomatis/*enzymology, Electron Spin Resonance Spectroscopy, Free Radicals/chemistry, Iron/*chemistry, Oxidation-Reduction, Ribonucleotide Reductases/*chemistry, Tyrosine/*chemistry
Identifiers
urn:nbn:se:umu:diva-6869 (URN)16777966 (PubMedID)
Available from: 2007-12-20 Created: 2007-12-20 Last updated: 2018-06-09Bibliographically approved
Håkansson, P., Dahl, L., Chilkova, O., Domkin, V. & Thelander, L. (2006). The Schizosaccharomyces pombe replication inhibitor Spd1 regulates ribonucleotide reductase activity and dNTPs by binding to the large Cdc22 subunit.. Journal of Biological Chemistry, 281(3), 1778-1783
Open this publication in new window or tab >>The Schizosaccharomyces pombe replication inhibitor Spd1 regulates ribonucleotide reductase activity and dNTPs by binding to the large Cdc22 subunit.
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2006 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 281, no 3, p. 1778-1783Article in journal (Refereed) Published
Keywords
Cell Cycle Proteins/genetics/*metabolism/*physiology, Cloning; Molecular, DNA Replication, Deoxyribonucleotides/*metabolism, Escherichia coli/genetics, G1 Phase, Kinetics, Protein Subunits/metabolism, Recombinant Proteins/isolation & purification/metabolism, Ribonucleotide Reductases/antagonists & inhibitors/genetics/*metabolism, Schizosaccharomyces/cytology/enzymology/*genetics, Schizosaccharomyces pombe Proteins/genetics/*metabolism/*physiology
Identifiers
urn:nbn:se:umu:diva-6344 (URN)10.1074/jbc.M511716200 (DOI)16317005 (PubMedID)
Available from: 2007-12-09 Created: 2007-12-09 Last updated: 2018-06-09Bibliographically approved
Chabes, A., Georgieva, B., Domkin, V., Zhao, X., Rothstein, R. & Thelander, L. (2003). Survival of DNA damage in yeast directly depends on increased dNTP levels allowed by relaxed feedback inhibition of ribonucleotide reductase.. Cell, 112(3), 391-401
Open this publication in new window or tab >>Survival of DNA damage in yeast directly depends on increased dNTP levels allowed by relaxed feedback inhibition of ribonucleotide reductase.
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2003 (English)In: Cell, ISSN 0092-8674, E-ISSN 1097-4172, Vol. 112, no 3, p. 391-401Article in journal (Refereed) Published
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.

Identifiers
urn:nbn:se:umu:diva-36449 (URN)12581528 (PubMedID)
Available from: 2010-09-30 Created: 2010-09-30 Last updated: 2018-06-08
Domkin, V., Thelander, L. & Chabes, A. (2002). Yeast DNA damage-inducible Rnr3 has a very low catalytic activity strongly stimulated after the formation of a cross-talking Rnr1/Rnr3 complex.. Journal of Biological Chemistry, 277(21), 18574-8
Open this publication in new window or tab >>Yeast DNA damage-inducible Rnr3 has a very low catalytic activity strongly stimulated after the formation of a cross-talking Rnr1/Rnr3 complex.
2002 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 277, no 21, p. 18574-8Article in journal (Refereed) Published
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.

Identifiers
urn:nbn:se:umu:diva-36445 (URN)10.1074/jbc.M201553200 (DOI)11893751 (PubMedID)
Available from: 2010-09-30 Created: 2010-09-30 Last updated: 2018-06-08
Zhao, X., Georgieva, B., Chabes, A., Domkin, V., Ippel, J. H., Schleucher, J., . . . Rothstein, R. (2000). Mutational and structural analyses of the ribonucleotide reductase inhibitor Sml1 define its Rnr1 interaction domain whose inactivation allows suppression of mec1 and rad53 lethality.. Mol Cell Biol, 20(23), 9076-83
Open this publication in new window or tab >>Mutational and structural analyses of the ribonucleotide reductase inhibitor Sml1 define its Rnr1 interaction domain whose inactivation allows suppression of mec1 and rad53 lethality.
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2000 (English)In: Mol Cell Biol, ISSN 0270-7306, Vol. 20, no 23, p. 9076-83Article in journal (Refereed) Published
Keywords
Cell Cycle Proteins, Chromosomes; Fungal, DNA Mutational Analysis, Enzyme Inhibitors, Fungal Proteins/*genetics, Humans, Mutation; Missense, Protein Binding, Protein Kinases/*genetics, Protein Structure; Secondary, Protein Structure; Tertiary, Protein-Serine-Threonine Kinases, Ribonucleotide Reductases/*antagonists & inhibitors, Saccharomyces cerevisiae Proteins, Solutions, Species Specificity, Suppression; Genetic, Two-Hybrid System Techniques
Identifiers
urn:nbn:se:umu:diva-10301 (URN)11074005 (PubMedID)
Available from: 2008-08-11 Created: 2008-08-11 Last updated: 2018-06-09Bibliographically approved
Chabes, A., Domkin, V., Larsson, G., Liu, A., Gräslund, A., Wijmenga, S. S. & Thelander, L. (2000). Yeast ribonucleotide reductase has a heterodimeric iron-radical-containing subunit. Proceedings of the National Academy of Sciences of the United States of America, 97(6), 2474-2479
Open this publication in new window or tab >>Yeast ribonucleotide reductase has a heterodimeric iron-radical-containing subunit
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2000 (English)In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 97, no 6, p. 2474-2479Article in journal (Refereed) Published
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.

Identifiers
urn:nbn:se:umu:diva-42939 (URN)10.1073/pnas.97.6.2474 (DOI)10716984 (PubMedID)
Available from: 2011-04-14 Created: 2011-04-14 Last updated: 2018-06-08Bibliographically approved
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