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DNA precursor biosynthesis-allosteric regulation and medical applications
Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
2008 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Ribonucleotide reductase (RNR) is a key enzyme for de novo dNTP biosynthesis. We have studied nucleotide-dependent oligomerization of the allosterically regulated mammalian RNR using a mass spectrometry–related technique called Gas-phase Electrophoretic Mobility Macromolecule Analysis (GEMMA). Our results showed that dATP and ATP induce the formation of an α6β2 protein complex. This complex can either be active or inactive depending on whether ATP or dATP is bound.

In order to understand whether formation of the large complexes is a general feature in the class Ia RNRs, we compared the mammalian RNR to the E. coli enzyme. The E. coli protein is regarded a prototype for all class Ia RNRs. We found that the E. coli RNR cycles between an active α2β2 form (in the presence of ATP, dTTP or dGTP) and an inactive α4β4 form in the presence of dATP or a combination of ATP with dTTP/dGTP. The E. coli R1 mutant (H59A) which needs higher dATP concentrations to be inhibited than the wild-type enzyme had decreased ability to form these complexes. It remains to be discovered how the regulation functions in the mammalian enzyme where both the active and inactive forms are α6β2 complexes.

An alternative way to produce dNTPs is via salvage biosynthesis where deoxyribonucleosides are taken up from outside the cell and phosphorylated by deoxyribonucleoside kinases. We have found that the pathogen Trypanosoma brucei, which causes African sleeping sickness, has a very efficient salvage of adenosine, deoxyadenosine and adenosine analogs such as adenine arabinoside (Ara-A). One of the conclusions made was that this nucleoside analog is phosphorylated by the T. brucei adenosine kinase and kills the parasite by causing nucleotide pool imbalances and by incorporation into nucleic acids. Ara-A-based therapies can hopefully be developed into new medicines against African sleeping sickness.

Generally, the dNTPs produced from the de novo and salvage pathways can be imported into mitochondria and participate in mtDNA replication. The minimal mtDNA replisome contains DNA polymerase γA, DNA polymerase γB, helicase (TWINKLE) and the mitochondrial single-stranded DNA-binding protein (mtSSB). Here, it was demonstrated that the primase-related domain (N-terminal region) of the TWINKLE protein lacked primase activity and instead contributes to single-stranded DNA binding and DNA helicase activities. This region is not absolutely required for mitochondrial DNA replisome function but is needed for the formation of long DNA products.

Place, publisher, year, edition, pages
Umeå: Medicinsk kemi och biofysik , 2008. , 29 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1175
Keyword [en]
Biochemistry, DNA biosynthesis, ribonucleotide reductase, allosteric regulation, Trypanosoma brucei, adenosine kinase, nucleoside analogs, mitochondrial DNA, TWINKLE
Keyword [sv]
Biokemi
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy) Biochemistry and Molecular Biology
Research subject
Medical Biochemistry
Identifiers
URN: urn:nbn:se:umu:diva-1678ISBN: 978-91-7264-558-5 (print)OAI: oai:DiVA.org:umu-1678DiVA: diva2:141806
Public defence
2008-06-13, KB3A9, KBC Huset, Umeå University, SE-90187, Umeå, 13:00 (English)
Opponent
Supervisors
Available from: 2008-05-27 Created: 2008-05-27 Last updated: 2014-01-22Bibliographically approved
List of papers
1. Enzymatically active mammalian ribonucleotide reductase exists primarily as an α6β2 octamer
Open this publication in new window or tab >>Enzymatically active mammalian ribonucleotide reductase exists primarily as an α6β2 octamer
2006 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 281, no 38, 27705-27711 p.Article in journal (Refereed) Published
Abstract [en]

Ribonucleotide reductase synthesizes deoxyribonucleotides, which are essential building blocks for DNA synthesis. The mammalian ribonucleotide reductase is described as an alpha(2)beta(2) complex consisting of R1 (alpha) and R2 (beta) proteins. ATP stimulates and dATP inhibits enzyme activity by binding to an allosteric site called the activity site on the R1 protein. Despite the opposite effects by ATP and dATP on enzyme activity, both nucleotides induce formation of R1 oligomers. By using a new technique termed Gas-phase Electrophoretic-Mobility Macromolecule Analysis (GEMMA), we have found that the ATP/dATP-induced R1 oligomers have a defined size (hexamers) and can interact with the R2 dimer to form an enzymatically active protein complex (alpha(6)beta(2)). The newly discovered alpha(6)beta(2) complex can either be in an active or an inhibited state depending on whether ATP or dATP is bound. Our results suggest that this protein complex is the major form of ribonucleotide reductase at physiological levels of R1-R2 protein and nucleotides.

National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-22397 (URN)10.1074/jbc.M605573200 (DOI)16861739 (PubMedID)
Available from: 2009-05-07 Created: 2009-05-07 Last updated: 2017-12-13Bibliographically approved
2. Oligomerization status directs overall activity regulation of the Escherichia coli class Ia ribonucleotide reductase
Open this publication in new window or tab >>Oligomerization status directs overall activity regulation of the Escherichia coli class Ia ribonucleotide reductase
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2008 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 283, no 51, 35310-35318 p.Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
American Society for Biochemistry and Molecular Biology, 2008
Keyword
E. coli ribonucleotide reductase, RNR, oligomerization, octamer, ATP, dATP
National Category
Biochemistry and Molecular Biology
Research subject
biological chemistry
Identifiers
urn:nbn:se:umu:diva-10704 (URN)10.1074/jbc.M806738200 (DOI)18835811 (PubMedID)
Funder
Swedish Cancer SocietySwedish Research Council
Available from: 2008-10-23 Created: 2008-10-23 Last updated: 2017-12-14Bibliographically approved
3. Adenosine kinase mediates high affinity adenosine salvage in Trypanosoma brucei
Open this publication in new window or tab >>Adenosine kinase mediates high affinity adenosine salvage in Trypanosoma brucei
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2008 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 283, no 9, 5380-5388 p.Article in journal (Refereed) Published
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-9175 (URN)10.1074/jbc.M705603200 (DOI)18167353 (PubMedID)
Available from: 2008-03-07 Created: 2008-03-07 Last updated: 2017-12-14Bibliographically approved
4. The N-terminal domain of TWINKLE contributes to single-stranded DNA binding and DNA helicase activities.
Open this publication in new window or tab >>The N-terminal domain of TWINKLE contributes to single-stranded DNA binding and DNA helicase activities.
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2008 (English)In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 36, no 2, 393-403 p.Article in journal (Refereed) Published
Identifiers
urn:nbn:se:umu:diva-6621 (URN)10.1093/nar/gkm1025 (DOI)18039713 (PubMedID)
Available from: 2008-03-07 Created: 2008-03-07 Last updated: 2017-12-14Bibliographically approved

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