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Assembly of a fragmented ribonucleotide reductase by protein interaction domains derived from a mobile genetic element
Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics. (Britt-Marie Sjöberg)
Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
Department of Biochemistry, Schulich School of Medicine and Dentistry, University of Western Ontario.
Department of Biochemistry, Schulich School of Medicine and Dentistry, University of Western Ontario.
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2011 (English)In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 39, no 4, 1381-1389 p.Article in journal (Refereed) Published
Abstract [en]

Ribonucleotide reductase (RNR) is a critical enzyme of nucleotide metabolism, synthesizing precursors for DNA replication and repair. In prokaryotic genomes, RNR genes are commonly targeted by mobile genetic elements, including free standing and intron-encoded homing endonucleases and inteins. Here, we describe a unique molecular solution to assemble a functional product from the RNR large subunit gene, nrdA that has been fragmented into two smaller genes by the insertion of mobE, a mobile endonuclease. We show that unique sequences that originated during the mobE insertion and that are present as C- and N-terminal tails on the split NrdA-a and NrdA-b polypeptides, are absolutely essential for enzymatic activity. Our data are consistent with the tails functioning as protein interaction domains to assemble the tetrameric (NrdA-a/NrdA-b)2 large subunit necessary for a functional RNR holoenzyme. The tails represent a solution distinct from RNA and protein splicing or programmed DNA rearrangements to restore function from a fragmented coding region and may represent a general mechanism to neutralize fragmentation of essential genes by mobile genetic elements.

Place, publisher, year, edition, pages
Oxford University Press , 2011. Vol. 39, no 4, 1381-1389 p.
National Category
Biochemistry and Molecular Biology
Research subject
Molecular Biology
Identifiers
URN: urn:nbn:se:su:diva-45737DOI: 10.1093/nar/gkq924ISI: 000288019400025OAI: oai:DiVA.org:su-45737DiVA: diva2:369446
Note
authorCount :6Available from: 2010-11-10 Created: 2010-11-10 Last updated: 2017-12-12Bibliographically approved
In thesis
1. Quaternary structure and interaction approaches to allosteric regulation of class I ribonucleotide reductases
Open this publication in new window or tab >>Quaternary structure and interaction approaches to allosteric regulation of class I ribonucleotide reductases
2010 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Deoxyribonucleic acid (DNA) chains in which our genetic blueprint is stored are built from four DNA precursors by DNA polymerases. The enzyme ribonucleotide reductase (RNR) provides the only de novo synthesis pathway of deoxyribonucleotides from ribonucleotides and is essential for nearly all organisms. All four ribonucleotides are substrates for RNR and key to this flexibility is a sophisticated allosteric regulation. Nucleotide effectors (ATP, dATP, dTTP or dGTP) binding to the allosteric specificity site determines substrate specificity for the active site. When present at high concentrations, dATP binds to the allosteric overall activity site and inhibits activity by an unknown mechanism. Three approaches, RNR activity measurements, subunit interaction studies and quaternary structure studies were applied to four different class I RNRs to address the allosteric overall regulation. We found that allosteric overall inhibition was closely linked to formation of tight and large RNR protein complexes; α4β4 complex for the Escherichia coli class Ia RNR and α6β2 for the Dictyostelium discoideum class Ia RNR with functional allosteric inhibitions. The Aeh1 phage class Ia RNR with a non-functional dATP inhibition showed weak remnant inhibition features, while the Bacillus anthracis class Ib RNR without the allosteric overall regulation domain lacked these features. In addition, we presented the first biochemical characterization of a mechanism to restore protein function after gene fragmentation, we showed that the B. anthracis class Ib RNR was most active when reconstituted with manganese and in the presence of a physiological redoxin protein and we found that the class Ia RNR is the principal RNR in D. discoideum, although the coexisting class II RNR could partly compensate class I RNR inhibition during axenic growth. Finally, our improved method for studying RNR interactions has potential for RNR inhibitor screening.

Place, publisher, year, edition, pages
Stockholm: Department of Molecular Biology and Functional Genomics, Stockholm University, 2010. 57 p.
Keyword
Ribonucleotide reductase, allosteric regulation, quaternary structure, subunit interactions, enzyme activity, SPR, GEMMA
National Category
Biochemistry and Molecular Biology
Research subject
Molecular Biology
Identifiers
urn:nbn:se:su:diva-45740 (URN)978-91-7447-186-1 (ISBN)
Public defence
2010-12-16, sal G, Arrheniuslaboratorierna, Svante Arrhenius väg 20 C, Stockholm, 13:00 (English)
Opponent
Supervisors
Note
At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 4: Manuscript. Paper 5: Manuscript.Available from: 2010-11-24 Created: 2010-11-10 Last updated: 2010-12-06Bibliographically approved

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