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Regulation of the Expression of Mouse Ribonucleotide Reductase Small Subunit at the Levels of Transcription and Protein Degradation
Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
2003 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Deoxyribonucleic acid (DNA) carries all the genetic information of a cell. Ribonucleotide reductase (RNR) provides balanced pools of all four dNTPs, the building blocks of DNA. These building blocks are needed during DNA synthesis and repair. A failure in the control of the dNTP levels and/or their relative amounts leads to cell death or genetic abnormalities. Because of its central role in dNTP metabolism, RNR is highly regulated on multiple levels.

The active RNR enzyme consists of two non-identical subunits called proteins R1 and R2. In mammalian cells, during an unperturbed cell cycle, the activity of RNR is highest during S and G2 phases. This is achieved by de novo synthesis of the limiting R2 protein at the onset of S phase, and by controlled degradation of the R2 protein during mitosis.

This thesis deals with both the S phase-specific transcription of the mouse R2 gene, and the M phase-specific degradation of the mouse R2 protein. Sequence comparison of the mouse R2 promoter to human and guinea pig R2 promoters revealed some conserved elements. These putative regulatory elements were tested in both in vitro and in vivo transcription assays. We demonstrated that the previously identified,

NF-Y binding CCAAT box is essential for high-level expression from the R2 promoter, but not for its S phase specificity. In addition, the conserved TATA box is dispensable both for basal and S phase-specific R2 transcription as long as the first 17 basepairs of the 5’ untranslated region are present. However, if this 5’ untranslated region is absent, the TATA box is needed for correct initiation of transcription.

Focusing on the S phase specificity of the R2 gene expression, we demonstrated that the S phase-specific activity of the mouse R2 promoter is dependent on a protein-binding region located ~500 basepairs upstream of the transcription start site and an E2F binding site close to the transcription start site. Deletion of the upstream activating region results in an inactive promoter. In contrast, mutation of the E2F site leads to premature promoter activation in G1 and increased overall promoter activity. However, if the activating mutation of the E2F site is combined with mutation of the upstream activating region, the promoter becomes inactive. These results suggest that the E2F-dependent regulation is important but not sufficient for cell-cycle specific R2 transcription, and that the upstream activating region is crucial for the overall R2 promoter activity.

In our studies of the M phase-specific R2 degradation, we found that it is dependent on a KEN sequence in the N-terminus of the R2 protein, recognized by the Cdh1-APC complex. Mutating the KEN box stabilizes the R2 protein during mitosis and G1 phase.

In summary, these studies further extend our understanding of the regulation of the limiting R2 subunit of the enzyme ribonucleotide reductase. The S phase-specific transcription of the R2 gene and the M phase-specific degradation of the R2 protein may serve as important

mechanisms to protect the cell against unscheduled DNA synthesis.

Place, publisher, year, edition, pages
Umeå: Medicinsk kemi och biofysik , 2003. , 34 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 847
Keyword [en]
Molecular biology, ribonucleotide reductase, transcription, protein degradation, cell cycle
Keyword [sv]
Molekylärbiologi
National Category
Biochemistry and Molecular Biology
Research subject
Medical Cell Biology
Identifiers
URN: urn:nbn:se:umu:diva-32ISBN: 91-7305-468-2 (print)OAI: oai:DiVA.org:umu-32DiVA: diva2:143087
Public defence
2003-06-13, KB3A9, KBC-huset, Umeå, 13:00 (English)
Opponent
Supervisors
Available from: 2003-06-04 Created: 2003-06-04 Last updated: 2015-01-19Bibliographically approved
List of papers
1. A mouse in vitro transcription system reconstituted from highly purified RNA polymerase II, TFIIH, and recombinant TBP, TFIIB, TFIIE and TFIIF.
Open this publication in new window or tab >>A mouse in vitro transcription system reconstituted from highly purified RNA polymerase II, TFIIH, and recombinant TBP, TFIIB, TFIIE and TFIIF.
Show others...
2001 (English)In: European Journal of Biochemistry, ISSN 0014-2956, E-ISSN 1432-1033, Vol. 268, no 16, 4527-4536 p.Article in journal (Refereed) Published
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.

Keyword
in vitro transcription, RNA polymerase II, ribonucleotide reductase, mouse general transcription factors
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:umu:diva-5476 (URN)10.1046/j.1432-1327.2001.02378.x (DOI)11502214 (PubMedID)
Available from: 2003-09-06 Created: 2003-09-06 Last updated: 2017-12-14Bibliographically approved
2. Sequences downstream of the transcription initiation site are important for proper initiation and regulation of mouse ribonucleotide reductase R2 gene transcription
Open this publication in new window or tab >>Sequences downstream of the transcription initiation site are important for proper initiation and regulation of mouse ribonucleotide reductase R2 gene transcription
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2003 (English)In: European Journal of Biochemistry, ISSN 0014-2956, E-ISSN 1432-1033, Vol. 270, no 8, 1791-1801 p.Article in journal (Refereed) Published
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.

Place, publisher, year, edition, pages
Blackwell Publishing, 2003
Keyword
in vitro transcription, ribonucleotide reductase, TAFs, TATA-box, transcription regulation
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:umu:diva-5477 (URN)10.1046/j.1432-1033.2003.03541.x (DOI)000182118800019 ()
Available from: 2003-09-06 Created: 2003-09-06 Last updated: 2017-12-14Bibliographically approved
3. 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.
Open this publication in new window or tab >>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.
(English)Manuscript (Other academic)
Identifiers
urn:nbn:se:umu:diva-4114 (URN)
Available from: 2003-06-04 Created: 2003-06-04 Last updated: 2011-03-30Bibliographically approved
4. Mouse ribonucleotide reductase R2 protein: A new target for anaphase-promoting complex-Cdh1-mediated proteolysis.
Open this publication in new window or tab >>Mouse ribonucleotide reductase R2 protein: A new target for anaphase-promoting complex-Cdh1-mediated proteolysis.
2003 (English)In: Proc. Natl. Acad. Sci. U S A., Vol. 100, no 7, 3925-3929 p.Article in journal (Refereed) Published
Identifiers
urn:nbn:se:umu:diva-4115 (URN)
Available from: 2003-06-04 Created: 2003-06-04 Last updated: 2011-03-30Bibliographically approved

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