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Transcriptional regulation of mouse ribonucleotide reductase
Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. (Lars Thelander)
2011 (English)Licentiate thesis, comprehensive summary (Other academic)
Abstract [en]

All living organisms are made of cells and they store their hereditary information in the form of double stranded DNA. In all organisms DNA replication and repair is essential for cell division and cell survival. These processes require deoxyribonucleotides (dNTPs), the building blocks of DNA. Ribonucleotide reductase (RNR) is catalyzing the rate limiting step in the de novo synthesis of dNTPs. Active RNR is a heterodimeric protein complex. In S phase cells, the mouse RNR consists of the R1 and the R2 proteins. The R1/R2 RNR-complex supplies the cell with dNTPs required for DNA replication. Outside S-phase or in non-proliferating cells RNR is composed of R1 and p53R2 proteins. The R1/p53R2 RNR-complex supplies cells with dNTPs required for mitochondrial DNA replication and for DNA repair. An undisturbed dNTP regulation is important since unbalanced dNTP pools results in DNA mutations and cell death. Since unbalanced pools are harmful to the cell, RNR activity is regulated at many levels. The aim of this thesis is to study how the mouse RNR genes are regulated at a transcriptional level. We have focused on the promoter regions of all three mouse RNR genes. Primer extension experiments show that the transcription start of the TATA-less p53R2 promoter colocalizes with an earlier unidentified initiator element (Inr-element). This element is similar to the known Inr-element in the mouse R1 promoter. Furthermore, functional studies of the R1 promoter revealed a putative E2F binding element. This result suggests that the S phase specific transcription of the R1 gene is regulated by a similar mechanism as the R2 promoter which contains an E2F binding site. Finally we have established a method to partially purify the transcription factor(s) binding the upstream activating region in the mouse R2 promoter by phosphocellulose chromatography and affinity purification using oligonucleotides immobilized on magnetic beads. This method will allow us to further study the transcription factors responsible for activating expression of the R2 protein. This method has a potential to be utilized as a general method when purifying unknown transcription factors.

Place, publisher, year, edition, pages
Umeå: Universitet , 2011. , 19 p.
Keyword [en]
Ribonucleotide reductase; promoters; cell cycle; transcriptional regulation; primer extension; protein purification; EMSA
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Research subject
Medical Biochemistry
Identifiers
URN: urn:nbn:se:umu:diva-41272ISBN: 978-91-7459-187-3OAI: oai:DiVA.org:umu-41272DiVA: diva2:405426
Presentation
2011-04-15, KB3B1, KBC-huset, Umeå, 09:00 (English)
Supervisors
Available from: 2011-03-24 Created: 2011-03-22 Last updated: 2011-03-24Bibliographically approved
List of papers
1. Characterization of the promoters of the mouse ribonucleotide reductase R1, R2 and p53R2-genes
Open this publication in new window or tab >>Characterization of the promoters of the mouse ribonucleotide reductase R1, R2 and p53R2-genes
(English)Manuscript (preprint) (Other academic)
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.

National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Research subject
Medical Biochemistry
Identifiers
urn:nbn:se:umu:diva-41400 (URN)
Available from: 2011-03-24 Created: 2011-03-24 Last updated: 2011-03-24Bibliographically approved

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