Mouse Ribonucleotide Reductase R2 Research Articles

The Involvement of Arg265 of Mouse Ribonucleotide Reductase R2 Protein in Proton Transfer and Catalysis

Ribonucleotide reductase class I enzymes consist of two non-identical subunits, R1 and R2, the latter containing a diiron carboxylate center and a stable tyrosyl radical (Tyr*), both essential for catalysis. Catalysis is known to involve highly conserved amino acid residues covering a range of approximately 35 A and a concerted mechanism involving long range electron transfer, probably coupled to proton transfer. A number of residues involved in electron transfer in both the R1 and R2 proteins have been identified, but no direct model has been presented regarding the proton transfer side of the process. Arg265 is conserved in all known sequences of class Ia R2. In this study we have used site-directed mutagenesis to gain insight into the role of this residue, which lies close to the catalytically essential Asp266 and Trp103. Mutants to Arg265 included replacement by Ala, Glu, Gln, and Tyr. All mutants of Arg265 were found to have no or low catalytic activity with the exception of Arg265 to Glu, which shows approximately 40% of the activity of native R2. We also found that the Arg mutants were capable of stable tyrosyl radical generation, with similar kinetics of radical formation and R1 binding as native R2. Our results, supported by molecular modeling, strongly suggest that Arg265 is involved in the proton-coupled electron transfer pathway and may act as a proton mediator during catalysis.

A Tri-nuclear metal cluster in reduced mouse ribonucleotide reductase R2 subunit

A Tri-nuclear metal cluster in reduced mouse ribonucleotide reductase R2 subunit

Loss of the tyrosyl radical in mouse ribonucleotide reductase by (−)-epicatechin

The flavonoid (−)-epicatechin was previously demonstrated to interfere with tyrosine nitration by peroxynitrite [Biochem. Biophys. Res. Commun. 285 (2001) 782]. This effect was hypothesized to be based upon an interaction of epicatechin with a transiently generated tyrosyl radical. In the present study, using electron paramagnetic resonance, we demonstrate that (−)-epicatechin is capable of destabilizing the tyrosyl radical of the mouse ribonucleotide reductase R2 component. First-order rate constants for the disappearance of tyrosyl radical signals were 1 × 10 −4 and 2 × 10 −4 s −1for epicatechin and hydroxyurea, a well-known tyrosyl radical scavenger, respectively. In keeping with scavenging the ribonucleotide reductase tyrosyl radical, cellular production of deoxyribonucleotides and DNA synthesis were impaired by (−)-epicatechin in normal human keratinocytes and in human squamous carcinoma cells.

S Phase-specific Transcription of the Mouse Ribonucleotide Reductase R2 Gene Requires Both a Proximal Repressive E2F-binding Site and an Upstream Promoter Activating Region

Ribonucleotide reductase is essential for supplying a balanced pool of the four deoxyribonucleotides required for DNA synthesis and repair. The active enzyme consists of two non-identical subunits called proteins R1 and R2. There are multiple levels of regulation of ribonucleotide reductase activity, which is highest during the S and G(2) phases of an unperturbed cell cycle in mammalian cells. Previous reports in the literature have indicated that the S phase-specific transcription of the mammalian R2 gene is regulated by a transcriptional block, is dependent on the transcription factor E2F1, or is simply regulated by proteins that bind to promoter CCAAT boxes plus the TATA box. Here, we demonstrate that the S phase-specific transcription of the mouse R2 gene is dependent on an upstream promoter activating region (located at nucleotides (nt) -672 to -527 from the transcription start site) and one proximal promoter repressive element (located at nt -112 to -107) that binds E2F4. Binding to the E2F site is modulated by binding of nuclear factor-Y to an adjacent CCAAT element (nt -79 to -75). The upstream activating region is crucial for overall R2 promoter activity. Mutation of the E2F-binding site leads to premature promoter activation in G(1) and increases overall promoter activity but only when the upstream activating region is present and intact. Therefore, E2F-dependent repression is essential for cell cycle-specific R2 transcription.

Mouse ribonucleotide reductase R2 protein: a new target for anaphase-promoting complex-Cdh1-mediated proteolysis.

Ribonucleotide reductase consists of two nonidentical proteins, R1 and R2, and catalyzes the rate-limiting step in DNA precursor synthesis: the reduction of ribonucleotides to deoxyribonucleotides. A strictly balanced supply of deoxyribonucleotides is essential for both accurate DNA replication and repair. Therefore, ribonucleotide reductase activity is under exquisite control both transcriptionally and posttranscriptionally. In proliferating mammalian cells, enzyme activity is regulated by control of R2 protein stability. This control, which responds to DNA damage, is effective until cells pass into mitosis. We demonstrate that the mitotic degradation and hence the overall periodicity of R2 protein levels depends on a KEN box sequence, recognized by the Cdh1-anaphase-promoting complex. The mouse R2 protein specifically binds Cdh1 and is polyubiquitinated in an in vitro ubiquitin assay system. Mutating the KEN signal stabilizes the R2 protein during mitosisG(1) in R2 protein-overexpressing cells. The degradation process, which blocks deoxyribonucleotide production during G(1), may be an important mechanism protecting the cell against unscheduled DNA synthesis. The newly discovered p53-induced p53R2 protein that lacks a KEN box may supply deoxyribonucleotides for DNA repair during G(0)G(1).

C-Myc initiates illegitimate replication of the ribonucleotide reductase R2 gene.

The mechanisms through which the oncoprotein c-Myc initiates locus-specific gene amplification are not understood. When analysing the initiation mechanism of c-Myc-dependent amplification of the mouse ribonucleotide reductase R2 (R2) gene, we observe c-Myc-dependent initiation of illegitimate DNA replication of the R2 gene. We demonstrate multiple simultaneous c-Myc-induced R2 replication forks, whereas R2 normally replicates with a single fork. In contrast, cyclin C replicates with only a single replication fork irrespective of c-Myc deregulation. In addition to de novo replication forks, c-Myc also initiates bi-allelic replication of R2, abrogating its normal mono-allelic replication pattern. Moreover, several chromosomal regions also display c-Myc-induced illegitimate replication profiles. Thus, c-Myc can act as an illegitimate replication-licensing factor that promotes de novo replication initiation and illegitimate replication timing that adversely impacts upon genomic stability.

A mouse in vitro transcription system reconstituted from highly purified RNA polymerase II, TFIIH and recombinant TBP, TFIIB, TFIIE and TFIIF.

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.

Characterization of the Promoter Region of the Mouse Ribonucleotide Reductase R2 Gene

Characterization of the Promoter Region of the Mouse Ribonucleotide Reductase R2 Gene

Evidence by Mutagenesis that Tyr370 of the Mouse Ribonucleotide Reductase R2 Protein Is the Connecting Link in the Intersubunit Radical Transfer Pathway

Ribonucleotide reductase catalyzes all de novo synthesis of deoxyribonucleotides. The mammalian enzyme consists of two non-identical subunits, the R1 and R2 proteins, each inactive alone. The R1 subunit contains the active site, whereas the R2 protein harbors a binuclear iron center and a tyrosyl free radical essential for catalysis. It has been proposed that the radical properties of the R2 subunit are transferred approximately 35 A to the active site of the R1 protein, through a coupled electron/proton transfer along a conserved hydrogen-bonded chain, i.e. a radical transfer pathway (RTP). To gain a better insight into the properties and requirements of the proposed RTP, we have used site-directed mutagenesis to replace the conserved tyrosine 370 in the mouse R2 protein with tryptophan or phenylalanine. This residue is located close to the flexible C terminus, known to be essential for binding to the R1 protein. Our results strongly indicate that Tyr(370) links the RTP between the R1 and R2 proteins. Interruption of the hydrogen-bonded chain in Y370F inactivates the enzyme complex. Alteration of the same chain in Y370W slows down the RTP, resulting in a 58 times lower specific activity compared with the native R2 protein and a loss of the free radical during catalysis.

Regeneration of the Tyrosyl Radical in Native orp-Butoxyphenol-Treated Mouse Ribonucleotide Reductase R2 Protein

The regeneration of the tyrosyl radical in chemically reduced native orp-butoxyphenol-treated radical free forms of mouse ribonucleotide reductase R2 protein has been studied. Chemical reduction has been achieved by treatment with light-activated flavin compounds: deazaflavin, flavin mononucleotide, or deazaflavin with methylviologen as mediator. The admission of air to the flavin reduced mouse R2 protein results in regeneration of up to 59% of the initial tyrosyl radical contents, whereas not more than 6% could be regenerated in thep-butoxyphenol-treated form. The mixed-valent EPR signal generated in thep-butoxyphenol-treated mouse R2 protein is different from the spectrum observed after flavin reduction in the native mouse R2 protein, indicating that treatment of the protein withp-butoxyphenol results in a structural rearrangement of the diferric/radical site. The presence of 0.1 mM Fe(II) in the anaerobic protein/buffer solution significantly improves the regeneration of tyrosyl radical upon admission of air to the flavin reduced mouse R2 protein, but less to the protein treated withp-butoxyphenol.

The Three-dimensional Structure of Mammalian Ribonucleotide Reductase Protein R2 Reveals a More-accessible Iron-radical Site than Escherichia coliR2

The three-dimensional structure of mouse ribonucleotide reductase R2 has been determined at 2.3 Å resolution using molecular replacement and refined to an R-value of 19.1% ( R free=25%) with good stereo-chemistry. The overall tertiary structure architecture of mouse R2 is similar to that from Escherichia coliR2. However, several important structural differences are observed. Unlike the E. coliprotein, the mouse dimer is completely devoid of β-strands. The sequences differ significantly between the mouse and E. coliR2s, but there is high sequence identity among the eukaryotic R2 proteins, and the identities are localized over the whole sequence. Therefore, the three-dimensional structures of other mammalian ribonucle otide reductase R2 proteins are expected to be very similar to that of the mouse enzyme. In mouse R2 a narrow hydrophobic channel leads to the proposed binding site for molecular oxygen near to the iron-radical site in the interior of the protein. In E. coliR2 this channel is blocked by the phenyl ring of a tyrosine residue, which in mouse R2 is a serine. These structural variations may explain the observed differences in sensitivity to radical scavengers. The structure determination is based on diffraction data from crystals grown at pH 4.7. Unexpectedly, the protein is not iron-free, but contains one iron ion bound at one of the dinuclear iron sites. This ferric ion is bound with partial occupancy and is coordinated by three glutamic acids (one bidentate) and one histidine in a bipyramidal coordination that has a free apical coordination position. Soaking of crystals in a solution of ferrous salt at pH 4.7 increased the occupancy on the already occupied site, but without any detectable binding at the second site.

Reversible Red-Ox Reactions of the Diiron Site in the Mouse Ribonucleotide Reductase R2 Protein

The red-ox reactions of the dinuclear iron center of mouse R2 protein upon interaction with different reductants (dithionite alone and with mediators) and oxidants (PES, methylene blue, hydrogen peroxide and para-benzoquinone) have been studied by EPR and optical spectroscopy. The obtained results indicate that the transitions between Fe(III)Fe(III), Fe(II)Fe(III), and Fe(II)Fe(II) states of the dinuclear iron center are reversible and the μ-oxo-bridge may be formed upon oxidation by non-oxygen oxidants. In contrast to the case for theE. coliR2 protein, dithionite alone reduces the tyrosyl radical and diiron center in mouse R2 protein.

Role of a Proximal NF-Y Binding Promoter Element in S Phase-specific Expression of Mouse Ribonucleotide Reductase R2 Gene

Cell cycle-regulated transcription of the R2 gene of mouse ribonucleotide reductase was earlier shown to be controlled at the level of elongation by an S phase-specific release from a transcriptional block. However, the R2 promoter is activated very early when quiescent cells start to proliferate, and this activation is dependent on three upstream sequences located nucleotide -672 to nucleotide -527 from the transcription start. In this study, we use R2-luciferase reporter gene constructs and gel shift assays to demonstrate that, in addition to the upstream sequences, a proximal CCAAT element specifically binding the transcription factor NF-Y is required for continuous activity of the R2 promoter through the S phase. When the CCAAT element is deleted or mutated, promoter activity induced by the upstream elements decays before cells enter S phase, and the transcriptional block is released. This is a clear example of how changing of a proximal sequence element can alter not only the quantitative but also the qualitative response to upstream transcription activation domains.

An S-phase specific release from a transcriptional block regulates the expression of mouse ribonucleotide reductase R2 subunit.

Ribonucleotide reductase (RR) activity in mammalian cells is closely linked to DNA synthesis. The RR enzyme is composed of two non-identical subunits, proteins R1 and R2. Both proteins are required for holoenzyme activity, which is regulated by S-phase specific de novo synthesis and breakdown of the R2 subunit. In quiescent cells stimulated to proliferate and in elutriated cell populations enriched in the various cell cycle phases the R2 protein levels are correlated to R2 mRNA levels that are low in G0/G1-phase cells but increase dramatically at the G1/S border. Using an R2 promoter-luciferase reporter gene construct we demonstrate an unexpected early activation of the R2 promoter as cells pass from quiescence to proliferation. However, due to a transcriptional block, this promoter activation only results in very short R2 transcripts until cells enter the S-phase, when full-length R2 transcripts start to appear. The position for the transcriptional block was localized to a nucleotide sequence approximately 87 bp downstream from the first exon/intron boundary by S1 nuclease mapping of R2 transcripts from modified in vitro nuclear run-on experiments. These results identify blocking of transcription as a mechanism to control cell cycle regulated gene expression.