Purification, characterization, and localization of subunit interaction area of recombinant mouse ribonucleotide reductase R1 subunit.

Davis, Rachael; Thelander, M; Mann, Graham J.; Behravan, Gity; Soucy, François; Beaulieu, Pierre L.; Lavallée, Pierre; Gräslund, Astrid; Thelander, Lars

doi:10.1016/s0021-9258(17)31635-6

Cited by 54 publications

(14 citation statements)

References 31 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…A homogeneous preparation of human MTH1 protein was obtained as described by Yakushiji et al (17). Mouse ribonucleotide reductase R1 and R2 proteins were prepared as described (18). Bovine guanylate kinase and P1 nuclease were purchased from Sigma and Boehringer Mannheim, respectively.…”

Section: Methodsmentioning

confidence: 99%

Metabolic Fate of Oxidized Guanine Ribonucleotides in Mammalian Cells

et al. 1999

View full text Add to dashboard Cite

8-Oxo-7,8-dihydroguanine- (8-oxoguanine-) containing nucleotides are generated in the cellular nucleotide pool by the action of oxygen radicals produced during normal cellular metabolism. We examined the interconversion and metabolic fate of 8-oxoguanine-containing ribonucleotides in mammalian cells. (1) 8-OxoGTP can be generated not only by direct oxidation of GTP but also by phosphorylation of 8-oxoGDP by nucleotide diphosphate kinase, and the 8-oxoGTP thus formed can serve as a substrate for RNA polymerase II to induce transcription errors. (2) MTH1 protein carrying intrinsic 8-oxo-dGTPase activity has the potential to hydrolyze 8-oxoGTP to 8-oxoGMP, thus preventing misincorporation of 8-oxoguanine into RNA. 8-OxoGMP, the degradation product, cannot be reutilized, since guanylate kinase, which has the potential to phosphorylate both GMP and dGMP, is inactive on 8-oxoGMP. (3) Ribonucleotide reductase, which catalyzes reduction of four naturally occurring ribonucleoside diphosphates, cannot convert 8-oxoguanine-containing ribonucleotide to the deoxyribonucleotide. This step appears to serve as a gatekeeper to prevent formation of mutagenic substrates for DNA synthesis from oxidized ribonucleotides.

show abstract

Section: Methodsmentioning

confidence: 99%

Metabolic Fate of Oxidized Guanine Ribonucleotides in Mammalian Cells

et al. 1999

View full text Add to dashboard Cite

show abstract

“…Mouse Protein Rl. Recombinant mouse Rl protein was expressed and purified to homogeneity as described (Davis et al, 1994). The protein concentration was determined by the Also absorbance using an E\ lm of 12 (Thelander et al, 1980).…”

Section: Methodsmentioning

confidence: 99%

“…To avoid possible formation of inclusion bodies, we choose to grow our mutant R2 expressing cells at 15 °C (cf. Davis et al, 1994). Instead of LB we used TB (terrific broth) medium which allows a much higher yield of bacteria per milliliter of medium.…”

Section: Construction Of Mouse Protein R2 Expression Plasmidsmentioning

confidence: 99%

Evidence by Site-Directed Mutagenesis Supports Long-Range Electron Transfer in Mouse Ribonucleotide Reductase

Rova¹,

Goodtzova²,

Ingemarson³

et al. 1995

Biochemistry

116

120

View full text Add to dashboard Cite

Mammalian ribonucleotide reductase consists of two nonidentical subunits, proteins R1 and R2, each inactive alone. The R1 protein binds the ribonucleotide substrates while the R2 protein contains a binuclear iron center and a tyrosyl free radical, essential for activity. The crystal structures of the corresponding Escherichia coli proteins suggest that the distance from the active site in R1 to the tyrosyl radical buried in R2 is about 35 A. Therefore, an electron pathway was suggested between the active site and the tyrosyl radical. Such a pathway could include a conserved tryptophan on the suggested R1 interaction surface of R2 and a conserved aspartic acid hydrogen bonded both to the tryptophan and to a histidine iron ligand. To find experimental support for such an electron pathway, we have replaced the conserved tryptophan in mouse R2 with phenylalanine or tyrosine and the aspartic acid with alanine. All the mutated R2 proteins were shown to bind metal with the same affinity as native R2 and to form the binuclear iron center. In addition, the W103Y and D266A proteins formed a normal tyrosyl free radical while only low amounts of radical were observed in the W103F protein. Neither the kinetic rate constants nor the equilibrium dissociation constant of the R1/R2 complex was affected by the mutations as shown by BIAcore biosensor technique. However, all mutant R2 proteins were completely inactive in the enzymatic assay, supporting the hypothesis that the tryptophan and aspartic acid residues are important links in an amino acid residue specific long-range electron transfer.

show abstract

“…Cloning, Expression, and Purification of Mouse R2. Mouse R2 was cloned, expressed, and purified based on previous protocols, 62,63 with some modifications. In brief, the R2 gene synthesized and cloned into a pET-3b plasmid was ordered from GenScript (GenScript USA Inc.).…”

Section: Inorganic Chemistrymentioning

confidence: 99%

New Iminodiacetate–Thiosemicarbazone Hybrids and Their Copper(II) Complexes Are Potential Ribonucleotide Reductase R2 Inhibitors with High Antiproliferative Activity

et al. 2017

View full text Add to dashboard Cite

As ribonucleotide reductase (RNR) plays a crucial role in nucleic acid metabolism, it is an important target for anticancer therapy. The thiosemicarbazone Triapine is an efficient R2 inhibitor, which has entered ∼20 clinical trials. Thiosemicarbazones are supposed to exert their biological effects through effectively binding transition-metal ions. In this study, six iminodiacetate-thiosemicarbazones able to form transition-metal complexes, as well as six dicopper(II) complexes, were synthesized and fully characterized by analytical, spectroscopic techniques (IR, UV-vis; H andC NMR), electrospray ionization mass spectrometry, and X-ray diffraction. The antiproliferative effects were examined in several human cancer and one noncancerous cell lines. Several of the compounds showed high cytotoxicity and marked selectivity for cancer cells. On the basis of this, and on molecular docking calculations one lead dicopper(II) complex and one thiosemicarbazone were chosen for in vitro analysis as potential R2 inhibitors. Their interaction with R2 and effect on the Fe(III)-Y· cofactor were characterized by microscale thermophoresis, and two spectroscopic techniques, namely, electron paramagnetic resonance and UV-vis spectroscopy. Our findings suggest that several of the synthesized proligands and copper(II) complexes are effective antiproliferative agents in several cancer cell lines, targeting RNR, which deserve further investigation as potential anticancer drugs.

show abstract

Purification, characterization, and localization of subunit interaction area of recombinant mouse ribonucleotide reductase R1 subunit.

Cited by 54 publications

References 31 publications

Metabolic Fate of Oxidized Guanine Ribonucleotides in Mammalian Cells

Metabolic Fate of Oxidized Guanine Ribonucleotides in Mammalian Cells

Evidence by Site-Directed Mutagenesis Supports Long-Range Electron Transfer in Mouse Ribonucleotide Reductase

New Iminodiacetate–Thiosemicarbazone Hybrids and Their Copper(II) Complexes Are Potential Ribonucleotide Reductase R2 Inhibitors with High Antiproliferative Activity

Contact Info

Product

Resources

About