This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Xue, L. Articles by Yen, Y. Search for Related Content PubMed PubMed Citation Articles by Xue, L. Articles by Yen, Y.

Structurally Dependent Redox Property of Ribonucleotide Reductase Subunit p53R2

¹ Department of Medical Oncology and Therapeutic Research, City of Hope National Medical Center, Duarte and ² Department of Chemistry, California State University, Carson, California

Requests for reprints: Yun Yen, Department of Medical Oncology and Therapeutic Research, City of Hope National Medical Center, 1500 East Duarte Road, Duarte, CA 91010. Phone: 626-359-8111, ext. 62867; Fax: 626-301-8233; E-mail: yyen{at}coh.org.

	Abstract

Top Abstract Introduction Materials and Methods Results and Discussion References

 
p53R2 is a newly identified small subunit of ribonucleotide reductase (RR) and plays a key role in supplying precursors for DNA repair in a p53-dependent manner. Currently, we are studying the redox property, structure, and function of p53R2. In cell-free systems, p53R2 did not oxidize a reactive oxygen species (ROS) indicator carboxy-H₂DCFDA, but another class I RR small subunit, hRRM2, did. Further studies showed that purified recombinant p53R2 protein has catalase activity, which breaks down H₂O₂. Overexpression of p53R2 reduced intracellular ROS and protected the mitochondrial membrane potential against oxidative stress, whereas overexpression of hRRM2 did not and resulted in a collapse of mitochondrial membrane potential. In a site-directed mutagenesis study, antioxidant activity was abrogated in p53R2 mutants Y331F, Y285F, Y49F, and Y241H, but not Y164F or Y164C. The fluorescence intensity in mutants oxidizing carboxy-H₂DCFDA, in order from highest to lowest, was Y331F > Y285F > Y49F > Y241H > wild-type p53R2. This indicates that Y331, Y285, Y49, and Y241 in p53R2 are critical residues involved in scavenging ROS. Of interest, the ability to oxidize carboxy-H₂DCFDA indicated by fluorescence intensity was negatively correlated with RR activity from wild-type p53R2, mutants Y331F, Y285F, and Y49F. Our findings suggest that p53R2 may play a key role in defending oxidative stress by scavenging ROS, and this antioxidant property is also important for its fundamental enzymatic activity. (Cancer Res 2006; 66(4): 1900-5)

	Introduction

Top Abstract Introduction Materials and Methods Results and Discussion References

 
It is well known that the basic structure and function of ribonucleotide reductase (RR) is closely linked to its redox state. In Escherichia coli, active B2 (RR small subunit) harbors the iron center and the radical in oxidized states (1). However, reduction and oxidation studies in E. coli and mouse showed the existence of a stable, fully reduced form of the B2 protein (2, 3). The B2 protein shows dynamic carboxylate, radical, and water shifts in different redox forms (4). Similarly, cytochrome c oxidase, which has redox active tyrosine in the binuclear center, participates in reducing oxygen in respiration (5, 6), suggesting that proteins which possess tyrosyl radicals with a binuclear center may be reducing or oxidizing reagents.

HRRM2 and p53R2, two small subunits of human RR, belong to class I RR, which have a fundamental function motif: a tyrosyl radical coupled with µ-oxo-diiron (III) cluster (4). Overexpression of hRRM2 could have significant effects on the biological properties of cells, tumor development, metastasis, and drug resistance (7), but hydroxyurea, an inhibitor of hRRM2, can protect the cell from H₂O₂-induced DNA damage through free radical scavenging activity (8). Conversely, p53R2, induced by tumor suppressor p53, may be important for supplying deoxyribonucleotides to the DNA damage repair system. For example, p53R2-deficient mice showed more susceptibility to oxidative stress than wild-type mice (9). Therefore, p53R2 may have a role that is distinct from the role of hRRM2 in regulating a cellular redox state, but the redox property and functions of p53R2 remains unclear. Here, we compare the redox properties of p53R2 and hRRM2 proteins, and clarify the structure and function of p53R2 relative to its redox properties.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Cell culture and transfections. Human oropharyngeal carcinoma KB cells and their derivative clones were cultured on tissue culture plates in RPMI 1640, supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin in a 5% CO₂ atmosphere at 37°C. KB cells were transfected with pcDNA3.1 vector (KB-vector), p53R2 sense (KB-p53R2S) or antisense (KB-p53R2AS), hRRM2 sense (KB-M2S) or antisense (KB-M2AS) cDNA as described (10). Exponentially growing cells were treated with indicated doses of H₂O₂ and incubated for 24 hours.

Site-directed mutagenesis. A site-specific mutation in p53R2 and hRRM2 was made by PCR, using pET28a-p53R2 and pET28a-hRRM2 as templates, respectively (11). The PCR products were digested with restriction enzyme DpnI, using QuikChange II XL site-directed mutagenesis kit (Stratagene, La Jolla, CA).

Protein expression and purification. Recombinant hRRM2, p53R2 or mutant p53R2, hRRM2 proteins were expressed and purified as described (11). Protein concentration was determined using a protein assay kit (Bio-Rad, Hercules, CA), and purity was determined by densitometric scanning of the Coomassie-stained SDS-PAGE gels.

Fluorescence assays of carboxy-H₂DCFDA oxidation. Carboxy-H₂DCFDA (Molecular Probes, Eugene, OR) is an indicator for reactive oxygen species (ROS) that do not fluoresce until hydrolyzed by esterases and oxidation occurs within the reaction system. For functional assays in a cell-free system, esterases inherent in mitochondrial extracts or commercial esterases (esterase from porcine liver, Sigma, St. Louis, MO) were added. All incubations contained 10 µmol/L carboxy-H₂DCFDA and 240 µg/mL of mitochondrial lysate or 75 µg/mL of esterase enzyme, and with wild-type p53R2, hRRM2, or mutant p53R2, hRRM2 proteins at 37°C for 30 minutes. For in vitro studies, 100 µL of 10⁵ cells/mL KB-vector and KB-p53R2S cells were plated in a 96-well cell culture plate and incubated at 37°C for 24 hours. Following incubation, cells were treated with H₂O₂ for another 24 hours. Cells were washed with HBSS without phenol red. Carboxy-H₂DCFDA (10 µmol/L) was added and cells were further incubated for 30 minutes. The fluorescence of the oxidized form of carboxy-H₂DCFDA was measured using a fMax microplate reader (Molecular Devices, Sunnyvale, CA) with a fluorescence excitation of 485 nm and emission at 538 nm.

Electron paramagnetic resonance spectra. X-band electron paramagnetic resonance (EPR) spectra of purified hRRM2 and p53R2 proteins were measured as described previously (11).

Determination of catalase activity. Catalase activities were detected using the Amplex red reagent-based assay kit (Molecular Probes) according to manufacturer protocols. We prepared catalase standard curve samples and recombinant purified p53R2 protein sample in 1x reaction buffer (0.1 mol/L Tris-HCl, pH 7.5); 25 µL of the above samples were delivered to separate wells of a 96-well microplate. The reaction was initiated in 20 µmol/L H₂O₂ in 1x reaction buffer and incubated for 30 minutes. The final reaction contained 50 µmol/L Amplex red reagent and 0.2 units/mL horseradish peroxidase and was incubated at 37°C. After 30 minutes, absorbance was measured in a microplate reader at 550 nm. Change in absorbance is reported as the observed absorbance intensity subtracted from a negative control. The absorbance of p53R2 was measured, and catalase-specific activity was calculated using the catalase standard curve.

Inner mitochondrial membrane potential analysis. JC-1 dye (Molecular Probes) was dissolved in DMSO to produce a 1 mg/mL stock solution in our study. Cells were loaded with 1 µg/mL JC-1 for 30 minutes at 37°C, trypsinized, resuspended in medium at a density of 10⁶/mL, and transferred on ice to a flow cytometer (MoFlo MLS, DakoCytomation, Inc., Carpinteria, CA). JC-1 was excited at 488 nm and the monomer signal (green) was analyzed at 525 nm. Simultaneously, the aggregate signal (red) was analyzed at 590 nm. The distribution of red and green fluorescence from JC-1 was displayed in two-color contour plots. The ratios between red and green signals were measured.

	Results and Discussion

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Recombinant purified p53R2 protein lacked the ability to oxidize carboxy-H₂DCFDA. The recombinant human p53R2 and hRRM2 proteins were purified and immediately used to evaluate their ROS-generating activity in a cell-free system containing mitochondrial extract. Mitochondrial extract was used to supply esterase; without esterase, carboxy-H₂DCFDA is not sensitive to oxidants (9). Therefore, the fluorescence intensity (FI) of oxidized carboxy-H₂DCF of both p53R2 and hRRM2 without mitochondrial extract was considered background (Fig. 1A). After mitochondrial extract was added, FI of p53R2 was unchanged at 4 µg/mL to 40 µg/mL concentrations whereas the FI of hRRM2 significantly increased. Furthermore, FI of p53R2 was much lower than hRRM2, at 6% and 20% of hRRM2 at 8 and 40 µg/mL, respectively (Fig. 1A). These results suggest that hRRM2 is able to generate ROS in the presence of mitochondrial extract (designated as assay 1), but p53R2 is not. Similarly, when mitochondrial extract was replaced with pure esterase (designated as assay 2), FI of p53R2 was consistently close to background and lower than hRRM2 (Fig. 1B), suggesting that the inability of p53R2 to oxidize carboxy-H₂DCF was inherent to p53R2. However, larger differences in FI were observed between hRRM2 and p53R2 in assay 1 than in assay 2, suggesting the mitochondrial environment may influence redox properties of p53R2 and hRRM2. This may be because mitochondria contain oxidants and antioxidants, whereas the tyrosyl radicals in B2 proteins are known to react with a large number of radical scavengers, antioxidants, and oxidants (1). Because p53R2 and hRRM2 have an identical tyrosyl radical, as shown in the EPR spectra (Fig. 1C), the difference in their ability to oxidize carboxy-H₂DCFDA cannot be attributed to the tyrosyl radical itself.

View larger version (21K): [in this window] [in a new window]   Figure 1. Recombinant purified p53R2 lacked the ability to oxidize carboxy-H2DCFDA. Carboxy-H2DCFDA (10 µmol/L) was incubated at 37°C with mitochondrial lysate of KB cells (A) or esterases purchased (B) for 30 minutes in the presence of purified proteins hRRM2 or p53R2. Control incubations contained hRRM2 or p53R2 but no mitochondrial lysate (A) or no commercial esterase enzyme (B). Carboxy-DCF generation was monitored fluorometrically. Points, mean of three independent experiments, each carried out in triplicate. MIT, mitochondrial extract; ES, esterase enzyme purchased. C, EPR spectra of the tyrosyl radical in human p53R2 and hRRM2 proteins.

View larger version (18K): [in this window] [in a new window]   Figure 2. p53R2 scavenged H2O2. A, p53R2 displayed catalase activity. Catalase activity was detected using the Amplex Red reagent-based assay. Initially, each reaction contained the catalase enzyme (for standard curve) or p53R2 protein and 20 µmol/L H2O2 in 1x reaction buffer and was incubated for 30 minutes. The final reaction contained 50 µmol/L Amplex Red reagent and 0.2 units/mL of horseradish peroxidase and was incubated at 37°C. After 30 minutes, absorbance was measured in a microplate reader at 550 nm. Change in absorbance is reported as the observed absorbance intensity subtracted from that of a no-catalase control. The absorbance of p53R2 was then changed to catalase-specific activity according to the catalase standard curve. Catalase activity, where 1 unit is defined as the amount of enzyme that will decompose 1.0 µmol of H2O2 per minute (pH 7.0) at 25°C. B, p53R2 reduced intracellular ROS induced by H2O2. KB-vector and KB-p53R2S cells were treated with indicated concentrations of H2O2 for 24 hours before staining with 10 µmol/L carboxy-H2DCFDA. Carboxy-DCF generation (FI) was monitored fluorometrically. Points, mean of three independent experiments, each carried out in quadruplicate; bars, ± SE.

p53R2 protected the mitochondrial membrane against oxidative stress. Because mitochondria are particularly susceptible to oxidative damage, leading to mitochondrial membrane potential (_mt) decrease (12), we investigated the influence of both p53R2 and hRRM2 on _mt in response to H₂O₂ in KB-wt, KB-p53R2S, KB-M2S, KB-p53R2AS, and KB-M2AS cells with JC-1. Expression of p53R2 increased 3-fold with KB-p53R2S compared with the KB-wt or KB-vector, whereas expression of p53R2 decreased with KB-p53R2AS compared with the control (Fig. 3A). hRRM2 protein decreased under isopropyl-L-thio-ß-D-galactopyranoside (IPTG) induction in KB-M2AS clone (Fig. 3B). KB-M2S clone showed overexpression of hRRM2 RNA and protein (data not shown). JC-1 is _mt sensitive probe, which forms monomers (green fluorescence) at a low _mt and J-aggregates (red fluorescence) at a higher _mt (13). To exclude the discrepancy of cell number from each sample, the change of _mt was indicated as the ratio of red/green fluorescence. In the KB-wt cells, treatment with 250 µmol/L H₂O₂ resulted in a shift of ratios toward lower values compared with untreated cells (Fig. 3C, KB-wt). A threshold ratio of 100 clearly divides the cells into two populations, one representing the untreated cells with a ratio >100 and the other representing cells with injured mitochondria with a ratio <100. On the basis of this clear distinction, we designated a threshold ratio of 100 to distinguish between cells with intact (high ratio, designated as R2 cells) and injured mitochondria (low ratio, designated as R1 cells). Treatment with 250 µmol/L of H₂O₂ resulted in a cell shift of R2 to R1 compared with the respective untreated clones, except KB-p53R2S clones, which shifted from R1 to R2 (Fig. 3C). To clearly show the effect of p53R2 and hRRM2 on _mt induced by H₂O₂, the mean fluorescence intensity (MFI) ratio of the red/green ratio was plotted as a function of H₂O₂ concentration (Fig. 3D). KB-p53R2S and KB-M2AS cells were more resistant to H₂O₂ attack than KB-wt cells, shown by MFI increase, whereas KB-p53R2AS and KB-M2S cells were more sensitive to H₂O₂ attack than KB-wt cells with MFI decrease. These results clearly show that p53R2 protected the mitochondrial membrane against oxidative stress. This result is consistent with a study using p53R2 knockout mouse embryonic fibroblasts (MEF) in which MEFs containing p53R2 (p532R2-wt) had higher viability in response to H₂O₂ attack than mutant MEFs (p53R2-ko; ref. 14), although the difference of viability between p53R2-wt and p53R2-ko cells was much higher than the difference of their DNA repair ability. Our current results may explain this variance, because p53R2 may protect cells against the loss of from H₂O₂ stress by decreasing ROS. This idea is supported by our results of catalase activity displayed by recombinant p53R2 protein (Fig. 2A) and intracellular ROS was reduced in p53R2-overexpressing cells compared with control cells in response to H₂O₂ (Fig. 2B). These findings are in agreement with another study demonstrating the catalase activity of B2 protein from E. coli (15).

View larger version (34K): [in this window] [in a new window]   Figure 3. p53R2 protected mitochondrial membrane from H2O2 attack. A, expression of p53R2 protein in KB-wt cell and KB-vector (pcDNA3.1 vector), KB-p53R2S, and KB-p53R2AS clones. B, expression of antisense hRRM2 was induced by 5 mmol/L of IPTG. The hRRM2 protein and hRRM2 AS RNA level were determined here with 18S RNA control. C, histogram distribution of red/green fluorescence ratios in KB-wt, KB-p53R2S, KB-p53R2AS, KB-M2S, and KB-M2AS cells after treatment with 250 µmol/L of H2O2 for 24 hours. Cells were stained with 1 µg/mL JC-1 for 30 minutes at 37°C before harvest. Histogram distribution showed two populations (R1 and R2) of cells with intact (ratio >100, designated as R2 cells) and injured mitochondria (low ratio <100, designated as R1 cells). D, variations of the red/green MFI ratio as a function of the H2O2 concentration. Points, mean of three independent experiments; bars, ± SE.

View larger version (34K): [in this window] [in a new window]   Figure 4. Y331, Y285, Y49, and Y241 are critical residues for p53R2's lack of ability to oxidize carboxy-H2DCFDA. A, the primer sequences used in site-directed mutagenesis for p53R2 and hRRM2 mutants. B, in vitro expression and purification of wild-type p53R2, wild-type hRRM2, and mutant p53R2, hRRM2. 10% SDS-PAGE analysis of the purified recombinant proteins by one-step Ni-resin affinity chromatography. C, mutations of Y331F, Y285F, Y49F, and Y241H rather than Y164F or Y164C of p53R2 resulted in p53R2 obtaining the ability to oxidize carboxy-H2DCFDA. Carboxy-H2DCFDA (10 µmol/L) was incubated at 37°C with mitochondrial lysate of KB cells for 30 minutes in the presence of purified proteins wild-type p53R2, wild-type hRRM2, and mutant p53R2, hRRM2. Carboxy-DCF generation was monitored fluorometrically as in Fig. 1A. Points, mean of three independent experiments, each carried out in triplicate. D, the negative relationship of FI and RR activity produced by wild-type p53R2 and its mutants Y331F, Y285F, and Y49F. The FI was measured as in (C). The RR activity of the protein was measured as relative RR activity of dC / (C + dC)%, formation of percentage of dCDP at 37°C (see Materials and Methods). Data was normalized against the wild-type proteins, which are considered to give 100% conversion of CDP to dCDP. Each value is the average of two determinations with deviations <0.3. The enzyme activity was measured in the presence of 5 µg of B1 protein and 5 µg of p53R2 wild-type or mutant proteins with a total sample volume of 100 µL. Correlation coefficient between FI and RR activity is –0.91.

In investigations of the crystal structure of B2 protein from E. coli and mouse, the tyrosyl radical was buried in a hydrophobic pocket, away from the B1 protein active site (4). Therefore, the RR enzymatic reaction may involve a coupled electron/proton transfer pathway from the tyrosyl radical in B2 to cysteine in B1. The COOH-terminal conserved tyrosine residues (356 in the E. coli B2 protein, and 370 in the mouse B2 protein) may be the connecting link between B1 and B2 (16–18). In human p53R2, this conserved residue corresponds to Y331. Substituting the p53R2 protein Y331 with phenylalanine dramatically increased FI (Fig. 4C), however, as in the B2 protein from E. coli and mouse, RR enzymatic activity was abolished (RRac, Fig. 4D). These results indicate that Y331 in p53R2 is an important residue that bridges p53R2 and B1 protein in the electron/proton transfer pathway. Abolishment of RR activity and increased FI occurred when Y331 was mutated to F331 in p53R2 (Fig. 4D), suggesting that efficient electron transfer through Y331 required the reduction potential of Y331. This is in agreement with the idea that the rate of radical transfer through position Y356 in E. coli is affected mainly by the reduction potential of the residue at that position (19). The Y285 residue is close to Y331. Y258 and Y49 residues in p53R2 have not yet been reported to involve electron/proton transfer, but according to the computer model, Y331, Y285, and Y49 residues localize on the surface of p53R2. RR activity was not abolished but decreased significantly after the Y49 or Y285 residues were replaced by phenylalanine (RRac, Fig. 4D), indicating that Y49 and Y285 residues may be partially involved in this transfer pathway.

ROS are potentially dangerous by-products of cellular metabolism that have direct effects on cell growth and development and cell survival, and have a significant role in the pathogenesis of cancer. To intercept toxic ROS and prevent the damage they may trigger, a goal for cancer prevention may be genetic mutation (20). The antioxidant ability of p53R2 implies that p53R2 plays an important role in preventing cancer because, not only can p53R2 supply deoxyribonucleotides to the DNA repair system (9), but it can also scavenge ROS (this report) to maintain genomic integrity in response to oxidative stress.

	Acknowledgments

 
Grant support: NCI R01 grant CA72767, partially supported by the Sino American Cancer Foundation.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Drs. Kristine A. Justus and Thomas LeBon for helpful revisions of this manuscript, and Leila Su for analyzing the geometric structure of RR small subunits using computer modeling.

Received 7/27/05. Revised 10/ 2/05. Accepted 12/20/05.

	References

Top Abstract Introduction Materials and Methods Results and Discussion References

 

1. Reichard P, Ehrenberg A. Ribonucleotide reductase—a radical enzyme. Science 1983;221:514–9.[Free Full Text]
2. Sahlin M, Graslund A, Petersson L, Ehrenberg A, Sjoberg BM. Reduced forms of the iron-containing small subunit of ribonucleotide reductase from Escherichia coli. Biochemistry 1989;28:2618–25.[CrossRef][Medline]
3. Davydov A, Schmidt PP, Graslund A. Reversible red-ox reactions of the diiron site in the mouse ribonucleotide reductase R2 protein. Biochem Biophys Res Commun 1996;219:213–8.[CrossRef][Medline]
4. Kolberg M, Strand KR, Graff P, Andersson KK. Structure, function, and mechanism of ribonucleotide reductases. Biochim Biophys Acta 2004;1699:1–34.[Medline]
5. Proshlyakov DA, Pressler MA, Babcock GT. Dioxygen activation and bond cleavage by mixed-valence cytochrome c oxidase. Proc Natl Acad Sci U S A 1998;95:8020–5.[Abstract/Free Full Text]
6. Proshlyakov DA, Pressler MA, DeMaso C, Leykam JF, DeWitt DL, Babcock GT. Oxygen activation and reduction in respiration: involvement of redox-active tyrosine 244. Science 2000;290:1588–91.[Abstract/Free Full Text]
7. Fan H, Villegas C, Huang A, Wright JA. The mammalian ribonucleotide reductase R2 component cooperates with a variety of oncogenes in mechanisms of cellular transformation. Cancer Res 1998;58:1650–3.[Abstract/Free Full Text]
8. Rauko P, Romanova D, Miadokova E, et al. DNA-protective activity of new ribonucleotide reductase inhibitors. Anticancer Res 1997;17:3437–40.[Medline]
9. Kimura T, Takeda S, Sagiya Y, Gotoh M, Nakamura Y, Arakawa H. Impaired function of p53R2 in Rrm2b-null mice causes severe renal failure through attenuation of dNTP pools. Nat Genet 2003;34:440–5.[CrossRef][Medline]
10. Zhou B, Liu X, Mo X, et al. The human ribonucleotide reductase subunit hRRM2 complements p53R2 in response to UV-induced DNA repair in cells with mutant p53. Cancer Res 2003;63:6583–94.[Abstract/Free Full Text]
11. Shao J, Zhou B, Zhu L, et al. In vitro characterization of enzymatic properties and inhibition of the p53R2 subunit of human ribonucleotide reductase. Cancer Res 2004;64:1–6.[Abstract/Free Full Text]
12. Sastre J, Pallardo FV, Vina J. The role of mitochondrial oxidative stress in aging. Free Radic Biol Med 2003;35:1–8.[Medline]
13. Smiley ST, Reers M, Mottola-Hartshorn C, et al. Intracellular heterogeneity in mitochondrial membrane potentials revealed by a J-aggregate-forming lipophilic cation JC-1. Proc Natl Acad Sci U S A 1991;88:3671–5.[Abstract/Free Full Text]
14. Tanaka H, Arakawa H, Yamaguchi T, et al. A ribonucleotide reductase gene involved in a p53-dependent cell-cycle checkpoint for DNA damage. Nature 2000;404:42–9.[CrossRef][Medline]
15. Sahlin M, Sjoberg BM, Backes G, Loehr T, Sanders-Loehr J. Activation of the iron-containing B2 protein of ribonucleotide reductase by hydrogen peroxide. Biochem Biophys Res Commun 1990;167:813–8.[CrossRef][Medline]
16. Climent I, Sjoberg BM, Huang CY. Site-directed mutagenesis and deletion of the carboxyl terminus of Escherichia coli ribonucleotide reductase protein R2. Effects on catalytic activity and subunit interaction. Biochemistry 1992;31:4801–7.[CrossRef][Medline]
17. Tong W, Burdi D, Riggs-Gelasco P, et al. Characterization of Y122F R2 of Escherichia coli ribonucleotide reductase by time-resolved physical biochemical methods and X-ray crystallography. Biochemistry 1998;37:5840–8.[CrossRef][Medline]
18. Rova U, Adrait A, Potsch S, Graslund A, Thelander L. Evidence by mutagenesis that Tyr(370) of the mouse ribonucleotide reductase R2 protein is the connecting link in the intersubunit radical transfer pathway. J Biol Chem 1999;274:23746–51.[Abstract/Free Full Text]
19. Chang MC, Yee CS, Nocera DG, Stubbe J. Site-specific replacement of a conserved tyrosine in ribonucleotide reductase with an aniline amino acid: a mechanistic probe for a redox-active tyrosine. J Am Chem Soc 2004;126:16702–3.[CrossRef][Medline]
20. Dreher D, Junod AF. Role of oxygen free radicals in cancer development. Eur J Cancer 1996;32A:30–8.[CrossRef]

This article has been cited by other articles:

				H. Muniyappa, S. Song, C. K. Mathews, and K. C. Das Reactive Oxygen Species-independent Oxidation of Thioredoxin in Hypoxia: INACTIVATION OF RIBONUCLEOTIDE REDUCTASE AND REDOX-MEDIATED CHECKPOINT CONTROL J. Biol. Chem., June 19, 2009; 284(25): 17069 - 17081. [Abstract] [Full Text] [PDF]

				L. Chang, B. Zhou, S. Hu, R. Guo, X. Liu, S. N. Jones, and Y. Yen ATM-mediated serine 72 phosphorylation stabilizes ribonucleotide reductase small subunit p53R2 protein against MDM2 to DNA damage PNAS, November 25, 2008; 105(47): 18519 - 18524. [Abstract] [Full Text] [PDF]

				X. Xu, J. L. Page, J. A. Surtees, H. Liu, S. Lagedrost, Y. Lu, R. Bronson, E. Alani, A. Yu. Nikitin, and R. S. Weiss Broad Overexpression of Ribonucleotide Reductase Genes in Mice Specifically Induces Lung Neoplasms Cancer Res., April 15, 2008; 68(8): 2652 - 2660. [Abstract] [Full Text] [PDF]

				X. Liu, B. Zhou, L. Xue, J. Shih, K. Tye, W. Lin, C. Qi, P. Chu, F. Un, W. Wen, et al. Metastasis-Suppressing Potential of Ribonucleotide Reductase Small Subunit p53R2 in Human Cancer Cells. Clin. Cancer Res., November 1, 2006; 12(21): 6337 - 6344. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Xue, L. Articles by Yen, Y. Search for Related Content PubMed PubMed Citation Articles by Xue, L. Articles by Yen, Y.