Inactivation Of Ribonucleotide Reductase Research Articles

De novo dNTP production is essential for normal postnatal murine heart development

The building blocks of DNA, dNTPs, can be produced de novo or can be salvaged from deoxyribonucleosides. However, to what extent the absence of de novo dNTP production can be compensated for by the salvage pathway is unknown. Here, we eliminated de novo dNTP synthesis in the mouse heart and skeletal muscle by inactivating ribonucleotide reductase (RNR), a key enzyme for the de novo production of dNTPs, at embryonic day 13. All other tissues had normal de novo dNTP synthesis and theoretically could supply heart and skeletal muscle with deoxyribonucleosides needed for dNTP production by salvage. We observed that the dNTP and NTP pools in WT postnatal hearts are unexpectedly asymmetric, with unusually high dGTP and GTP levels compared with those in whole mouse embryos or murine cell cultures. We found that RNR inactivation in heart led to strongly decreased dGTP and increased dCTP, dTTP, and dATP pools; aberrant DNA replication; defective expression of muscle-specific proteins; progressive heart abnormalities; disturbance of the cardiac conduction system; and lethality between the second and fourth weeks after birth. We conclude that dNTP salvage cannot substitute for de novo dNTP synthesis in the heart and that cardiomyocytes and myocytes initiate DNA replication despite an inadequate dNTP supply. We discuss the possible reasons for the observed asymmetry in dNTP and NTP pools in WT hearts.

Triapine Radiochemotherapy in Advanced Stage Cervical Cancer

Clinical ribonucleotide reductase (RNR) inhibitors have reinvigorated enthusiasm for radiochemotherapy treatment of patients with regionally advanced stage cervical cancers. About two-thirds of patients outlive their cervical cancer (1), even though up to half of their tumors retain residual microscopic disease (2). The National Cancer Institute Cancer Therapy Evaluation Program conducted two prospective trials of triapine–cisplatin–radiation to improve upon this finding by precisely targeting cervical cancer’s overactive RNR. Triapine’s potent inactivation of RNR arrests cells at the G1/S cell cycle restriction checkpoint and enhances cisplatin–radiation cytotoxicity. In this article, we provide perspective on challenges encountered in and future potential of clinical development of a triapine–cisplatin–radiation combination for patients with regionally advanced cervical cancer. New trial results and review presented here suggest that a triapine–cisplatin–radiation combination may offer molecular cell cycle target control to maximize damage in cancers and to minimize injury to normal cells. A randomized trial now accrues patients with regionally advanced stage cervical cancer to evaluate triapine’s contribution to clinical benefit after cisplatin–radiation (clinicaltrials.gov, NCT02466971).

Replication Rapidly Recovers and Continues in the Presence of Hydroxyurea in Escherichia coli.

In both prokaryotes and eukaryotes, hydroxyurea is suggested to inhibit DNA replication by inactivating ribonucleotide reductase and depleting deoxyribonucleoside triphosphate pools. In this study, we show that the inhibition of replication in Escherichia coli is transient even at concentrations of 0.1 M hydroxyurea and that replication rapidly recovers and continues in its presence. The recovery of replication does not require the alternative ribonucleotide reductases NrdEF and NrdDG or the translesion DNA polymerases II (Pol II), Pol IV, and Pol V. Ribonucleotides are incorporated at higher frequencies during replication in the presence of hydroxyurea. However, they do not contribute significantly to the observed synthesis or toxicity. Hydroxyurea toxicity was observed only under conditions where the stability of hydroxyurea was compromised and by-products known to damage DNA directly were allowed to accumulate. The results demonstrate that hydroxyurea is not a direct or specific inhibitor of DNA synthesis in vivo and that the transient inhibition observed is most likely due to a general depletion of iron cofactors from enzymes when 0.1 M hydroxyurea is initially applied. Finally, the results support previous studies suggesting that hydroxyurea toxicity is mediated primarily through direct DNA damage induced by the breakdown products of hydroxyurea, rather than by inhibition of replication or depletion of deoxyribonucleotide levels in the cell.IMPORTANCE Hydroxyurea is commonly suggested to function by inhibiting DNA replication through the inactivation of ribonucleotide reductase and depleting deoxyribonucleoside triphosphate pools. Here, we show that hydroxyurea only transiently inhibits replication in Escherichia coli before replication rapidly recovers and continues in the presence of the drug. The recovery of replication does not depend on alternative ribonucleotide reductases, translesion synthesis, or RecA. Further, we show that hydroxyurea toxicity is observed only in the presence of toxic intermediates that accumulate when hydroxyurea breaks down, damage DNA, and induce lethality. The results demonstrate that hydroxyurea toxicity is mediated indirectly by the formation of DNA damage, rather than by inhibition of replication or depletion of deoxyribonucleotide levels in the cell.

Abstract 847: Suppression of p53R2 but not R2 radiosensitizes mutant p53 tumor cells

Abstract Gemcitabine (2’,2’-difluroro-2’-deoxycytidine;dFdCyd) is a potent radiosensitizer in tumor cells in vitro and in vivo. dFdCyd elicits cytotoxicity primarily via incorporation of its triphosphate, dFdCTP, into DNA, whereas inhibition of ribonucleotide reductase (RR) by dFdCDP produces a profound depletion of dATP which correlates to radiosensitization. We have demonstrated that dNTP imbalances generated by dFdCyd produce mismatches in DNA, which augment sensitivity to subsequent ionizing radiation (IR) but are not required to elicit cytotoxicity. We have proposed that RR suppression would be as effective as dFdCyd for radiosensitization. RR is a heterodimeric tetramer composed of the regulatory and active site subunit R1 paired with either R2 or its p53-inducible homolog, p53R2, as the catalytic and rate-limiting subunit. We used a RNAi approach to suppress either R2 or p53R2 (≥ 90% suppression of either R2 or p53R2, with little effect (≤20%) on expression of its homolog) in two p53 wild type cell lines, MCF7 breast carcinoma and A549 lung carcinoma. This approach produced equivalent radiosensitization, dATP depletion, cytotoxicity and increase in DNA mismatches compared to inactivation of RR by dFdCyd (IC50). These results reinforce our prior finding that radiosensitization with dFdCyd is the result of inhibition of RR and not incorporation into DNA or cytotoxicity. We then proposed that R2 but not p53R2 suppression would radiosensitize mutant p53 tumor cells. Interestingly, R2 shRNA suppression did not radiosensitize mutant p53 MCF7/ADR cells (radiation enhancement ratio (RER) = 1.07 ± 0.06). Despite the mutant p53 status of MCF7/ADR cells, p53R2 was elevated (≥ 50%) following suppression of R2 compared to untreated cells (no shRNA). We hypothesized that this increase in p53R2 permits RR to continue producing dNTPs for DNA replication and repair in the absence of R2, thus preventing radiosensitization. Indeed, simultaneous suppression of R2 and p53R2 produced excellent radiosensitization (RER = 1.68 ± 0.04 (shRNAs) vs. 1.6 ± 0.01 (dFdCyd (IC50)), and similar cytotoxicity (surviving fraction (SF) = 76.5 ± 8.3% (shRNA) vs. 71.3 ± 6.7% (dFdCyd)). Impressively, suppression of p53R2 alone produced excellent radiosensitization (RER= 1.76 ± 0.30) with similar cytotoxicity (SF = 84 ± 20%) compared to the double knockdown or dFdCyd. Considering that most solid tumors express mutant p53, suppression of p53R2 instead of R2 may be a more effective method for radiosensitization. These studies suggest that deoxynucleotide biosynthesis is regulated differently in p53 wild type compared to mutant p53 tumor cells, and may be used to maintain DNA replication and repair and prevent anticancer efficacy after damage induced by IR or other DNA damaging agents. The mechanism by which mutant p53 tumor cells upregulate p53R2 warrants further investigation. Note: This abstract was not presented at the meeting. Citation Format: Sheryl A. Flanagan, Jeffrey J. Ackroyd, Sudha Mannava, Mikhail A. Nikiforov, Donna S. Shewach. Suppression of p53R2 but not R2 radiosensitizes mutant p53 tumor cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 847. doi:10.1158/1538-7445.AM2014-847

Tangled Up in Knots: Structures of Inactivated Forms of E. coli Class Ia Ribonucleotide Reductase

Ribonucleotide reductases (RNRs) provide the precursors for DNA biosynthesis and repair and are successful targets for anticancer drugs such as clofarabine and gemcitabine. Recently, we reported that dATP inhibits E. coli class Ia RNR by driving formation of RNR subunits into α4β4 rings. Here, we present the first X-ray structure of a gemcitabine-inhibited E. coli RNR and show that the previously described α4β4 rings can interlock to form an unprecedented (α4β4)2 megacomplex. This complex is also seen in a higher-resolution dATP-inhibited RNR structure presented here, which employs a distinct crystal lattice from that observed in the gemcitabine-inhibited case. With few reported examples of protein catenanes, we use data from small-angle X-ray scattering and electron microscopy to both understand the solution conditions that contribute to concatenation in RNRs as well as present a mechanism for the formation of these unusual structures.

Ribonucleotide Reductase Inhibition Enhances Chemoradiosensitivity of Human Cervical Cancers

For repair of damaged DNA, cells increase de novo synthesis of deoxyribonucleotide triphosphates through the rate-limiting, p53-regulated ribonucleotide reductase (RNR) enzyme. In this study we investigated whether pharmacological inhibition of RNR by 3-aminopyridine-2-carboxaldehyde thiosemicarbazone (3-AP, NSC #663249) enhanced chemoradiation sensitivity through a mechanism involving sustained DNA damage. RNR inactivation by 3-AP and resulting chemoradiosensitization were evaluated in human cervical (CaSki, C33-a) cancer cells through study of DNA damage (γ-H2AX signal) by flow cytometry, RNR subunit p53R2 and p21 protein steady-state levels by Western blot analysis and laser scanning imaging cytometry, and cell survival by colony formation assays. 3-AP treatment led to sustained radiation- and cisplatin-induced DNA damage (i.e. increased γ-H2AX signal) in both cell lines through a mechanism of inhibited RNR activity. Radiation, cisplatin and 3-AP exposure resulted in significantly elevated numbers and persistence of γ-H2AX foci that were associated with reduced clonogenic survival. DNA damage was associated with a rise in p53R2 but not p21 protein levels 6 h after treatment with radiation and/or cisplatin plus 3-AP. We conclude that blockage of RNR activity by 3-AP impairs DNA damage responses that rely on deoxyribonucleotide production and thereby may substantially increase chemoradiosensitivity of human cervical cancers.

Ribonucleotide Reductase: A Mechanistic Portrait of Substrate Analogues Inhibitors

Ribonucleotide reductase (RNR) is the key enzyme in the biosynthesis of deoxyribonucleotides. Several different strategies for inactivation of RNRs have been reported, including the use of substrate analogues as mechanism-based inhibitors. This article undergoes a critical analysis on the current status of ribonucleotide reductase inhibitory mechanisms by substrate analogues highlighting experimental and theoretical/computational approaches. We have summarized a general portrait of the inhibitory mechanisms and classified the nucleoside analogue inhibitors in three main classes. The critical analysis undertaken will contribute in finding new and more effective ways of inhibiting RNR.

Understanding the Mechanism for Ribonucleotide Reductase Inactivation by 2'- Deoxy-2'-methylenecytidine-5'-diphosphate.

Ribonucleotide reductase (RNR) is the key enzyme in the biosynthesis of deoxyribonucleotides. The enzyme has thus an attractive target for chemotherapies that fight proliferation-based diseases. 2'-Deoxy-2'methylenecytidine-5'-diphosphate (CH2dCDP) is a potent mechanism-based inhibitor of the enzyme RNR, which decomposes to an active alkylating furanone specie. The details of the inhibition mechanism are unknown, and experimental studies have indicated that some properties of the inactivation are dissimilar to those observed with a number of 2'-substituted 2'-deoxynucleotides mechanism-based inhibitors. To disclose the mechanism involved in RNR inactivation by CH2dCDP we explored the potential-energy surface in two different models of the system with different objectives in mind. In order to conveniently explore the reactional space, i.e. to study the possible reactions between the CH2dCDP and the RNR, we used a small model representing the active site of RNR with CH2dCDP using DFT. To provide further insights and efficiently account for the long-range RNR-CH2dCDP interactions and the stereochemical strain imposed by the protein scaffold we performed theoretical calculations on the more promising reactions using hybrid QM/MM calculations on a larger model system. We used quantum mechanics for the active-site region (CH2dCDP and active-site residues) and molecular mechanics for the surroundings (6373 atoms of the R1 monomer). The results obtained led us to understand the correct mechanism for RNR inactivation by CH2dCDP, and the furanone species formed presumably explains the dissimilarities observed with a number of 2'-substituted 2'-deoxynucleotides.

Inactivation of Lactobacillus leichmannii ribonucleotide reductase by 2',2'-difluoro-2'-deoxycytidine 5'-triphosphate: covalent modification.

Ribonucleotide reductase (RNR) from Lactobacillus leichmannii, a 76 kDa monomer using adenosylcobalamin (AdoCbl) as a cofactor, catalyzes the conversion of nucleoside triphosphates to deoxynucleotides and is rapidly (<30 s) inactivated by 1 equiv of 2',2'-difluoro-2'-deoxycytidine 5'-triphosphate (F(2)CTP). [1'-(3)H]- and [5-(3)H]F(2)CTP were synthesized and used independently to inactivate RNR. Sephadex G-50 chromatography of the inactivation mixture revealed that 0.47 equiv of a sugar was covalently bound to RNR and that 0.71 equiv of cytosine was released. Alternatively, analysis of the inactivated RNR by SDS-PAGE without boiling resulted in 33% of RNR migrating as a 110 kDa protein. Inactivation of RNR with a mixture of [1'-(3)H]F(2)CTP and [1'-(2)H]F(2)CTP followed by reduction with NaBH(4), alkylation with iodoacetamide, trypsin digestion, and HPLC separation of the resulting peptides allowed isolation and identification by MALDI-TOF mass spectrometry (MS) of a (3)H/(2)H-labeled peptide containing C(731) and C(736) from the C-terminus of RNR accounting for 10% of the labeled protein. The MS analysis also revealed that the two cysteines were cross-linked to a furanone species derived from the sugar of F(2)CTP. Incubation of [1'-(3)H]F(2)CTP with C119S-RNR resulted in 0.3 equiv of sugar being covalently bound to the protein, and incubation with NaBH(4) subsequent to inactivation resulted in trapping of 2'-fluoro-2'-deoxycytidine. These studies and the ones in the preceding paper (DOI: 10.1021/bi9021318 ) allow proposal of a mechanism of inactivation of RNR by F(2)CTP involving multiple reaction pathways. The proposed mechanisms share many common features with F(2)CDP inactivation of the class I RNRs.

Histological and genetic markers for non-small-cell lung cancer: Customizing treatment based on individual tumor biology

To describe how molecular and genetic markers influence the response to therapy in patients with advanced non-small-cell lung cancer (NSCLC). Cisplatin resistance has been associated with high rates of expression of endonucleases, a family of DNA repair proteins that includes ERCC1 and BRCA1. Gemcitabine is thought to produce its antitumor effects by several mechanisms, including inhibition of ribonucleotide conversion to deoxyribonucleotide through the inactivation of ribonucleotide reductase. One type of gemcitabine resistance occurs when the inactivation of ribonucleotide reductase decreases, which correlates with increased expression of the M1 subunit of ribonucleotide reductase. Taxanes produce their anti-tumor effects by binding to and stabilizing intracellular microtubules, which are necessary for DNA replication and cell division. High expression of beta-tubulin III, a microtubule subunit with low taxane binding affinity, appears to confer resistance to taxane therapy. High expression of thymidylate synthase, an enzyme that is important in purine synthesis and DNA replication, has been associated with decreased response to uracil antimetabolites, and may also be an important prognostic factor in patients receiving pemetrexed. The epidermal growth factor receptor (EGFR) is a cell- surface receptor with an intracellular tyrosine kinase domain that is thought to modulate numerous cellular functions that contribute to tumorigenicity, tumor invasiveness, and resistance to therapy. Recent clinical trials have demonstrated that treatment response to small-molecule and monoclonal antibody inhibitors of EGFR is greatest for patients who have a higher EGFR gene copy number. Several genetic, molecular, and histological markers may affect treatment response in patients with NSCLC. Evaluating patients for such markers is important not only for treatment efficacy, but also to improve safety and tolerability by avoiding exposure to treatments that are unlikely to produce significant benefits. Nearly all of the available information regarding the predictive value of these markers has been derived from retrospective studies. Prospective clinical trials are important to validate marker evaluation methodology and the prospective utility of biomarkers in clinical decision making.

Insight into the Mechanism of Inactivation of Ribonucleotide Reductase by Gemcitabine 5′-Diphosphate in the Presence or Absence of Reductant

Gemcitabine 5'-diphosphate (F(2)CDP) is a potent inhibitor of ribonucleotide reductases (RNRs), enzymes that convert nucleotides (NDPs) to deoxynucleotides and are essential for DNA replication and repair. The Escherichia coli RNR, an alpha2beta2 complex, when incubated with 1 equiv of F(2)CDP catalyzes the release of two fluorides and cytosine concomitant with enzyme inactivation. In the presence of reductant (thioredoxin/thioredoxin reductase/NADPH or DTT), the enzyme inactivation results from its covalent labeling of alpha with the sugar of F(2)CDP (one label/alpha2beta2). SDS-PAGE analysis of the inactivated RNR without boiling of the sample reveals that alpha migrates as an 87 and 110 kDa protein in a ratio of 0.6:0.4. When the reductant is omitted, RNR is inactivated by loss of the essential tyrosyl radical and formation of a new radical. Inactivation studies with C225S-alpha in the presence or absence of reductants, reveal it behaves like wt-RNR in the absence of reductant. Inactivated C225S-alpha migrates as an 87 kDa protein and is not covalently modified. C225 is one of the cysteines in RNR's active site that supplies reducing equivalents to make dNDPs. To identify the new radical formed, [1'-(2)H]-F(2)CDP was studied with wt- and C225S-RNR by 9 and 140 GHz EPR spectroscopy. These studies revealed that the new radical is a nucleotide derived with g values of g(x) 2.00738, g(y) 2.00592, and g(z) 2.00230 and with altered hyperfine interactions (apparent triplet collapsed to a doublet) relative to [1'-(1)H]-F(2)CDP. The EPR features are very similar to those we recently reported for the nucleotide radical generated with CDP and E441Q-RNR.

Inactivation of Ribonucleotide Reductase by gemcitabine‐5′‐diphosphate

Ribonucleotide reductases (RNRs) catalyze the conversion of nucleotides to deoxynucleotides in all organisms supplying the monomeric precursors required for DNA replication and repair. The relative ratio of the deoxynucleotide (dNTP) pools and the absolute amounts of dNTPs govern the fidelity of these processes. RNRs are composed of two types of subunits: α and β. α contains the binding site for nucleoside diphosphates (NDPs) that are reduced and the allosteric effectors (ATP and dNTPs) that control the specificity and rate of NDP reduction. β houses the essential diferric‐tyrosyl radical (Y•) cofactor. The central role of RNRs in nucleic acid metabolism has made them the successful target of drugs that are used clinically. Gemzar, 2′, 2″‐difluorodeoxycytidine (F2C) is used clinically in the treatment of non small cell lung carcinomas and advanced pancreatic cancer. While the mode of cytotoxicity is complex, one its targets is RNR. Synthesis of [5′‐3H]‐F2C CDP has shown that 1 equivalent per α2 is sufficient to inactivate the E. coli and human RNRs. Studies on the mechanism of inactivation will be presented.

Understanding Ribonucleotide Reductase Inactivation by Gemcitabine

This paper focuses on the inhibition of ribonucleotide reductase (RNR) by gemcitabine, 2',2'-difluoro-2'-deoxycytidine (dFdC), a deoxycytidine analogue that is a very active drug against solid tumors and is currently commercialized as gemzar. RNR inactivation is reductant-dependent and occurs in a very different way from that of other known substrate analogues. In the presence of reductants monomer R1 of RNR is inhibited, whereas in the absence of reductants the radical is lost and monomer R2 is inhibited. As inside the cell reductants are available, it is likely that R1 inactivation is the most favorable mechanism responsible for drug cytotoxicity. This inhibition pathway has been unknown to date, but we have conducted a theoretical study that has led us to the first proposal of a mechanism for RNR inhibition by dFdC in the presence of reductants.

Hydroxyurea Associated Leg Ulcer Succesfully Treated with Hyperbaric Oxygen in a Diabetic Patient

Oxygen tension in healing tissues is heterogeneous. Increased oxygen mostly stimulates repair mechanisms and enhances tissue healing. Hyperbaric oxygen therapy increases blood and tissue oxygen content and may help maintain cellular integrity and function. Hydroxyurea (HU) is a cytotoxic agent, which leads to inactivation of ribonucleotide reductase, inhibition of cellular DNA synthesis, and cell death in the S phase. HU induced leg ulcers occur after use of this agent for a long time and at higher cumulative doses. Here we describe a diabetic patient with foot ulcer associated with HU treatment for polycythemia vera, who was treated successfully with hyperbaric oxygen and general wound care after discontinuation of HU. Faster improvement of leg ulcer in our patient compared to literature regarding HU withdrawal as single therapy suggests that hyperbaric oxygen may be helpful in the management of HU associated leg ulcers, especially in diabetic subjects.

Theoretical Study on the Inhibition of Ribonucleotide Reductase by 2‘-Mercapto-2‘-deoxyribonucleoside-5‘-diphosphates

Ribonucleotide reductase (RNR) is responsible for the reduction of ribonucleotides into the correspondent 2'-deoxyribonucleotides in the only physiological process that yields the monomers of DNA. The enzyme has thus become an attractive target for chemotherapies that fight proliferation-based diseases, specifically cancer and infections by some viruses and parasites. 2'-Mercapto-2'-deoxyribonucleoside-5'-diphosphates (SHdNDP) are mechanism-based inhibitors of RNR and therefore potential chemotherapeutic agents for those indications. Previous experimental studies established the in vitro and in vivo activity of SHdNDP. In the in vitro studies, it was observed that the activity was dependent on the oxidative status of the medium, with the inactivation of RNR only occurring when molecular oxygen was available. To better understand the mechanism involved in RNR inactivation by SHdNDP, we performed theoretical calculations on the possible reactions between the inhibitors and the RNR active site. As a result, we propose the possible mechanistic pathways for the chemical events that occur in the absence and in the presence of O2. They correspond to a refinement and a complement of those proposed in the literature.

Mechanism for ribonucleotide reductase inactivation by the anticancer drug gemcitabine.

Gemcitabine (2',2'-difluoro-2'-deoxycytidine, dFdC) is a very promising anticancer drug, already approved for clinical use in three therapeutic indications. It is metabolized intracellularly to 5'-diphosphate (dFdCDP), which is known to be a potent inhibitor of ribonucleotide reductase (RNR). Although several nucleotide analogs show in vitro capacity of RNR inactivation, none has shown the in vivo efficacy of dFdCDP. Accordingly, the experimental data suggests that its mechanism of inhibition is different from the other known RNR suicide inhibitors. Enzyme inhibition in the absence of reductive species leads to complete loss of the essential radical in subunit R2, and formation of a new nucleotide-based radical. Interestingly, however, the presence of the reductants does not prevent inhibition--the radical is not lost but the targeted subunit of RNR becomes R1, which is inactivated possibly by alkylation. We have conducted a theoretical study, which led us to the first proposal of a possible mechanism for RNR inhibition by dFdCDP in the absence of reductants. This mechanism turned out to be very similar to the natural substrate reduction pathway and only deviates from the natural course after the formation of the well-known disulphide bridge. This deviation is caused precisely by the F atom in the beta-face, only present in this inhibitor. The essential radical in R2 is lost, and so is the enzyme catalytic activity. The nucleotide-based radical that constitutes the end product of our mechanism has been suggested in the literature as a possible candidate for the one detected experimentally. In fact, all experimental data available has been reproduced by the theoretical calculations performed here.

Radiation Inactivation of Ribonucleotide Reductase, an Enzyme with a Stable Free Radical

Herpes simplex virus ribonucleotide reductase (RR) is a tetrameric enzyme composed of two homodimers of large R1 and small R2 subunits with a tyrosyl free radical located on the small subunit. Irradiation of the holoenzyme yielded simple exponential decay curves and an estimated functional target size of 315 kDa. Western blot analysis of irradiated holoenzyme R1 and R2 yielded target sizes of 281 kDa and 57 kDa (approximately twice their expected size). Irradiation of free R1 and analysis by all methods yielded a single exponential decay with target sizes ranging from 128–153 kDa. For free R2, quantitation by enzyme activity and Western blot analyses yielded simple inactivation curves but considerably different target sizes of 223 kDa and 19 kDa, respectively; competition for radioligand binding in irradiated R2 subunits yielded two species, one with a target size of ∼210 kDa and the other of ∼20 kDa. These results are consistent with a model in which there is radiation energy transfer between the two monomers of both R1 and R2 only in the holoenzyme, a radiation-induced loss of free radical only in the isolated R2, and an alteration of the tertiary structure of R2.

Liz1p, a novel fission yeast membrane protein, is required for normal cell division when ribonucleotide reductase is inhibited.

Ribonucleotide reductase activity is required for generating deoxyribonucleotides for DNA replication. Schizosaccharomyces pombe cells lacking ribonucleotide reductase activity arrest during S phase of the cell cycle. In a screen for hydroxyurea-sensitive mutants in S. pombe, we have identified a gene, liz1(+), which when mutated reveals an additional, previously undescribed role for ribonucleotide reductase activity during mitosis. Inactivation of ribonucleotide reductase, by either hydroxyurea or a cdc22-M45 mutation, causes liz1(-) cells in G2 to undergo an aberrant mitosis, resulting in chromosome missegregation and late mitotic arrest. liz1(+) encodes a 514-amino acid protein with strong similarity to a family of transmembrane transporters, and localizes to the plasma membrane of the cell. These results reveal an unexpected G2/M function of ribonucleotide reductase and establish that defects in a transmembrane protein can affect cell cycle progression.

Detection of a new substrate-derived radical during inactivation of ribonucleotide reductase from Escherichia coli by gemcitabine 5'-diphosphate.

Ribonucleotide reductases (RNRs) play a central role in replication and repair by catalyzing the conversion of nucleotides to deoxynucleotides. Gemcitabine 5'-diphosphate (F2CDP), the nucleoside of which was recently approved by the FDA for treatment of pancreatic cancer, is a potent mechanism-based inhibitor of class I and II RNRs. Inactivation of the Eschericia coli class I RNR is accompanied by loss of two fluorides and one cytosine. This RNR is composed of two homodimeric subunits: R1 and R2. R1 is the site of nucleotide reduction, and R2 contains the essential diferric-tyrosyl radical cofactor. The mechanism of inactivation depends on the availability of reductant. In the presence of reductant [thioredoxin (TR)/thioredoxin reductase (TRR)/NADPH or dithiothreitol], inhibition results from R1 inactivation. In the absence of reductant with prereduced R1 and R2, inhibition results from loss of the essential tyrosyl radical in R2. The same result is obtained with C754S/C759S-R1 in the presence of TR/TRR/NADPH. In both cases, tyrosyl radical loss is accompanied by formation of a new stable radical (0.15-0.25 equiv/RNR). EPR studies in 2H2O, with [U-2H]R1, and examination of the microwave power saturation of the observed signal, indicate by process of elimination that this new radical is nucleotide-based. In contrast to all previously investigated 2'-substituted nucleotide inhibitors of RNR, inactivation is not accompanied by formation of a new protein-associated chromophore under any conditions. The requirement for reductant in the R1 inactivation pathway, the lack of chromophore on the protein, the loss of two fluoride ions, and the stoichiometry of the inactivation all suggest a unique mechanism of RNR inactivation not previously observed with other 2'-substituted nucleotide inhibitors of RNR. This unique mode of inactivation is proposed to be responsible for its observed clinical efficacy.

Characterization of a Substrate-Derived Radical Detected during the Inactivation of Ribonucleotide Reductase fromEscherichia coliby 2‘-Fluoromethylene-2‘-deoxycytidine 5‘-Diphosphate

Ribonucleotide reductase (RDPR) from E. coli catalyzes the conversion of nucleotides to deoxynucleotides and contains an unusual tyrosyl radical−diferric cluster cofactor. (E)-2‘-Fluoromethylene-2‘-deoxycytidine 5‘-diphosphate [(E)-1] obtained by phosphorylation of the clinically promising antitumor agent MDL 101,731, is a potent time-dependent inactivator of this protein. Electron paramagnetic resonance (EPR) spectroscopy reveals that inactivation is accompanied by loss of the essential tyrosyl radical cofactor and formation of a new radical species. The 9.4-GHz EPR spectrum of this new radical reveals two hyperfine splittings of approximately 1.5 mT producing a triplet-like signal. Incubation of the enzyme with [6‘-2H]-(E)-1 alters this EPR spectrum, providing the first evidence for a nucleotide-based radical species generated by RDPR. The observed spectrum is a 1:1 composite of a doublet and a triplet signal, the latter being identical to that obtained with unlabeled (E)-1. Studies with (E)-1 in 2H2O also produce a 1:1 mixture of these two radical signals. The results of these isotope labeling experiments suggest wash-out or wash-in of ∼0.5 equiv of deuterium at the 6‘ position, respectively. Incubation of the enzyme with [6‘-2H]-(E)-1 in 2H2O produced only the doublet as would be expected on the basis of this hypothesis. EPR (139.5 GHz) spectra established the principal g values of the new radical species. Simulation of the 9.4- and 139.5-GHz EPR spectra yield a self-consistent set of principal hyperfine values. A structure is proposed for the radical intermediate that is consistent with the EPR data and kinetic data on release of the fluoride ion that accompanies inactivation. The proposed structure and a postulated mechanism for its formation provide further support for the hypothesis that catalysis is initiated by 3‘-hydrogen atom abstraction from the nucleotide substrate.