Search

Deoxynucleoside triphosphates (dNTPs) are crucial for the replication and maintenance of genomic information within cells. The balance of the dNTP pool involves several cellular enzymes, including dihydrofolate reductase (DHFR), ribonucleotide reductase (RNR), and SAM and HD domain-containing protein 1 (SAMHD1), among others. DHFR is vital for the de novo synthesis of purines and deoxythymidine monophosphate, which are necessary for DNA synthesis. SAMHD1, a ubiquitously expressed deoxynucleotide triphosphohydrolase, converts dNTPs into deoxynucleosides and inorganic triphosphates. This process counteracts the de novo dNTP synthesis primarily carried out by RNR and cellular deoxynucleoside kinases, which are most active during the S phase of the cell cycle. The intracellular levels of dNTPs can influence various viral infections. This review provides a concise summary of the interactions between different viruses and the genes involved in dNTP metabolism. [ABSTRACT FROM AUTHOR]

Ribonucleotide reductase (RNR) is the rate-limiting enzyme in the synthesis of deoxyribonucleotides and the target of multiple chemotherapy drugs, including gemcitabine. We previously identified that inhibition of RNR in Ewing sarcoma tumors upregulates the expression levels of multiple members of the activator protein-1 (AP-1) transcription factor family, including c-Jun and c-Fos, and downregulates the expression of c-Myc. However, the broader functions and downstream targets of AP-1, which are highly context- and cell-dependent, are unknown in Ewing sarcoma tumors. Consequently, in this work, we used genetically defined models, transcriptome profiling, and gene-set -enrichment analysis to identify that AP-1 and EWS-FLI1, the driver oncogene in most Ewing sarcoma tumors, reciprocally regulate the expression of multiple extracellular-matrix proteins, including fibronectins, integrins, and collagens. AP-1 expression in Ewing sarcoma cells also drives, concurrent with these perturbations in gene and protein expression, changes in cell morphology and phenotype. We also identified that EWS-FLI1 dysregulates the expression of multiple AP-1 proteins, aligning with previous reports demonstrating genetic and physical interactions between EWS-FLI1 and AP-1. Overall, these results provide novel insights into the distinct, EWS-FLI1-dependent features of Ewing sarcoma tumors and identify a novel, reciprocal regulation of extracellular-matrix components by EWS-FLI1 and AP-1. [ABSTRACT FROM AUTHOR]

Proteasome inhibitors are used in the therapy of several cancers, and clinical trials are underway for their use in the treatment of glioblastoma (GBM). However, GBM becomes resistant to chemotherapy relatively rapidly. Recently, the overexpression of ribonucleotide reductase (RNR) genes was found to mediate therapy resistance in GBM. The use of combinations of chemotherapeutic agents is considered a promising direction in cancer therapy. The present work aimed to evaluate the efficacy of the combination of proteasome and RNR inhibitors in yeast and GBM cell models. We have shown that impaired proteasome function results in increased levels of RNR subunits and increased enzyme activity in yeast. Co-administration of the proteasome inhibitor bortezomib and the RNR inhibitor hydroxyurea was found to significantly reduce the growth rate of S. cerevisiae yeast. Accordingly, the combination of bortezomib and another RNR inhibitor gemcitabine reduced the survival of DBTRG-05MG compared to the HEK293 cell line. Thus, yeast can be used as a simple model to evaluate the efficacy of combinations of proteasome and RNR inhibitors. [ABSTRACT FROM AUTHOR]

The DnaA protein has long been considered to play the key role in the initiation of chromosome replication in modern bacteria. Many questions about this role, however, remain unanswered. Here, we raise these questions within a framework based on the dynamics of hyperstructures, alias large assemblies of molecules and macromolecules that perform a function. In these dynamics, hyperstructures can (1) emit and receive signals or (2) fuse and separate from one another. We ask whether the DnaA-based initiation hyperstructure acts as a logic gate receiving information from the membrane, the chromosome, and metabolism to trigger replication; we try to phrase some of these questions in terms of DNA supercoiling, strand opening, glycolytic enzymes, SeqA, ribonucleotide reductase, the macromolecular synthesis operon, post-translational modifications, and metabolic pools. Finally, we ask whether, underpinning the regulation of the cell cycle, there is a physico-chemical clock inherited from the first protocells, and whether this clock emits a single signal that triggers both chromosome replication and cell division. [ABSTRACT FROM AUTHOR]

Purpose: Ewing's sarcoma is a highly malignant childhood tumour whose outcome has hardly changed over the past two decades despite numerous attempts at chemotherapy intensification. It is therefore essential to identify new treatment options. The present study was conducted to explore the effectiveness of combined inhibition of two promising targets, ATR and ribonucleotide reductase (RNR), in Ewing's sarcoma cells. Methods: Effects of the ATR inhibitor VE821 in combination with the RNR inhibitors triapine and didox were assessed in three Ewing's sarcoma cell lines with different TP53 status (WE-68, SK-ES-1, A673) by flow cytometric analysis of cell death, mitochondrial depolarisation and cell cycle distribution as well as by caspase 3/7 activity determination, by immunoblotting and by real-time RT-PCR. Interactions between inhibitors were evaluated by combination index analysis. Results: Single ATR or RNR inhibitor treatment produced small to moderate effects, while their combined treatment produced strong synergistic ones. ATR and RNR inhibitors elicited synergistic cell death and cooperated in inducing mitochondrial depolarisation, caspase 3/7 activity and DNA fragmentation, evidencing an apoptotic form of cell death. All effects were independent of functional p53. In addition, VE821 in combination with triapine increased p53 level and induced p53 target gene expression (CDKN1A, BBC3) in p53 wild-type Ewing's sarcoma cells. Conclusion: Our study reveals that combined targeting of ATR and RNR was effective against Ewing's sarcoma in vitro and thus rationalises an in vivo exploration into the potential of combining ATR and RNR inhibitors as a new strategy for the treatment of this challenging disease. [ABSTRACT FROM AUTHOR]

The four-celled stomatal complex consists of a pair of guard cells (GCs) and two subsidiary cells (SCs) in grasses, which supports a fast adjustment of stomatal aperture. The formation and development of SCs are thus important for stomatal functionality. Here, we report a maize lost subsidiary cells (lsc) mutant, with many stomata lacking one or two SCs. The loss of SCs is supposed to have resulted from impeded subsidiary mother cell (SMC) polarization and asymmetrical division. Besides the defect in SCs, the lsc mutant also displays a dwarf morphology and pale and striped newly-grown leaves. LSC encodes a large subunit of ribonucleotide reductase (RNR), an enzyme involved in deoxyribonucleotides (dNTPs) synthesis. Consistently, the concentration of dNTPs and expression of genes involved in DNA replication, cell cycle progression, and SC development were significantly reduced in the lsc mutant compared with the wild-type B73 inbred line. Conversely, overexpression of maize LSC increased dNTP synthesis and promoted plant growth in both maize and Arabidopsis. Our data indicate that LSC regulates dNTP production and is required for SMC polarization, SC differentiation, and growth of maize. [ABSTRACT FROM AUTHOR]

The APOBEC3 family of DNA cytosine deaminases comprises an important arm of the innate antiviral defense system. The gamma-herpesviruses Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus and the alpha-herpesviruses herpes simplex virus (HSV)-1 and HSV-2 have evolved an efficient mechanism to avoid APOBEC3 restriction by directly binding to APOBEC3B and facilitating its exclusion from the nuclear compartment. The only viral protein required for APOBEC3B relocalization is the large subunit of the ribonucleotide reductase (RNR). Here, we ask whether this APOBEC3B relocalization mechanism is conserved with the beta-herpesvirus human cytomegalovirus (HCMV). Although HCMV infection causes APOBEC3B relocalization from the nucleus to the cytoplasm in multiple cell types, the viral RNR (UL45) is not required. APOBEC3B relocalization occurs rapidly following infection suggesting the involvement of an immediate early or early (IE/E) viral protein. In support of this possibility, genetic (IE1 mutant) and pharmacologic (cycloheximide) strategies that prevent the expression of IE/E viral proteins also block APOBEC3B relocalization. In comparison, the treatment of infected cells with phosphonoacetic acid, which interferes with viral late protein expression, still permits A3B relocalization. These results combine to indicate that the beta-herpesvirus HCMV uses an RNR-independent, yet phenotypically similar, molecular mechanism to antagonize APOBEC3B. [ABSTRACT FROM AUTHOR]

This article reports on the 3-formylchromone N(4)-substituted thiosemicarbazone's coordination behavior towards transition metal ions. CHNS analysis, magnetic moment, thermal analysis, IR, UV, NMR, and EPR spectroscopy were used to characterize the compounds. Moreover, DFT, insilico ADME, and docking studies of the ligand were done. Based on the spectral results, copper, cobalt, and nickel complexes had square planar geometry. Conversely, the manganese complex has octahedral geometry. The Gaussian 09 program is utilized for DFT studies. The GLIDE program was used for docking calculations. As receptor molecules, histone deacetylase, ribonucleotide reductase, and DNA methyltransferase were chosen. The SwissADME tool was used to evaluate the pharmacokinetics of the ligand. Based on Lipinski's rules, the estimation of insilico ADMET discovered that ligands procure significant drug-likeness characteristics. Its ability to block histone deacetylase 8 (PDB ID 1T69) protein is shown by docking calculations, and its proper action can be further confirmed in wet lab tests. [ABSTRACT FROM AUTHOR]

Ribonucleotide reductases (RRs or RNRs) catalyze the reduction of the OH group on the 2nd carbon of ribose, reducing four ribonucleotides (NTPs) to the corresponding deoxyribonucleotides (dNTPs) to promote DNA synthesis. Large DNA viruses, such as herpesviruses and poxviruses, could benefit their replication through increasing dNTPs via expression of viral RRs. Little is known regarding the relationship between cellular RRs and RNA viruses. Mammalian RRs contain two subunits of ribonucleotide reductase M1 polypeptide (RRM1) and two subunits of ribonucleotide reductase M2 polypeptide (RRM2). In this study, expression of cellular RRMs, including RRM1 and RRM2, is found to be down-regulated in hepatitis C virus (HCV)-infected Huh7.5 cells and Huh7 cells with HCV subgenomic RNAs (HCVr). As expected, the NTP/dNTP ratio is elevated in HCVr cells. Compared with that of the control Huh7 cells with sh-scramble, the NTP/dNTP ratio of the RRM-knockdown cells is elevated. Knockdown of RRM1 or RRM2 increases HCV replication in HCV replicon cells. Moreover, inhibitors to RRMs, including Didox, Trimidox and hydroxyurea, enhance HCV replication. Among various HCV viral proteins, the NS5A and/or NS3/4A proteins suppress the expression of RRMs. When these are taken together, the results suggest that HCV down-regulates the expression of RRMs in cultured cells to promote its replication. [ABSTRACT FROM AUTHOR]

Ferroptosis is an iron-dependent regulated cell death that suppresses tumor growth. It is activated by extensive peroxidation of membrane phospholipids caused by oxidative stress. GPX4, an antioxidant enzyme, reduces these peroxidized membrane phospholipids thereby inhibiting ferroptosis. This enzyme has two distinct subcellular localization; the cytosol and mitochondria. Dihydroorotate dehydrogenase (DHODH) complements mitochondrial GPX4 in reducing peroxidized membrane phospholipids. It is the rate-limiting enzyme in de novo pyrimidine nucleotide biosynthesis. Its role in ferroptosis inhibition suggests that DHODH inhibitors could have two complementary mechanisms of action against tumors; inhibiting de novo pyrimidine nucleotide biosynthesis and enhancing ferroptosis. However, the link between mitochondrial function and ferroptosis, and the involvement of DHODH in the ETC suggests that its role in ferroptosis could be modulated by the Warburg effect. Therefore, we reviewed relevant literature to get an insight into the possible effect of this metabolic reprogramming on the role of DHODH in ferroptosis. Furthermore, an emerging link between DHODH and cellular GSH pool has also been highlighted. These insights could contribute to the rational design of ferroptosis-based anticancer drugs. 8YjiDgdRtHZ2Rfs-RM3Nkf Video Abstract [ABSTRACT FROM AUTHOR]

Introduction: Ribonucleotide reductase (RR) is essential for the replication of the double-stranded DNA virus CyHV-2 due to its ability to catalyze the conversion of ribonucleotides to deoxyribonucleotides, and is a potential target for the development of antiviral drugs to control CyHV-2 infection. Methods: Bioinformatic analysis was conducted to identify potential homologues of RR in CyHV-2. The transcription and translation levels of ORF23 and ORF141, which showed high homology to RR, were measured during CyHV-2 replication in GICF. Co-localization experiments and immunoprecipitation were performed to investigate the interaction between ORF23 and ORF141. siRNA interference experiments were conducted to evaluate the effect of silencing ORF23 and ORF141 on CyHV-2 replication. The inhibitory effect of hydroxyurea, a nucleotide reductase inhibitor, on CyHV-2 replication in GICF cells and RR enzymatic activity in vitro was also evaluated. Results: ORF23 and ORF141 were identified as potential viral ribonucleotide reductase homologues in CyHV-2, and their transcription and translation levels increased with CyHV-2 replication. Co-localization experiments and immunoprecipitation suggested an interaction between the two proteins. Simultaneous silencing of ORF23 and ORF141 effectively inhibited the replication of CyHV-2. Additionally, hydroxyurea inhibited the replication of CyHV-2 in GICF cells and the in vitro enzymatic activity of RR. Conclusion: These results suggest that the CyHV-2 proteins ORF23 and ORF141 function as viral ribonucleotide reductase and their function makes an effect to CyHV-2 replication. Targeting ribonucleotide reductase could be a crucial strategy for developing new antiviral drugs against CyHV-2 and other herpesviruses. [ABSTRACT FROM AUTHOR]

Various external and internal factors damaging DNA constantly disrupt the stability of the genome. Cells use numerous dedicated DNA repair systems to detect damage and restore genomic integrity in a timely manner. Ribonucleotide reductase (RNR) is a key enzyme providing dNTPs for DNA repair. Molecular mechanisms of indirect regulation of yeast RNR activity are well understood, whereas little is known about its direct regulation. The study was aimed at elucidation of the proteasome-dependent mechanism of direct regulation of RNR subunits in Saccharomyces cerevisiae. Proteome analysis followed byWestern blot, RT-PCR, and yeast plating analysis showed that upregulation of RNR by proteasome deregulation is associated with yeast hyper resistance to 4-nitroquinoline-1-oxide (4-NQO), a UV-mimetic DNA-damaging drug used in animal models to study oncogenesis. Inhibition of RNR or deletion of RNR regulatory proteins reverses the phenotype of yeast hyper resistance to 4-NQO. We have shown for the first time that the yeast Rnr1 subunit is a substrate of the proteasome, which suggests a common mechanism of RNR regulation in yeast and mammals. [ABSTRACT FROM AUTHOR]

Background and Aims Ribonucleotide reductase (RNR), functioning in the de novo synthesis of deoxyribonucleoside triphosphates (dNTPs), is crucial for DNA replication and cell cycle progression. In most plants, the large subunits of RNR have more than one homologous gene. However, the different functions of these homologous genes in plant development remain unknown. In this study, we obtained the mutants of two large subunits of RNR in tomato and studied their functions. Methods The mutant ylc1 was obtained by ethyl methyl sulfonate (EMS) treatment. Through map-based cloning, complementation and knock-out experiments, it was confirmed that YLC1 encodes a large subunit of RNR (SlRNRL1). The expression level of the genes related to cell cycle progression, chloroplast biogenesis and photosynthesis was assessed by RNA-sequencing. In addition, we knocked out SlRNRL2 (a SlRNRL1 homologue) using CRISPR-Cas9 technology in the tomato genome, and we down-regulated SlRNRL2 expression in the genetic background of slrnrl1-1 using a tobacco rattle virus-induced gene silencing (VIGS) system. Key results The mutant slrnrl1 exhibited dwarf stature, chlorotic young leaves and smaller fruits. Physiological and transcriptomic analyses indicated that SlRNRL1 plays a crucial role in the regulation of cell cycle progression, chloroplast biogenesis and photosynthesis in tomato. The slrnrl2 mutant did not exhibit any visible phenotype. SlRNRL2 has a redundant function with SlRNRL1, and the double mutant slrnrl1slrnrl2 is lethal. Conclusions SlRNRL1 is essential for cell cycle progression, chloroplast biogenesis and photosynthesis. In addition, SlRNRL1 and SlRNRL2 possess redundant functions and at least one of these RNRLs is required for tomato survival, growth and development. [ABSTRACT FROM AUTHOR]

Ribonucleotide reductase (RR) is an essential multi-subunit enzyme found in all living organisms; it catalyzes the rate-limiting step in dNTP synthesis, namely, the conversion of ribonucleoside diphosphates to deoxyribonucleoside diphosphates. As expression levels of human RR (hRR) are high during cell replication, hRR has long been considered an attractive drug target for a range of proliferative diseases, including cancer. While there are many excellent reviews regarding the structure, function, and clinical importance of hRR, recent years have seen an increase in novel approaches to inhibiting hRR that merit an updated discussion of the existing inhibitors and strategies to target this enzyme. In this review, we discuss the mechanisms and clinical applications of classic nucleoside analog inhibitors of hRRM1 (large catalytic subunit), including gemcitabine and clofarabine, as well as inhibitors of the hRRM2 (free radical housing small subunit), including triapine and hydroxyurea. Additionally, we discuss novel approaches to targeting RR and the discovery of new classes of hRR inhibitors. [ABSTRACT FROM AUTHOR]

Background: Aberrant DNA repair pathways contribute to malignant transformation or disease progression and the acquisition of drug resistance in multiple myeloma (MM); therefore, these pathways could be therapeutically exploited. Ribonucleotide reductase (RNR) is the rate-limiting enzyme for the biosynthesis of deoxyribonucleotides (dNTPs), which are essential for DNA replication and DNA damage repair. In this study, we explored the efficacy of the novel RNR inhibitor, 4-hydroxysalicylanilide (HDS), in myeloma cells and xenograft model. In addition, we assessed the clinical activity and safety of HDS in patients with MM. Methods: We applied bioinformatic, genetic, and pharmacological approaches to demonstrate that HDS was an RNR inhibitor that directly bound to RNR subunit M2 (RRM2). The activity of HDS alone or in synergy with standard treatments was evaluated in vitro and in vivo. We also initiated a phase I clinical trial of single-agent HDS in MM patients (ClinicalTrials.gov: NCT03670173) to assess safety and efficacy. Results: HDS inhibited the activity of RNR by directly targeting RRM2. HDS decreased the RNR-mediated dNTP synthesis and concomitantly inhibited DNA damage repair, resulting in the accumulation of endogenous unrepaired DNA double-strand breaks (DSBs), thus inhibiting MM cell proliferation and inducing apoptosis. Moreover, HDS overcame the protective effects of IL-6, IGF-1 and bone marrow stromal cells (BMSCs) on MM cells. HDS prolonged survival in a MM xenograft model and induced synergistic anti-myeloma activity in combination with melphalan and bortezomib. HDS also showed a favorable safety profile and demonstrated clinical activity against MM. Conclusions: Our study provides a rationale for the clinical evaluation of HDS as an anti-myeloma agent, either alone or in combination with standard treatments for MM. Trial registration: ClinicalTrials.gov, NCT03670173, Registered 12 September 2018. [ABSTRACT FROM AUTHOR]

Herpesviruses have been reported to be able to encode and express functional viral microRNAs that target both viral and cellular transcripts. In our previous studies, we found a new miRNA miR‐KT‐635 encoded by Cyprinid herpesvirus 2, which is predicted to target viral genes and cellular genes involved in innate immune signalling pathway and apoptosis. However, the function and target gene of miR‐KT‐635 are not proved. In this study, the regulating target gene of miR‐KT‐635 was proved as the viral gene ORF23 directly, the target point sequence on gene was verified and miR‐KT‐635 was identified to regulate the expression of ORF23 protein. According to the bioinformatics analysis, the tRNA domain and ribosome domain in the protein sequence of ORF23 were found to share high homology with R2i and P53R2i, which are related to the ribonucleotide reductase small subunit in the host (transform NTP to dNTP). Within expectations, silencing of viral ORF23 or transfecting miR‐KT‐635 mimics in Carassius auratus gibelio caudal fin cell line (GiCF) could suppress viral propagation significantly. [ABSTRACT FROM AUTHOR]

Schizosaccharomyces pombe is an established yeast model for studying the cellular mechanisms conserved in humans, such as the DNA replication checkpoint. The replication checkpoint deals with replication stress caused by numerous endogenous and exogenous factors that perturb fork movement. If undealt with, perturbed forks collapse, causing chromosomal DNA damage or cell death. Hydroxyurea (HU) is an inhibitor of ribonucleotide reductase (RNR) commonly used in checkpoint studies. It produces replication stress by depleting dNTPs, which slows the movement of ongoing forks and thus activates the replication checkpoint. However, HU also causes side effects such as oxidative stress, particularly under chronic exposure conditions, which complicates the studies. To find a drug that generates replication stress more specifically, we tested three other RNR inhibitors gemcitabine, guanazole and triapine in S. pombe under various experimental conditions. Our results show that guanazole and triapine can produce replication stress more specifically than HU under chronic, not acute drug treatment conditions. Therefore, using the two drugs in spot assay, the method commonly used for testing drug sensitivity in yeasts, should benefit the checkpoint studies in S. pombe and likely the research in other model systems. [ABSTRACT FROM AUTHOR]

The E. coli ribonucleotide reductase (RNR), a paradigm for class Ia enzymes including human RNR, catalyzes the biosynthesis of DNA building blocks and requires a di‐iron tyrosyl radical (Y122.) cofactor for activity. The knowledge on the in vitro Y122. structure and its radical distribution within the β2 subunit has accumulated over the years; yet little information exists on the in vivo Y122.. Here, we characterize this essential radical in whole cells. Multi‐frequency EPR and electron‐nuclear double resonance (ENDOR) demonstrate that the structure and electrostatic environment of Y122. are identical under in vivo and in vitro conditions. Pulsed dipolar EPR experiments shed light on a distinct in vivo Y122. per β2 distribution, supporting the key role of Y. concentrations in regulating RNR activity. Additionally, we spectroscopically verify the generation of an unnatural amino acid radical, F3Y122. in whole cells, providing a crucial step towards unique insights into the RNR catalysis under physiological conditions. [ABSTRACT FROM AUTHOR]

Ribonucleotide reductase (RNR) is the key enzyme that catalyzes the production of deoxyribonucleotides (dNTPs) for DNA replication and it is also essential for cancer cell proliferation. As the RNR inhibitor, Gemcitabine is widely used in cancer therapies, however, resistance limits its therapeutic efficacy and curative potential. Here, we identified that mTORC2 is a main driver of gemcitabine resistance in non-small cell lung cancers (NSCLC). Pharmacological or genetic inhibition of mTORC2 greatly enhanced gemcitabine induced cytotoxicity and DNA damage. Mechanistically, mTORC2 directly interacted and phosphorylated RNR large subunit RRM1 at Ser 631. Ser631 phosphorylation of RRM1 enhanced its interaction with small subunit RRM2 to maintain sufficient RNR enzymatic activity for efficient DNA replication. Targeting mTORC2 retarded DNA replication fork progression and improved therapeutic efficacy of gemcitabine in NSCLC xenograft model in vivo. Thus, these results identified a mechanism through mTORC2 regulating RNR activity and DNA replication, conferring gemcitabine resistance to cancer cells. [ABSTRACT FROM AUTHOR]

Distinct mutation signatures arise from environmental exposures and/or from defects in metabolic pathways that promote genome stability. The presence of a particular mutation signature can therefore predict the underlying mechanism of mutagenesis. These insults to the genome often alter dNTP pools, which itself impacts replication fidelity. Therefore, the impact of altered dNTP pools should be considered when making mechanistic predictions based on mutation signatures. We developed a targeted deep-sequencing approach on the CAN1 gene in Saccharomyces cerevisiae to define information-rich mutational profiles associated with distinct rnr1 backgrounds. Mutations in the activity and selectivity sites of rnr1 lead to elevated and/or unbalanced dNTP levels, which compromises replication fidelity and increases mutation rates. The mutation spectra of rnr1Y285F and rnr1Y285A alleles were characterized previously; our analysis was consistent with this prior work but the sequencing depth achieved in our study allowed a significantly more robust and nuanced computational analysis of the variants observed, generating profiles that integrated information about mutation spectra, position effects, and sequence context. This approach revealed previously unidentified, genotype-specific mutation profiles in the presence of even modest changes in dNTP pools. Furthermore, we identified broader sequence contexts and nucleotide motifs that influenced variant profiles in different rnr1 backgrounds, which allowed specific mechanistic predictions about the impact of altered dNTP pools on replication fidelity. [ABSTRACT FROM AUTHOR]

RNA/DNA hybrids are processed by RNases H1 and H2, while single ribonucleoside-monophosphates (rNMPs) embedded in genomic DNA are removed by the error-free, RNase H2-dependent ribonucleotide excision repair (RER) pathway. In the absence of RER, however, topoisomerase 1 (Top1) can cleave single genomic rNMPs in a mutagenic manner. In RNase H2-deficient mice, the accumulation of genomic rNMPs above a threshold of tolerance leads to catastrophic genomic instability that causes embryonic lethality. In humans, deficiencies in RNase H2 induce the autoimmune disorders Aicardi–Goutières syndrome and systemic lupus erythematosus, and cause skin and intestinal cancers. Recently, we reported that in Saccharomyces cerevisiae, the depletion of Rnr1, the major catalytic subunit of ribonucleotide reductase (RNR), which converts ribonucleotides to deoxyribonucleotides, leads to cell lethality in absence of RNases H1 and H2. We hypothesized that under replicative stress and compromised DNA repair that are elicited by an insufficient supply of deoxyribonucleoside-triphosphates (dNTPs), cells cannot survive the accumulation of persistent RNA/DNA hybrids. Remarkably, we found that cells lacking RNase H2 accumulate ~ 5-fold more genomic rNMPs in absence than in presence of Rnr1. When the load of genomic rNMPs is further increased in the presence of a replicative DNA polymerase variant that over-incorporates rNMPs in leading or lagging strand, cells missing both Rnr1 and RNase H2 suffer from severe growth defects. These are reversed in absence of Top1. Thus, in cells lacking RNase H2 and containing a limiting supply of dNTPs, there is a threshold of tolerance for the accumulation of genomic ribonucleotides that is tightly associated with Top1-mediated DNA damage. In this mini-review, we describe the implications of the loss of RNase H2, or RNases H1 and H2, on the integrity of the nuclear genome and viability of budding yeast cells that are challenged with a critically low supply of dNTPs. We further propose that our findings in budding yeast could pave the way for the study of the potential role of mammalian RNR in RNase H2-related diseases. [ABSTRACT FROM AUTHOR]

Background: Deficiency in thymidine kinase 2 (TK2) or p53 inducible ribonucleotide reductase small subunit (p53R2) is associated with tissue specific mitochondrial DNA (mtDNA) depletion. To understand the mechanisms of the tissue specific mtDNA depletion we systematically studied key enzymes in dTMP synthesis in mitochondrial and cytosolic extracts prepared from adult rat tissues. Results: In addition to mitochondrial TK2 a cytosolic isoform of TK2 was characterized, which showed similar substrate specificity to the mitochondrial TK2. Total TK activity was highest in spleen and lowest in skeletal muscle. Thymidylate synthase (TS) was detected in cytosols and its activity was high in spleen but low in other tissues. TS protein levels were high in heart, brain and skeletal muscle, which deviated from TS activity levels. The p53R2 proteins were at similar levels in all tissues except liver where it was ~ 6-fold lower. Our results strongly indicate that mitochondria in most tissues are capable of producing enough dTTP for mtDNA replication via mitochondrial TK2, but skeletal muscle mitochondria do not and are most likely dependent on both the salvage and de novo synthesis pathways. Conclusion: These results provide important information concerning mechanisms for the tissue dependent variation of dTTP synthesis and explained why deficiency in TK2 or p53R2 leads to skeletal muscle dysfunctions. Furthermore, the presence of a putative cytosolic TK2-like enzyme may provide basic knowledge for the understanding of deoxynucleoside-based therapy for mitochondrial disorders. [ABSTRACT FROM AUTHOR]

The main curative treatments for hepatocellular carcinoma (HCC) are surgical resection and liver transplantation, which only benefits 15% to 25% of patients. In addition, HCC is highly refractory and resistant to cytotoxic chemotherapy. Although several multi-kinase inhibitors, such as sorafenib, regorafenib, and lenvatinib, have been approved for treating advanced HCC, only a short increase of median overall survival in HCC patients was achieved. Therefore, there is an urgent need to design more effective strategies for advanced HCC patients. Human ribonucleotide reductase is responsible for the conversion of ribonucleoside diphosphate to 20-deoxyribonucleoside diphosphate to maintain the homeostasis of nucleotide pools. In this study, mining the cancer genomics and proteomics data revealed that ribonucleotide reductase regulatory subunit M2 (RRM2) serves as a prognosis biomarker and a therapeutic target for HCC. The RNA sequencing (RNA-Seq) analysis and public microarray data mining found that RRM2 was a novel molecular target of sorafenib in HCC cells. In vitro experiments validated that sorafenib inhibits RRM2 expression in HCC cells, which is positively associated with the anticancer activity of sorafenib. Although both RRM2 knockdown and sorafenib induced autophagy in HCC cells, restoration of RRM2 expression did not rescue HCC cells from sorafenib-induced autophagy and growth inhibition. However, long-term colony formation assay indicated that RRM2 overexpression partially rescues HCC cells from the cytotoxicity of sorafenib. Therefore, this study identifies that RRM2 is a novel target of sorafenib, partially contributing to its anticancer activity in HCC cells. [ABSTRACT FROM AUTHOR]

Ribonucleotide reductase (RR) catalyses the rate-limiting step of dNTP synthesis, establishing it as an important cancer target. While RR is traditionally inhibited by nucleoside-based antimetabolites, we recently discovered a naphthyl salicyl acyl hydrazone-based inhibitor (NSAH) that binds reversibly to the catalytic site (C-site). Here we report the synthesis and in vitro evaluation of 13 distinct compounds (TP1-13) with improved binding to hRR over NSAH (TP8), with lower KD's and more predicted residue interactions. Moreover, TP6 displayed the greatest growth inhibiting effect in the Panc1 pancreatic cancer cell line with an IC50 of 0.393 µM. This represents more than a 2-fold improvement over NSAH, making TP6 the most potent compound against pancreatic cancer emerging from the hydrazone inhibitors. NSAH was optimised by the addition of cyclic and polar groups replacing the naphthyl moiety, which occupies the phosphate-binding pocket in the C-site, establishing a new direction in inhibitor design. [ABSTRACT FROM AUTHOR]

An integral part of the antiviral innate immune response is the APOBEC3 family of single-stranded DNA cytosine deaminases, which inhibits virus replication through deamination-dependent and -independent activities. Viruses have evolved mechanisms to counteract these enzymes, such as HIV-1 Vif-mediated formation of a ubiquitin ligase to degrade virus-restrictive APOBEC3 enzymes. A new example is Epstein-Barr virus (EBV) ribonucleotide reductase (RNR)-mediated inhibition of cellular APOBEC3B (A3B). The large subunit of the viral RNR, BORF2, causes A3B relocalization from the nucleus to cytoplasmic bodies and thereby protects viral DNA during lytic replication. Here, we use coimmunoprecipitation and immunofluorescence microscopy approaches to ask whether this mechanism is shared with the closely related gammaherpesvirus Kaposi’s sarcoma-associated herpesvirus (KSHV) and the more distantly related alphaherpesvirus herpes simplex virus 1 (HSV-1). The large RNR subunit of KSHV, open reading frame 61 (ORF61), coprecipitated multiple APOBEC3s, including A3B and APOBEC3A (A3A). KSHV ORF61 also caused relocalization of these two enzymes to perinuclear bodies (A3B) and to oblong cytoplasmic structures (A3A). The large RNR subunit of HSV-1, ICP6, also coprecipitated A3B and A3A and was sufficient to promote the relocalization of these enzymes from nuclear to cytoplasmic compartments. HSV-1 infection caused similar relocalization phenotypes that required ICP6. However, unlike the infectivity defects previously reported for BORF2-null EBV, ICP6 mutant HSV-1 showed normal growth rates and plaque phenotypes. Combined, these results indicate that both gamma- and alphaherpesviruses use a conserved RNR-dependent mechanism to relocalize A3B and A3A and furthermore suggest that HSV-1 possesses at least one additional mechanism to neutralize these antiviral enzymes. IMPORTANCE The APOBEC3 family of DNA cytosine deaminases constitutes a vital innate immune defense against a range of different viruses. A novel counterrestriction mechanism has recently been uncovered for the gammaherpesvirus EBV, in which a subunit of the viral protein known to produce DNA building blocks (ribonucleotide reductase) causes A3B to relocalize from the nucleus to the cytosol. Here, we extend these observations with A3B to include a closely related gammaherpesvirus, KSHV, and a more distantly related alphaherpesvirus, HSV-1. These different viral ribonucleotide reductases also caused relocalization of A3A, which is 92% identical to A3B. These studies are important because they suggest a conserved mechanism of APOBEC3 evasion by large double-stranded DNA herpesviruses. Strategies to block this host-pathogen interaction may be effective for treating infections caused by these herpesviruses. [ABSTRACT FROM AUTHOR]

Mining cancer-omics databases deepens our understanding of cancer biology and can lead to potential breakthroughs in cancer treatment. Here, we propose an integrative analytical approach to reveal across-cancer expression patterns and identify potential clinical impacts for genes of interest from five representative public databases. Using ribonucleotide reductase (RR), a key enzyme in DNA synthesis and cancer-therapeutic targeting, as an example, we characterized the mRNA expression profiles and inter-component associations of three RR subunit genes and assess their differing pathological and prognostic significance across over 30-types of cancers and their related subtypes. Findings were validated by immunohistochemistry with clinical tissue samples (n = 211) collected from multiple cancer centers in China and with clinical follow-up. Underlying mechanisms were further explored and discussed using co-expression gene network analyses. This framework represents a simple, efficient, accurate, and comprehensive approach for cancer-omics resource analysis and underlines the necessity to separate the tumors by their histological or pathological subtypes during the clinical evaluation of molecular biomarkers. [ABSTRACT FROM AUTHOR]

Ribonucleotide reductase (RNR) has been extensively probed as a target enzyme in the search for selective antibiotics. Here we report on the mechanism of inhibition of nine compounds, serving as representative examples of three different inhibitor classes previously identified by us to efficiently inhibit RNR. The interaction between the inhibitors and Pseudomonas aeruginosa RNR was elucidated using a combination of electron paramagnetic resonance spectroscopy and thermal shift analysis. All nine inhibitors were found to efficiently quench the tyrosyl radical present in RNR, required for catalysis. Three different mechanisms of radical quenching were identified, and shown to depend on reduction potential of the assay solution and quaternary structure of the protein complex. These results form a good foundation for further development of P. aeruginosa selective antibiotics. Moreover, this study underscores the complex nature of RNR inhibition and the need for detailed spectroscopic studies to unravel the mechanism of RNR inhibitors. [ABSTRACT FROM AUTHOR]

Outside of the photosynthetic machinery, high-valent manganese cofactors are rare in biology. It was proposed that a recently discovered subclass of ribonucleotide reductase (RNR), class Id, is dependent on a Mn2(IV,III) cofactor for catalysis. Class I RNRs consist of a substrate-binding component (NrdA) and a metal-containing radical-generating component (NrdB). Herein we utilize a combination of EPR spectroscopy and enzyme assays to underscore the enzymatic relevance of the Mn2(IV,III) cofactor in class Id NrdB from Facklamia ignava. Once formed, the Mn2(IV,III) cofactor confers enzyme activity that correlates well with cofactor quantity. Moreover, we present the X-ray structure of the apo- and aerobically Mn-loaded forms of the homologous class Id NrdB from Leeuwenhoekiella blandensis, revealing a dimanganese centre typical of the subclass, with a tyrosine residue maintained at distance from the metal centre and a lysine residue projected towards the metals. Structural comparison of the apo- and metal-loaded forms of the protein reveals a refolding of the loop containing the conserved lysine and an unusual shift in the orientation of helices within a monomer, leading to the opening of a channel towards the metal site. Such major conformational changes have not been observed in NrdB proteins before. Finally, in vitro reconstitution experiments reveal that the high-valent manganese cofactor is not formed spontaneously from oxygen, but can be generated from at least two different reduced oxygen species, i.e. H2O2 and superoxide (O2·−). Considering the observed differences in the efficiency of these two activating reagents, we propose that the physiologically relevant mechanism involves superoxide. [ABSTRACT FROM AUTHOR]

The molecular chaperones Hsp70 and Hsp90 bind and fold a significant proportion of the proteome. They are responsible for the activity and stability of many disease-related proteins including those in cancer. Substantial effort has been devoted to developing a range of chaperone inhibitors for clinical use. Recent studies have identified the oncogenic ribonucleotide reductase (RNR) complex as an interactor of chaperones. While several generations of RNR inhibitor have been developed for use in cancer patients, many of these produce severe side effects such as nausea, vomiting and hair loss. Development of more potent, less patient-toxic anti-RNR strategies would be highly desirable. Inhibition of chaperones and associated co-chaperone molecules in both cancer and model organisms such as budding yeast result in the destabilization of RNR subunits and a corresponding sensitization to RNR inhibitors. Going forward, this may form part of a novel strategy to target cancer cells that are resistant to standard RNR inhibitors. [ABSTRACT FROM AUTHOR]

Ribonucleotide reductases (RNR) are essential enzymes for all known life forms. Their current taxonomic distribution suggests extensive horizontal gene transfer e.g., by processes involving viruses. To improve our understanding of the underlying processes, we characterized a monomeric class II RNR (NrdJm) enzyme from a Thermus virus, a subclass not present in any sequenced Thermus spp. genome. Phylogenetic analysis revealed a distant origin of the nrdJm gene with the most closely related sequences found in mesophiles or moderate thermophiles from the Firmicutes phylum. GCcontent, codon usage and the ratio of coding to non-coding substitutions (dN/dS) suggest extensive adaptation of the gene in the virus in terms of nucleotide composition and amino acid sequence. The NrdJm enzyme is a monomeric B12-dependent RNR with nucleoside triphosphate specificity. It exhibits a temperature optimum at 60-70 °C, which is in the range of the growth optimum of Thermus spp. Experiments in combination with the Thermus thermophilus thioredoxin system show that the enzyme is able to retrieve electrons from the host NADPH pool via host thioredoxin and thioredoxin reductases. This is different from other characterized viral RNRs such as T4 phage RNR, where a viral thioredoxin is present. We hence show that the monomeric class II RNR, present in Thermus viruses, was likely transferred from an organism phylogenetically distant from the one they were isolated from, and adapted to the new host in genetic signature and amino acids sequence. [ABSTRACT FROM AUTHOR]

Ribonucleotide reductases (RNRs) are ancient enzymes that catalyze the reduction of ribonucleotides to deoxyribonucleotides. They are required for virtually all cellular life and are prominent within viral genomes. RNRs share a common ancestor and must generate a protein radical for direct ribonucleotide reduction. The mechanisms by which RNRs produce radicals are diverse and divide RNRs into three major classes and several subclasses. The diversity of radical generation methods means that cellular organisms and viruses typically contain the RNR best-suited to the environmental conditions surrounding DNA replication. However, such diversity has also fostered high rates of RNR misannotation within subject sequence databases. These misannotations have resulted in incorrect translative presumptions of RNR biochemistry and have diminished the utility of this marker gene for ecological studies of viruses. We discovered a misannotation of the RNR gene within the Prochlorococcus phage P-SSP7 genome, which caused a chain of misannotations within commonly observed RNR genes from marine virioplankton communities. These RNRs are found in marine cyanopodo- and cyanosiphoviruses and are currently misannotated as Class II RNRs, which are O2-independent and require cofactor B12. In fact, these cyanoviral RNRs are Class I enzymes that are O2-dependent and may require a di-metal cofactor made of Fe, Mn, or a combination of the two metals. The discovery of an overlooked Class I β subunit in the P-SSP7 genome, together with phylogenetic analysis of the α and β subunits confirms that the RNR from P-SSP7 is a Class I RNR. Phylogenetic and conserved residue analyses also suggest that the P-SSP7 RNR may constitute a novel Class I subclass. The reannotation of the RNR clade represented by P-SSP7 means that most lytic cyanophage contain Class I RNRs, while their hosts, B12-producing Synechococcus and Prochlorococcus , contain Class II RNRs. By using a Class I RNR, cyanophage avoid a dependence on host-produced B12, a more effective strategy for a lytic virus. The discovery of a novel RNR β subunit within cyanopodoviruses also implies that some unknown viral genes may be familiar cellular genes that are too divergent for homology-based annotation methods to identify. [ABSTRACT FROM AUTHOR]

The activity of ribonucleotide reductase (RNR), which catalyses the transformation of four ribonucleoside diphosphates (NDPs) to their corresponding deoxyribonucleoside diphosphates (dNDPs), is the main determiner of the cellular concentration of dNTP pools and should be tightly coordinated with DNA synthesis and cell-cycle progression. Constitutively increased or decreased RNR activity interferes with DNA replication and leads to arrested cell cycle progression; however, the mechanisms underlying these disruptive effects in higher plants remain to be uncovered. In this study, we identified a RNR large subunit mutant, sistl1, in Setaria italica (foxtail millet), which exhibited growth retardation as well as striped leaf phenotype, i.e. irregularly reduced leaf vein distances and decreased chloroplast biogenesis. We determined that a Gly737 to Glu substitution occurring in the C-terminus of the SiSTL1 protein slightly affected its optimal function, leading in turn to the reduced expression of genes variously involved in the assembly and activation of the DNA pre-replicative complex, elongation of replication forks and S phase entry. Our study provides new insights into how SiSTL1 regulates plant growth, chloroplast biogenesis, and cell cycle progression in Poaceae crops. [ABSTRACT FROM AUTHOR]

[Display omitted] • Inhibition of P53R2 leads to a decrease in the IC50 of daunorubicin in T-cell Acute Lymphoblastic Leukemia cells. • Inhibition of P53R2 leads to an increase in double-strand breaks induced by daunorubicin in T-cell Acute Lymphoblastic Leukemia cells. • p53R2 siRNA should be further investigated as a potential adjuvant therapy in combination with daunorubicin in T-cell ALL. p53R2 is a p53-inducible protein that, as one of the subunits of ribonucleotide reductase, plays an important role in providing dNTPs for DNA repair. Although p53R2 is associated with cancer progression, its role in T-cell acute lymphoblastic leukemia (T-ALL) cells is unknown. Therefore, in this study, we evaluated the effect of p53R2 silencing on double-stranded DNA breaks, apoptosis and cell cycle of T-ALL cells treated with Daunorubicin. Transfection was performed using Polyethyleneimine (PEI). Gene expression was measured using real-time PCR and protein expression was evaluated using Western blotting. Cell metabolic activity and IC50 were calculated using MTT assay, formation of double-stranded DNA breaks was checked using immunohistochemistry for γH2AX, and cell cycle and apoptosis were evaluated using flow cytometry. We found that p53 silencing synergistically inhibited the growth of T-ALL cells by Daunorubicin. p53R2 siRNA in combination with Daunorubicin but not alone increases the rate of DNA double-strand breaks in T-ALL cells. In addition, p53R2 siRNA significantly increased Daunorubicin-induced apoptosis. p53R2 siRNA also caused a non-significant increase in cells in G2 phase. The results of the present study showed that silencing of p53R2 using siRNA can significantly increase the antitumor effects of Daunorubicin on T-ALL cells. Therefore, p53R2 siRNA has the potential to be used as an adjuvant therapy in combination with Daunorubicin in T-ALL. [ABSTRACT FROM AUTHOR]

Glioblastoma (GBM) is an aggressive malignant primary brain tumor. Radioresistance largely contributes to poor clinical outcomes in GBM patients. We targeted ribonucleotide reductase subunit 2 (RRM2) with triapine to radiosensitize GBM. We found RRM2 is associated with increasing tumor grade, is overexpressed in GBM over lower grade gliomas and normal tissue, and is associated with worse survival. We found silencing or inhibition of RRM2 by siRNA or triapine sensitized GBM cells to ionizing radiation (IR) and delayed resolution of IR-induced γ-H2AX nuclear foci. In vivo , triapine and IR reduced tumor growth and increased mouse survival. Intriguingly, triapine led to RRM2 upregulation and CHK1 activation, suggesting a CHK1-dependent RRM2 upregulation following RRM2 inhibition. Consistently, silencing or inhibition of CHK1 with rabusertib abolished the triapine-induced RRM2 upregulation. Accordingly, combining rabusertib and triapine resulted in synthetic lethality in GBM cells. Collectively, our results suggest RRM2 is a promising therapeutic target for GBM, and targeting RRM2 with triapine sensitizes GBM cells to radiation and independently induces synthetic lethality of GBM cells with CHK1 inhibition. Our findings suggest combining triapine with radiation or rabusertib may improve therapeutic outcomes in GBM. • RRM2 is overexpressed in GBM and associates with poor prognosis. • Triapine inhibition of RRM2 sensitizes GBM to ionizing radiation treatment. • Simultaneous RRM2 and CHK1 inhibition induce synthetic lethality in GBM. [ABSTRACT FROM AUTHOR]

R2-like ligand-binding oxidases contain a dinuclear metal cofactor which can consist either of two iron ions or one manganese and one iron ion, but the heterodinuclear Mn/Fe cofactor is the preferred assembly in the presence of MnII and FeII in vitro. We have previously shown that both types of cofactor are capable of catalyzing formation of a tyrosine-valine ether cross-link in the protein scaffold. Here we demonstrate that Mn/Fe centers catalyze cross-link formation more efficiently than Fe/Fe centers, indicating that the heterodinuclear cofactor is the biologically relevant one. We further explore the chemical potential of the Mn/Fe cofactor by introducing mutations at the cross-linking valine residue. We find that cross-link formation is possible also to the tertiary beta-carbon in an isoleucine, but not to the secondary beta-carbon or tertiary gamma-carbon in a leucine, nor to the primary beta-carbon of an alanine. These results illustrate that the reactivity of the cofactor is highly specific and directed. [ABSTRACT FROM AUTHOR]

Telomeres, the nucleoprotein complexes at the end of eukaryotic chromosomes, protect them from degradation and ensure the replicative capacity of cells. In most human tumors and in budding yeast, telomere length is maintained by the activity of telomerase, an enzyme that adds dNTPs according to an internal RNA template. The dNTPs are generated with the help of the ribonucleotide reductase (RNR) complex. We have recently generated strains lacking the large subunit of RNR, Rnr1, which were kept viable by the expression of RNR complexes containing the Rnr1 homolog, Rnr3. Interestingly, we found that these Rnr1-deficient strains have short telomeres that are stably maintained, but cannot become efficiently elongated by telomerase. Thus, a basic maintenance of short telomeres is possible under conditions, where Rnr1 activity is absent, but a sustained elongation of short telomeres fully depends on Rnr1 activity. We show that Rnr3 cannot compensate for this telomeric function of Rnr1 even when overall cellular dNTP values are restored. This suggests that Rnr1 plays a role in telomere elongation beyond increasing cellular dNTP levels. Furthermore, our data indicate that telomerase may act in two different modes, one that is capable of coping with the “end-replication problem” and is functional even in the absence of Rnr1 and another required for the sustained elongation of short telomeres, which fully depends on the presence of Rnr1. Supply of dNTPs for telomere elongation is provided by the Mec1ATR checkpoint, both during regular DNA replication and upon replication fork stalling. We discuss the implications of these results on telomere maintenance in yeast and cancer cells. [ABSTRACT FROM AUTHOR]

The high fidelity of DNA replication and repair is attributable, in part, to the allosteric regulation of ribonucleotide reductases (RNRs) that maintains proper deoxynucleotide pool sizes and ratios in vivo. In class Ia RNRs, ATP (stimulatory) and dATP (inhibitory) regulate activity by binding to the ATP-cone domain at the N terminus of the large a subunit and altering the enzyme's quaternary structure. Class Ib RNRs, in contrast, have a partial cone domain and have generally been found to be insensitive to dATP inhibition. An exception is the Bacillus subtilis Ib RNR, which we recently reported to be inhibited by physiological concentrations of dATP. Here, we demonstrate that the a subunit of this RNR contains tightly bound deoxyadenosine 5'-monophosphate (dAMP) in its N-terminal domain and that dATP inhibition of CDP reduction is enhanced by its presence. X-ray crystallography reveals a previously unobserved (noncanonical) α2 dimer with its entire interface composed of the partial N-terminal cone domains, each binding a dAMP molecule. Using small-angle X-ray scattering (SAXS), we show that this noncanonical α2 dimer is the predominant form of the dAMP-bound a in solution and further show that addition of dATP leads to the formation of larger oligomers. Based on this information, we propose a model to describe the mechanism by which the noncanonical α2 inhibits the activity of the B. subtilis Ib RNR in a dATP- and dAMP-dependent manner. [ABSTRACT FROM AUTHOR]

The term hypoxia refers to any condition where insufficient oxygen is available and therefore encompasses a range of actual oxygen concentrations. The regions of tumours adjacent to necrotic areas are at almost anoxic levels and are known to be extremely therapy resistant (radiobiological hypoxia). The biological response to radiobiological hypoxia includes the rapid accumulation of replication stress and subsequent DNA damage response, including both ATR‐ and ATM‐mediated signalling, despite the absence of detectable DNA damage. The causes and consequences of hypoxia‐induced replication stress will be discussed. [ABSTRACT FROM AUTHOR]

A tightly controlled cellular deoxyribonucleotide (deoxynucleoside triphosphate [dNTP]) pool is critical for maintenance of genome integrity. One mode of dNTP pool regulation is through subcellular localization of ribonucleotide reductase (RNR), the enzyme that catalyzes the rate-limiting step of dNTP biosynthesis. In Saccharomyces cerevisiae, the RNR small subunit, Rnr2-Rnr4, is localized to the nucleus, whereas the large subunit, Rnr1, is cytoplasmic. As cells enter S phase or encounter DNA damage, Rnr2-Rnr4 relocalizes to the cytoplasm to form an active holoenzyme complex with Rnr1. Although the DNA damage-induced relocalization requires the checkpoint kinases Mec1-Rad53-Dun1, the S-phase-specific redistribution does not. Here, we report that the S-phase cyclin-cyclin-dependent kinase (CDK) complex Clb6-Cdc28 controls Rnr2-Rnr4 relocalization in S phase. Rnr2 contains a consensus CDK site and exhibits Clb6- dependent phosphorylation in S phase. Deletion of CLB6 or removal of the CDK site results in an increased association of Rnr2 with its nuclear anchor Wtm1, nuclear retention of Rnr2-Rnr4, and an enhanced sensitivity to the RNR inhibitor hydroxyurea. Thus, we propose that Rnr2-Rnr4 redistribution in S phase is triggered by Clb6-Cdc28-mediated phosphorylation of Rnr2, which disrupts the Rnr2-Wtm1 interaction and promotes the release of Rnr2-Rnr4 from the nucleus. [ABSTRACT FROM AUTHOR]

Ribonucleotide reductase (RNR) catalyses the rate limiting step of DNA synthesis utilising a mechanism that requires a tyrosyl radical. We have previously shown that superoxide can quench protein tyrosyl radicals in vitro , either by oxidative addition, or reduction of the radical to tyrosine. Here, we observe that Saccharomyces cerevisiae strains lacking either copper-zincSOD (SOD1) or manganese SOD (SOD2) had decreased RNR activity compared to SOD-competent yeast. When superoxide production was increased by treatment with paraquat, RNR activity was further decreased, with yeast lacking SOD1 being the most sensitive. The growth of yeast lacking SOD1 was also the most sensitive to paraquat treatment. Using expressed recombinant RNR, superoxide addition was not detectable using mass-spectrometry. This suggests that oxidative addition is not the major route of inhibition in our system, but does not rule out reduction by superoxide as a possible mechanism. Our results demonstrate that protection of RNR from inactivation by superoxide is an important function of SOD, particularly cytoplasmic SOD1. [ABSTRACT FROM AUTHOR]

The non-appropriate conditions faced by nutritionally stressed bacteria propitiate error-prone repair events underlying stationary-phase- or stress-associated mutagenesis (SPM). The genetic and molecular mechanisms involved in SPM have been deeply studied but the biochemical aspects of this process have so far been less explored. Previous evidence showed that under conditions of nutritional stress, non-dividing cells of strain B. subtilis YB955 overexpressing ribonucleotide reductase (RNR) exhibited a strong propensity to generate true reversions in the hisC952 (amber), metB5 (ochre) and leuC425 (missense) mutant alleles. To further advance our knowledge on the metabolic conditions underlying this hypermutagenic phenotype, a high-throughput LC-MS/MS proteomic analysis was performed in non-dividing cells of an amino acid-starved strain, deficient for NrdR, the RNR repressor. Compared with the parental strain, the level of 57 proteins was found to increase and of 80 decreases in the NrdR-deficient strain. The proteomic analysis revealed an altered content in proteins associated with the stringent response, nucleotide metabolism, DNA repair, and cell signaling in amino acid-starved cells of the ∆ nrdR strain. Overall, our results revealed that amino acid-starved cells of strain B. subtilis ∆ nrdR that escape from growth-limiting conditions exhibit a complex proteomic pattern reminiscent of a disturbed metabolism. Future experiments aimed to understand the consequences of disrupting the cell signaling pathways unveiled in this study, will advance our knowledge on the genetic adaptations deployed by bacteria to escape from growth-limiting environments. [ABSTRACT FROM AUTHOR]

Nonheme oxoiron(IV) complexes can serve as synthons for generating heterobimetallic oxo-bridged dimetal complexes by reaction with divalent metal complexes. The formation of Fe-O-Cr and Fe-O-Mn complexes is described herein. The latter complexes may serve as models for the Fe-X-Mn active sites of an emerging class of Fe/Mn enzymes represented by the Class 1c ribonucleotide reductase from Chlamydia trachomatis and the R2-like ligand-binding oxidase (R2lox) found in Mycobacterium tuberculosis. These synthetic complexes have been characterized by UV-Vis, resonance Raman, and X-ray absorption spectroscopy, as well as electrospray mass spectrometry. The Fe-O-Cr complexes exhibit a three-band UV-Vis pattern that differs from the simpler features associated with Fe-O-Fe complexes. The positions of these features are modulated by the nature of the supporting polydentate ligand on the iron center, and their bands intensify dramatically in two examples upon the binding of an axial cyanate or thiocyanate ligand trans to the oxo bridge. In contrast, the Fe-O-Mn complexes resemble Fe-O-Fe complexes more closely. Resonance Raman characterization of the Fe-O-M complexes reveals an O-sensitive vibration in the range of 760-890 cm. This feature has been assigned to the asymmetric Fe-O-M stretching mode and correlates reasonably with the Fe-O bond distance determined by EXAFS analysis. The likely binding of an acetate as a bridging ligand to the Fe-O-Mn complex 12 lays the foundation for further efforts to model the heterobimetallic active sites of Fe/Mn enzymes. [ABSTRACT FROM AUTHOR]

Cyclin-dependent kinase 9 (CDK9) inhibitors are a novel category of anticancer treatment for cancers. However, their effects on hepatocellular carcinoma (HCC) are rarely investigated. Human ribonucleotide reductase (RR, which consists of RRM1 and RRM2 subunits) catalyzes the conversion of ribonucleoside diphosphate into 2′-deoxyribonucleoside diphosphate to maintain the homeostasis of nucleotide pools, which play essential roles in DNA synthesis and DNA repair. In this study, we identified that CDK9 protein expression in adjacent non-tumor tissues predicted HCC patients' overall and progression-free survivals. The anticancer activity of a CDK9-selective inhibitor, LDC000067, on HCC cells was positively associated with its ability to inhibit the expression of RRM1 and RRM2. LDC000067 downregulated RRM1 and RRM2 expression through post-transcriptional pathway. Specifically, LDC000067 triggered RRM2 protein degradation via multiple pathways, including proteasome-, lysosome-, and calcium-dependent pathways. Furthermore, CDK9 positively correlates with RRM1 or RRM2 expression in HCC patients, and the expressions of these three genes were associated with the higher infiltration of immune cells in HCC. Taken together, this study identified the prognostic relevance of CDK9 in HCC and the molecular mechanism for the anticancer effect of CDK9 inhibitors on HCC. [Display omitted] • High CDK9 protein in adjacent non-tumor tissues predicts a worse prognosis. • LDC000067's anticancer activity is linked to RRM1 and RRM2 inhibition. • LDC000067 inhibits RRM1 and RRM2 expression through post-transcriptional pathway. • LDC000067 triggers RRM2 protein degradation via multiple pathways. [ABSTRACT FROM AUTHOR]

Cellular growth inhibition exerted by thiosemicarbazones is mainly attributed to down-regulation of ribonucleotide reductase (RNR) activity, with RNR being responsible for the rate-limiting step of de novo DNA synthesis. In this study, we investigated the antineoplastic effects of three newly synthesized thiosemicarbazone derivatives, thiazolyl hydrazones, in human HL-60 promyelocytic leukemia cells. The cytotoxicity of compounds alone and in combination with arabinofuranosylcytosine (AraC) was determined by growth inhibition assays. Effects on deoxyribonucleoside triphosphate (dNTP) concentrations were quantified by HPLC, and the incorporation of radio-labeled 14 C-cytidine into nascent DNA was measured using a beta counter. Cell cycle distribution was analyzed by FACS, and protein levels of RNR subunits and checkpoint kinases were evaluated by Western blotting. VG12, VG19, and VG22 dose-dependently decreased intracellular dNTP concentrations, impaired cell cycle progression and, consequently, inhibited the growth of HL-60 cells. VG19 also lowered the protein levels of RNR subunits R1 and R2 and significantly diminished the incorporation of radio-labeled 14 C-cytidine, being equivalent to an inhibition of DNA synthesis. Combination of thiazolyl hydrazones with AraC synergistically potentiated the antiproliferative effects seen with each drug alone and might therefore improve conventional chemotherapeutic regimens for the treatment of human malignancies such as acute promyelocytic or chronic myelogenous leukemia. [ABSTRACT FROM AUTHOR]

Heme oxygenases (HO) catalyze the breakdown of heme, aiding the recycling of its components. Several other enzymes have homologous tertiary structures to HOs, while sharing little sequence homology. These homologues include thiaminases, the hydroxylase component of methane monooxygenases, and the R2 component of Class I ribonucleotide reductases (RNR). This study compared these structural homologues of HO, using a large number of protein sequences for each homologue. Alignment of a total of 472 sequences showed little sequence conservation, with no residues having conservation in more than 80% of aligned sequences and only five residues conserved in at least 60% of the sequences. Fourteen additional positions, most of which were critical for hydrophobic packing, displayed amino acid similarity of 60% or higher. Ten conserved sequence motifs were identified in HOs and RNRs. Phylogenetic analysis verified the existence of the four distinct groups of HO homologues, which were then analyzed by group entropy analysis to identify residues critical to the unique function of each enzyme. Other methods for determining functional residues were also performed. Several common index positions identified represent critical evolutionary changes that resulted in the unique function of each enzyme, suggesting potential targets for site-directed mutagenesis. These positions included residues that coordinate ligands, form the active sites, and maintain enzyme structure. Enzymes Heme oxygenase (), methane monooxygenase (), ribonucleotide reductase (), thiaminase II (). [ABSTRACT FROM AUTHOR]

Tumor suppressor genes can be silenced genetically as well as epigenetically. One approach to reversing epigenetic suppression of tumor suppressor genes is to inhibit DNA methyl transferase. 5-aza-20',20'-diflurorodeoxycytidine (NUC013) is a novel DNA methyl transferase and ribonucleotide reductase inhibitor that is a more potent inhibitor of growth than decitabine in the NCI 60 cancer cell line panel. NUC013 is more active than decitabine against p53-null/mutant cancer cell lines (p = 0.027) but is even more so against p53 wild-type (WT) cell lines (p = 0.0025). The maximum tolerated dose in mice of NUC013 is greater than 120 mg/kg administered intravenously for three consecutive days a week for three weeks. With this regimen and a dose of 20 mg/kg in a human leukemia HL-60 (p53-null) NCr-nu/nu mouse xenograft model (n = 10/group), NUC013 demonstrated a survival benefit (saline median survival (MS) = 26.5 days, NUC013 MS = 32 days and hazard ratio (HR) = 0.26 (p = 0.032)). In a colon cancer LoVo (TP53 WT) xenograft, mice treated with decitabine at 5 mg/kg had worse survival than saline controls (decitabine MS = 31 days, saline MS > 60 days and HR = 26.89 (p < 0.0001)). At a dose of 20 mg/kg NUC013, mean tumor volume in the LoVo xenografts was lower than controls by 50.9% and at 40 mg/kg by 53.7% (both p < 0.0001). [ABSTRACT FROM AUTHOR]