Deoxyribonucleoside Triphosphate Pools Research Articles

Altered dNTP pools accelerate tumor formation in mice.

Alterations in deoxyribonucleoside triphosphate (dNTP) pools have been linked to increased mutation rates and genome instability in unicellular organisms and cell cultures. However, the role of dNTP poolchanges in tumor development in mammals remains unclear. In this study, we present a mouse model with a point mutation at the allosteric specificity site of ribonucleotide reductase, RRM1-Y285A. This mutation reduced ribonucleotide reductase activity, impairing the synthesis of deoxyadenosine triphosphate (dATP) and deoxyguanosinetriphosphate (dGTP). Heterozygous Rrm1+/Y285A mice exhibited distinct alterations in dNTP pools across various organs, shorter lifespans and earlier tumor onset compared with wild-type controls. Mutational spectrum analysis of tumors revealed two distinct signatures, one resembling a signature extracted from a human cancer harboring a mutation of the same amino acid residue in ribonucleotide reductase, RRM1Y285C. Our findings suggest that mutations in enzymes involved in dNTP metabolism can serve as drivers of cancer development.

Aicardi-Goutières syndrome with SAMHD1 deficiency can be diagnosed by unscheduled DNA synthesis test.

Aicardi-Goutières syndrome (AGS) is a rare genetic disorder characterised by progressive encephalopathy, involving microcephaly, intracranial calcification, and cerebrospinal fluid lymphocytosis with increased interferon-α concentrations. The clinical features of AGS overlap with fetal cerebral anomalies caused by congenital infections, such as TORCH (toxoplasmosis, other, rubella, cytomegalovirus, and herpes), or with those of other genetic disorders showing neonatal microcephaly, including Cockayne syndrome (CS) with transcription-coupled DNA repair deficiency, and Seckel syndrome (SS) showing aberrant cell-cycle checkpoint signaling. Therefore, a differential diagnosis to confirm the genetic cause or a proof of infection should be considered. In this report, we describe an individual who showed primordial dwarfism and encephalopathy, and whose initial diagnosis was CS. First, we conducted conventional DNA repair proficiency tests for the patient derived fibroblast cells. Transcription-coupled nucleotide excision repair (TC-NER) activity, which is mostly compromised in CS cases, was slightly reduced in the patient's cells. However, unscheduled DNA synthesis (UDS) was significantly diminished. These cellular traits were inconsistent with the diagnosis of CS. We further performed whole exome sequencing for the case and identified a compound heterozygous loss-of-function variants in the SAMHD1 gene, mutations in which are known to cause AGS. As SAMHD1 encodes deoxyribonucleoside triphosphate triphosphohydrolase, we reasoned that the deoxyribonucleoside triphosphate (dNTP) pool size in the patient's cells was elevated, and the labeling efficiency of UDS-test was hindered due to the reduced concentration of phosphorylated ethynyl deoxyuridine (EdU), a nucleoside analogue used for the assay. In conclusion, UDS assay may be a useful diagnostic tool to distinguish between AGS with SAMHD1 mutations and other related diseases.

Complex mutation profiles in mismatch repair and ribonucleotide reductase mutants reveal novel repair substrate specificity of MutS homolog (MSH) complexes.

Determining mutation signatures is standard for understanding the etiology of human tumors and informing cancer treatment. Multiple determinants of DNA replication fidelity prevent mutagenesis that leads to carcinogenesis, including the regulation of free deoxyribonucleoside triphosphate pools by ribonucleotide reductase and repair of replication errors by the mismatch repair system. We identified genetic interactions between rnr1 alleles that skew and/or elevate deoxyribonucleoside triphosphate levels and mismatch repair gene deletions. These defects indicate that the rnr1 alleles lead to increased mutation loads that are normally acted upon by mismatch repair. We then utilized a targeted deep-sequencing approach to determine mutational profiles associated with mismatch repair pathway defects. By combining rnr1 and msh mutations to alter and/or increase deoxyribonucleoside triphosphate levels and alter the mutational load, we uncovered previously unreported specificities of Msh2-Msh3 and Msh2-Msh6. Msh2-Msh3 is uniquely able to direct the repair of G/C single-base deletions in GC runs, while Msh2-Msh6 specifically directs the repair of substitutions that occur at G/C dinucleotides. We also identified broader sequence contexts that influence variant profiles in different genetic backgrounds. Finally, we observed that the mutation profiles in double mutants were not necessarily an additive relationship of mutation profiles in single mutants. Our results have implications for interpreting mutation signatures from human tumors, particularly when mismatch repair is defective.

A Yeast-Based Repurposing Approach for the Treatment of Mitochondrial DNA Depletion Syndromes Led to the Identification of Molecules Able to Modulate the dNTP Pool.

Mitochondrial DNA depletion syndromes (MDS) are clinically heterogenous and often severe diseases, characterized by a reduction of the number of copies of mitochondrial DNA (mtDNA) in affected tissues. In the context of MDS, yeast has proved to be both an excellent model for the study of the mechanisms underlying mitochondrial pathologies and for the discovery of new therapies via high-throughput assays. Among the several genes involved in MDS, it has been shown that recessive mutations in MPV17 cause a hepatocerebral form of MDS and Navajo neurohepatopathy. MPV17 encodes a non selective channel in the inner mitochondrial membrane, but its physiological role and the nature of its cargo remains elusive. In this study we identify ten drugs active against MPV17 disorder, modelled in yeast using the homologous gene SYM1. All ten of the identified molecules cause a concomitant increase of both the mitochondrial deoxyribonucleoside triphosphate (mtdNTP) pool and mtDNA stability, which suggests that the reduced availability of DNA synthesis precursors is the cause for the mtDNA deletion and depletion associated with Sym1 deficiency. We finally evaluated the effect of these molecules on mtDNA stability in two other MDS yeast models, extending the potential use of these drugs to a wider range of MDS patients.

Hydroxyurea Induces a Stress Response That Alters DNA Replication and Nucleotide Metabolism in Bacillus subtilis.

Hydroxyurea (HU) is classified as a ribonucleotide reductase (RNR) inhibitor and has been widely used to stall DNA replication by depleting deoxyribonucleoside triphosphate (dNTP) pools. Recent evidence in Escherichia coli shows that HU readily forms breakdown products that damage DNA directly, indicating that toxicity is a result of secondary effects. Because HU is so widely used in the laboratory and as a clinical therapeutic, it is important to understand its biological effects. To determine how Bacillus subtilis responds to HU-induced stress, we performed saturating transposon insertion mutagenesis followed by deep sequencing (Tn-seq), transcriptome sequencing (RNA-seq) analysis, and measurement of replication fork progression. Our data show that B. subtilis cells elongate, and replication fork progression is slowed, following HU challenge. The transcriptomic data show that B. subtilis cells initially mount a metabolic response likely caused by dNTP pool depletion before inducing the DNA damage response (SOS) after prolonged exposure. To compensate for reduced nucleotide pools, B. subtilis upregulates the purine and pyrimidine biosynthetic machinery and downregulates the enzymes producing ribose 5-phosphate. We show that overexpression of the RNR genes nrdEF suppresses the growth interference caused by HU, suggesting that RNR is an important target of HU in B. subtilis. Although genes involved in nucleotide and carbon metabolism showed considerable differential expression, we also find that genes of unknown function (y-genes) represent the largest class of differentially expressed genes. Deletion of individual y-genes caused moderate growth interference in the presence of HU, suggesting that cells have several ways of coping with HU-induced metabolic stress. IMPORTANCE Hydroxyurea (HU) has been widely used as a clinical therapeutic and an inhibitor of DNA replication. Some evidence suggests that HU inhibits ribonucleotide reductase, depleting dNTP pools, while other evidence shows that toxic HU breakdown products are responsible for growth inhibition and genotoxic stress. Here, we use multiple, complementary approaches to characterize the response of Bacillus subtilis to HU. B. subtilis responds by upregulating the expression of purine and pyrimidine biosynthesis. We show that HU challenge reduced DNA replication and that overexpression of the ribonucleotide reductase operon suppressed growth interference by HU. Our results demonstrate that HU targets RNR and several other metabolic enzymes contributing to toxicity in bacteria.

The Lon Protease Links Nucleotide Metabolism with Proteotoxic Stress

During proteotoxic stress, bacteria maintain critical processes like DNA replication while removing misfolded proteins, which are degraded by the Lon protease. Here, we show that in Caulobacter crescentus Lon controls deoxyribonucleoside triphosphate (dNTP) pools during stress through degradation of the transcription factor CcrM. Elevated dNTP/nucleotide triphosphate (NTP) ratios in Δlon cells protects them from deletion of otherwise essential deoxythymidine triphosphate (dTTP)-producing pathways and shields them from hydroxyurea-induced loss of dNTPs. Increased dNTP production in Δlon results from higher expression of ribonucleotide reductase driven by increased CcrM. We show that misfolded proteins can stabilize CcrM by competing for limited protease and that Lon-dependent control of dNTPs improves fitness during protein misfolding conditions. We propose that linking dNTP production with availability of Lon allows Caulobacter to maintain replication capacity when misfolded protein burden increases, such as during rapid growth. Because Lon recognizes misfolded proteins regardless of the stress, this mechanism allows for response to a variety of unanticipated conditions.

AICAr suppresses cell proliferation by inducing NTP and dNTP pool imbalances in acute lymphoblastic leukemia cells.

It has been shown that 5-amino-4-imidazolecarboxamide riboside (AICAr) can inhibit cell proliferation and induce apoptosis in childhood acute lymphoblastic leukemia (ALL) cells. Although AICAr could regulate cellular energy metabolism by activating AMPK, the cytotoxic mechanisms of AICAr are still unclear. Here, we knocked out TP53 or PRKAA1 gene (encoding AMPKα1) in NALM-6 and Reh cells by using the clustered regularly interspaced short palindromic repeats/Cas9 system and found that AICAr-induced proliferation inhibition was independent of AMPK activation but dependent on p53. Liquid chromatography-mass spectrometry analysis of nucleotide metabolites indicated that AICAr caused an increase in adenosine triphosphate, deoxyadenosine triphosphate, and deoxyguanosine triphosphate levels by up-regulating purine biosynthesis, while AICAr led to a decrease in cytidine triphosphate, uridine triphosphate, deoxycytidine triphosphate, and deoxythymidine triphosphate levels because of reduced phosphoribosyl pyrophosphate production, which consequently impaired the pyrimidine biosynthesis. Ribonucleoside triphosphate (NTP) pool imbalances suppressed the rRNA transcription efficiency. Furthermore, deoxy-ribonucleoside triphosphate (dNTP) pool imbalances induced DNA replication stress and DNA double-strand breaks, followed by cell cycle arrest and apoptosis in ALL cells. Exogenous uridine could rebalance the NTP and dNTP pools by supplementing pyrimidine and then attenuate AICAr-induced cytotoxicity. Our data indicate that RNA transcription inhibition and DNA replication stress induced by NTP and dNTP pool imbalances might play a key role in AICAr-mediated cytotoxic effects on ALL cells, suggesting a potential clinical application of AICAr in future ALL therapy.-Du, L., Yang, F., Fang, H., Sun, H., Chen, Y., Xu, Y., Li, H., Zheng, L., Zhou, B.-B. S. AICAr suppresses cell proliferation by inducing NTP and dNTP pool imbalances in acute lymphoblastic leukemia cells.

ERK Inhibitor Enhances Everolimus Efficacy through the Attenuation of dNTP Pools in Renal Cell Carcinoma

The clinical efficiency of everolimus, an mammalian target of rapamycin (mTOR) inhibitor, is palliative as sequential or second-line therapy for renal cell carcinoma (RCC). However, the limited response of everolimus in RCC remains uncertain. In the present study, everolimus-resistant RCC models were established to understand the mechanisms and to seek combination approaches. Consequently, the activation of ERK was found to contribute toward everolimus-acquired resistance and poor prognosis in patients with RCC. In addition, the efficacy and mechanism of combination treatment underlying RCC using everolimus and ERK inhibitors was investigated. The ERK inhibitor in combination with everolimus synergistically inhibited the proliferation of RCC cells by arresting the cell cycle in the G1 phase. The combination treatment markedly attenuated the deoxyribonucleoside triphosphate (dNTP) pools by downregulating the mRNA expression of RRM1 and RRM2 through E2F1. The overexpression of E2F1 or supplementation of dNTP rescued the anti-proliferation activity of the everolimus-SCH772984 combination. The antitumor efficacy of combination therapy was reiterated in RCC xenograft models. Thus, the current findings provided evidence that the everolimus-ERK inhibitor combination is a preclinical therapeutic strategy for RCC.

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.

Hydroxyurea-Mediated Cytotoxicity Without Inhibition of Ribonucleotide Reductase

SummaryIn many organisms, hydroxyurea (HU) inhibits class I ribonucleotide reductase, leading to lowered cellular pools of deoxyribonucleoside triphosphates. The reduced levels for DNA precursors is believed to cause replication fork stalling. Upon treatment of the hyperthermophilic archaeon Sulfolobus solfataricus with HU, we observe dose-dependent cell cycle arrest, accumulation of DNA double-strand breaks, stalled replication forks, and elevated levels of recombination structures. However, Sulfolobus has a HU-insensitive class II ribonucleotide reductase, and we reveal that HU treatment does not significantly impact cellular DNA precursor pools. Profiling of protein and transcript levels reveals modulation of a specific subset of replication initiation and cell division genes. Notably, the selective loss of the regulatory subunit of the primase correlates with cessation of replication initiation and stalling of replication forks. Furthermore, we find evidence for a detoxification response induced by HU treatment.

CyclinA2-Cyclin-dependent Kinase Regulates SAMHD1 Protein Phosphohydrolase Domain

SAMHD1 is a nuclear deoxyribonucleoside triphosphate triphosphohydrolase that contributes to the control of cellular deoxyribonucleoside triphosphate (dNTP) pool sizes through dNTP hydrolysis and modulates the innate immune response to viruses. CyclinA2-CDK1/2 phosphorylates SAMHD1 at Thr-592, but how this modification controls SAMHD1 functions in proliferating cells is not known. Here, we show that SAMHD1 levels remain relatively unchanged during the cell division cycle in primary human T lymphocytes and in monocytic cell lines. Inactivation of the bipartite cyclinA2-CDK-binding site in the SAMHD1 C terminus described herein abolished SAMHD1 phosphorylation on Thr-592 during S and G2 phases thus interfering with DNA replication and progression of cells through S phase. The effects exerted by Thr-592 phosphorylation-defective SAMHD1 mutants were associated with activation of DNA damage checkpoint and depletion of dNTP concentrations to levels lower than those seen upon expression of wild type SAMHD1 protein. These disruptive effects were relieved by either mutation of the catalytic residues of the SAMHD1 phosphohydrolase domain or by a Thr-592 phosphomimetic mutation, thus linking the Thr-592 phosphorylation state to the control of SAMHD1 dNTPase activity. Our findings support a model in which phosphorylation of Thr-592 by cyclinA2-CDK down-modulates, but does not inactivate, SAMHD1 dNTPase in S phase, thereby fine-tuning SAMHD1 control of dNTP levels during DNA replication.

Novel immunosuppressive agent caerulomycin A exerts its effect by depleting cellular iron content.

Recently, we have described the use of caerulomycin A (CaeA) as a potent novel immunosuppressive agent. Immunosuppressive drugs are crucial for long-term graft survival following organ transplantation and treatment of autoimmune diseases, inflammatory disorders, hypersensitivity to allergens, etc. The objective of this study was to identify cellular targets of CaeA and decipher its mechanism of action. Jurkat cells were treated with CaeA and cellular iron content, iron uptake/release, DNA content and deoxyribonucleoside triphosphate pool determined. Activation of MAPKs; expression level of transferrin receptor 1, ferritin and cell cycle control molecules; reactive oxygen species (ROS) and cell viability were measured using Western blotting, qRT-PCR or flow cytometry. CaeA caused intracellular iron depletion by reducing its uptake and increasing its release by cells. CaeA caused cell cycle arrest by (i) inhibiting ribonucleotide reductase (RNR) enzyme, which catalyses the rate-limiting step in the synthesis of DNA; (ii) stimulating MAPKs signalling transduction pathways that play an important role in cell growth, proliferation and differentiation; and (iii) by targeting cell cycle control molecules such as cyclin D1, cyclin-dependent kinase 4 and p21(CIP1/WAF1) . The effect of CaeA on cell proliferation was reversible. CaeA exerts its immunosuppressive effect by targeting iron. The effect is reversible, which makes CaeA an attractive candidate for development as a potent immunosuppressive drug, but also indicates that iron chelation can be used as a rationale approach to selectively suppress the immune system, because compared with normal cells, rapidly proliferating cells require a higher utilization of iron.

From the covalent linkage of drugs to novel inhibitors of ribonucleotide reductase: Synthesis and biological evaluation of valproic esters of 3′-C-methyladenosine

We synthesized a series of serum-stable covalently linked drugs derived from 3′-C-methyladenosine (3′-Me-Ado) and valproic acid (VPA), which are ribonucleotide reductase (RR) and histone deacetylase (HDAC) inhibitors, respectively. While the combination of free VPA and 3′-Me-Ado resulted in a clear synergistic apoptotic effect, the conjugates had lost their HDAC inhibitory effect as well as the corresponding apoptotic activity. Two of the analogs, 2′,5′-bis-O-valproyl-3′-C-methyladenosine (A160) and 5′-O-valproyl-3′-C-methyladenosine (A167), showed promising cytotoxic activities against human hematological and solid cancer cell lines. A167 was less potent than A160 but had interesting features as an RR inhibitor. It inhibited RR activity by competing with ATP as an allosteric effector and concomitantly reduced the intracellular deoxyribonucleoside triphosphate (dNTP) pools. A167 represents a novel lead compound, which in contrast to previously used RR nucleoside analogs does not require intracellular kinases for its activity and therefore holds promise against drug resistant tumors with downregulated nucleoside kinases.

The role of the Mpv17 protein mutations of which cause mitochondrial DNA depletion syndrome (MDDS): lessons from homologs in different species.

Mitochondrial DNA depletion syndromes (MDDS) are severe pediatric diseases with diverse clinical manifestations. Gene mutations that underlie MDDS have been associated with alterations in the mitochondrial DNA (mtDNA) replication machinery or in mitochondrial deoxyribonucleoside triphosphate pools. However, the nuclear gene MPV17, whose mutated forms are associated with hepatocerebral MDDS in humans, plays a so-far unknown role in mtDNA maintenance. A high degree of conservation has been determined between MPV17 and its mouse (Mpv17), zebrafish (tra) and yeast (SYM1) homologs, respectively, whereby mutants in these cause very different phenotypes. While dysfunction in this gene in humans causes fatal liver disease, kidney pathology is induced in mice. Moreover, in zebrafish inactivation of the Mpv17 homolog was detected as a viable dyscolouration mutant. Knock out of the yeast ortholog results in a temperature-sensitive metabolic growth phenotype. Detailed analyses on common denominators between these different phenotypes strengthen the hypothesis that the Mpv17 protein forms a channel in the inner mitochondrial membrane, allowing small molecules - in vertebrates probably nucleotides, and in yeast probably intermediates of the tricarboxylic acid cycle - to pass. Moreover, a function modifying the pathologic manifestations of MPV17-related disease in mice has been identified. This signaling pathway remarkably involves the non-mitochondrial catalytic subunit of DNA-dependent protein kinase (PRKDC), important in double-strand break repair resistance against reactive oxygen-induced genotoxic stress.

Ribonucleotide reductase and cancer: biological mechanisms and targeted therapies

Accurate DNA replication and repair is essential for proper development, growth and tumor-free survival in all multicellular organisms. A key requirement for the maintenance of genomic integrity is the availability of adequate and balanced pools of deoxyribonucleoside triphosphates (dNTPs), the building blocks of DNA. Notably, dNTP pool alterations lead to genomic instability and have been linked to multiple human diseases, including mitochondrial disorders, susceptibility to viral infection and cancer. In this review, we discuss how a key regulator of dNTP biosynthesis in mammals, the enzyme ribonucleotide reductase (RNR), impacts cancer susceptibility and serves as a target for anti-cancer therapies. Because RNR-regulated dNTP production can influence DNA replication fidelity while also supporting genome-protecting DNA repair, RNR has complex and stage-specific roles in carcinogenesis. Nevertheless, cancer cells are dependent on RNR for de novo dNTP biosynthesis. Therefore, elevated RNR expression is a characteristic of many cancers, and an array of mechanistically distinct RNR inhibitors serve as effective agents for cancer treatment. The dNTP metabolism machinery, including RNR, has been exploited for therapeutic benefit for decades and remains an important target for cancer drug development.

Deoxynucleoside stress exacerbates the phenotype of a mouse model of mitochondrial neurogastrointestinal encephalopathy.

Balanced pools of deoxyribonucleoside triphosphate precursors are required for DNA replication, and alterations of this balance are relevant to human mitochondrial diseases including mitochondrial neurogastrointestinal encephalopathy. In this disease, autosomal recessive TYMP mutations cause severe reductions of thymidine phosphorylase activity; marked elevations of the pyrimidine nucleosides thymidine and deoxyuridine in plasma and tissues, and somatic multiple deletions, depletion and site-specific point mutations of mitochondrial DNA. Thymidine phosphorylase and uridine phosphorylase double knockout mice recapitulated several features of these patients including thymidine phosphorylase activity deficiency, elevated thymidine and deoxyuridine in tissues, mitochondrial DNA depletion, respiratory chain defects and white matter changes. However, in contrast to patients with this disease, mutant mice showed mitochondrial alterations only in the brain. To test the hypothesis that elevated levels of nucleotides cause unbalanced deoxyribonucleoside triphosphate pools and, in turn, pathogenic mitochondrial DNA instability, we have stressed double knockout mice with exogenous thymidine and deoxyuridine, and assessed clinical, neuroradiological, histological, molecular, and biochemical consequences. Mutant mice treated with exogenous thymidine and deoxyuridine showed reduced survival, body weight, and muscle strength, relative to untreated animals. Moreover, in treated mutants, leukoencephalopathy, a hallmark of the disease, was enhanced and the small intestine showed a reduction of smooth muscle cells and increased fibrosis. Levels of mitochondrial DNA were depleted not only in the brain but also in the small intestine, and deoxyribonucleoside triphosphate imbalance was observed in the brain. The relative proportion, rather than the absolute amount of deoxyribonucleoside triphosphate, was critical for mitochondrial DNA maintenance. Thus, our results demonstrate that stress of exogenous pyrimidine nucleosides enhances the mitochondrial phenotype of our knockout mice. Our mouse studies provide insights into the pathogenic role of thymidine and deoxyuridine imbalance in mitochondrial neurogastrointestinal encephalopathy and an excellent model to study new therapeutic approaches.

The NTP pyrophosphatase DCTPP1 contributes to the homoeostasis and cleansing of the dNTP pool in human cells

The size and composition of dNTP (deoxyribonucleoside triphosphate) pools influence the accuracy of DNA synthesis and consequently the genetic stability of nuclear and mitochondrial genomes. In order to keep the dNTP pool in balance, the synthesis and degradation of DNA precursors must be precisely regulated. One such mechanism involves catabolic activities that convert deoxynucleoside triphosphates into their monophosphate form. Human cells possess an all-α NTP (nucleoside triphosphate) pyrophosphatase named DCTPP1 [dCTP pyrophosphatase 1; also known as XTP3-TPA (XTP3-transactivated protein A)]. In the present study, we provide an extensive characterization of this enzyme which is ubiquitously distributed in the nucleus, cytosol and mitochondria. Interestingly, we found that in addition to dCTP, methyl-dCTP and 5-halogenated nucleotides, DCTPP1 hydrolyses 5-formyl-dCTP very efficiently and with the lowest Km value described so far. Because the biological function of mammalian all-α NTP pyrophosphatases remains uncertain, we examined the role of DCTPP1in the maintenance of pyrimidine nucleotide pools and cellular sensitivity to pyrimidine analogues. DCTPP1-deficient cells accumulate high levels of dCTP and are hypersensitive to exposure to the nucleoside analogues 5-iodo-2'-deoxycytidine and 5-methyl-2'-deoxycytidine. The results of the present study indicate that DCTPP1 has a central role in the balance of dCTP and the metabolism of deoxycytidine analogues, thus contributing to the preservation of genome integrity.

Ribonucleotide Reductase Association with Mammalian Liver Mitochondria

Deoxyribonucleoside triphosphate pools in mammalian mitochondria are highly asymmetric, and this asymmetry probably contributes to the elevated mutation rate for the mitochondrial genome as compared with the nuclear genome. To understand this asymmetry, we must identify pathways for synthesis and accumulation of dNTPs within mitochondria. We have identified ribonucleotide reductase activity specifically associated with mammalian tissue mitochondria. Examination of immunoprecipitated proteins by mass spectrometry revealed R1, the large ribonucleotide reductase subunit, in purified mitochondria. Significant enzymatic and immunological activity was seen in rat liver mitochondrial nucleoids, isolated as described by Wang and Bogenhagen (Wang, Y., and Bogenhagen, D. F. (2006) J. Biol. Chem. 281, 25791-25802). Moreover, incubation of respiring rat liver mitochondria with [(14)C]cytidine diphosphate leads to accumulation of radiolabeled deoxycytidine and thymidine nucleotides within the mitochondria. Comparable results were seen with [(14)C]guanosine diphosphate. Ribonucleotide reduction within the mitochondrion, as well as outside the organelle, needs to be considered as a possibly significant contributor to mitochondrial dNTP pools.

Ribonucleotide reductase and thymidylate synthase or exogenous deoxyribonucleosides reduce DNA damage and senescence caused by C-MYC depletion

The down-regulation of dominant oncogenes, including C-MYC, in tumor cells often leads to the induction of senescence via mechanisms that are not completely identified. In the current study, we demonstrate that MYC-depleted melanoma cells undergo extensive DNA damage that is caused by the underexpression of thymidylate synthase (TS) and ribonucleotide reductase (RR) and subsequent depletion of deoxyribonucleoside triphosphate pools. Simultaneous genetic inhibition of TS and RR in melanoma cells induced DNA damage and senescence phenotypes very similar to the ones caused by MYC-depletion. Reciprocally, overexpression of TS and RR in melanoma cells or addition of deoxyribo-nucleosides to culture media substantially inhibited DNA damage and senescence-associated phenotypes caused by C-MYC depletion. Our data demonstrate the essential role of TS and RR in C-MYC-dependent suppression of senescence in melanoma cells.

Replication Fork Collapse and Genome Instability in a Deoxycytidylate Deaminase Mutant

Ribonucleotide reductase (RNR) and deoxycytidylate deaminase (dCMP deaminase) are pivotal allosteric enzymes required to maintain adequate pools of deoxyribonucleoside triphosphates (dNTPs) for DNA synthesis and repair. Whereas RNR inhibition slows DNA replication and activates checkpoint responses, the effect of dCMP deaminase deficiency is largely unknown. Here, we report that deleting the Schizosaccharomyces pombe dcd1(+) dCMP deaminase gene (SPBC2G2.13c) increases dCTP ∼30-fold and decreases dTTP ∼4-fold. In contrast to the robust growth of a Saccharomyces cerevisiae dcd1Δ mutant, fission yeast dcd1Δ cells delay cell cycle progression in early S phase and are sensitive to multiple DNA-damaging agents, indicating impaired DNA replication and repair. DNA content profiling of dcd1Δ cells differs from an RNR-deficient mutant. Dcd1 deficiency activates genome integrity checkpoints enforced by Rad3 (ATR), Cds1 (Chk2), and Chk1 and creates critical requirements for proteins involved in recovery from replication fork collapse, including the γH2AX-binding protein Brc1 and Mus81 Holliday junction resolvase. These effects correlate with increased nuclear foci of the single-stranded DNA binding protein RPA and the homologous recombination repair protein Rad52. Moreover, Brc1 suppresses spontaneous mutagenesis in dcd1Δ cells. We propose that replication forks stall and collapse in dcd1Δ cells, burdening DNA damage and checkpoint responses to maintain genome integrity.