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The Escherichia coli dnaE gene encodes the α‐catalytic subunit (pol IIIα) of DNA polymerase III, the cell’s main replicase. Like all high‐fidelity DNA polymerases, pol III possesses stringent base and sugar discrimination. The latter is mediated by a so‐called “steric gate” residue in the active site of the polymerase that physically clashes with the 2′‐OH of an incoming ribonucleotide. Our structural modeling data suggest that H760 is the steric gate residue in E.coli pol IIIα. To understand how H760 and the adjacent S759 residue help maintain genome stability, we generated DNA fragments in which the codons for H760 or S759 were systematically changed to the other nineteen naturally occurring amino acids and attempted to clone them into a plasmid expressing pol III core (α‐θ‐ε subunits). Of the possible 38 mutants, only nine were successfully sub‐cloned: three with substitutions at H760 and 6 with substitutions at S759. Three of the plasmid‐encoded alleles, S759C, S759N, and S759T, exhibited mild to moderate mutator activity and were moved onto the chromosome for further characterization. These studies revealed altered phenotypes regarding deoxyribonucleotide base selectivity and ribonucleotide discrimination. We believe that these are the first dnaE mutants with such phenotypes to be reported in the literature., The accurate replication of every living organisms’ genome is achieved by high fidelity DNA polymerases. The enzymes not only need to choose the nucleotide with the correctly paired base (G, A, T or C), but also the right sugar (deoxyribonucleotide, not ribonucleotide). We report here, the construction and characterization of three novel active site mutants of the α‐catalytic subunit of Escherichia coli DNA polymerase III that have altered phenotypes with regard to base and sugar discrimination.

The artificial nucleobase 1,3-diaza-2-oxophenoxazine (tC

Due to the extreme infectivity of Yersinia pestis it poses a serious threat as a potential biowarfare agent, which can be rapidly and facilely disseminated. A cost-effective and specific method for its rapid detection at extremely low levels is required, in order to facilitate a timely intervention for containment. Here, we report an ultrasensitive method exploiting a combination of isothermal nucleic acid amplification with a tailed forward primer and biotinylated dNTPs, which is performed in less than 30 min. The polymerase chain reaction (PCR) and enzyme linked oligonucleotide assay (ELONA) were used to optimise assay parameters for implementation on the LFA, and achieved detection limits of 45 pM and 940 fM using SA-HRP and SA-polyHRP, respectively. Replacing PCR with isothermal amplification, namely recombinase polymerase amplification, similar signals were obtained (314 fM), with just 15 min of amplification. The lateral flow detection of the isothermally amplified and labelled amplicon was then explored and detection limits of 7 fM and 0.63 fg achieved for synthetic and genomic DNA, respectively. The incorporation of biotinylated dNTPs and their exploitation for the ultrasensitive molecular detection of a nucleic acid target has been demonstrated and this generic platform can be exploited for a multitude of diverse real life applications.

POLD1 encodes the catalytic subunit of DNA polymerase delta (Polδ), the major lagging strand polymerase, which also participates in DNA repair. Mutations affecting the exonuclease domain increase the risk of various cancers, while mutations that change the polymerase active site cause a progeroid syndrome called mandibular hypoplasia, deafness, progeroid features, and lipodystrophy (MDPL) syndrome. We generated a set of catalytic subunit of human telomerase (hTERT)-immortalized human fibroblasts expressing wild-type or mutant POLD1 using the retroviral LXSN vector system. In the resulting cell lines, expression of endogenous POLD1 was suppressed in favor of the recombinant POLD1. The siRNA screening of DNA damage-related genes revealed that fibroblasts expressing D316H and S605del POLD1 were more sensitive to knockdowns of ribonuclease reductase (RNR) components, RRM1 and RRM2 in the presence of hydroxyurea (HU), an RNR inhibitor. On the contrary, SAMHD1 siRNA, which increases the concentration of dNTPs, increased growth of wild type, D316H, and S605del POLD1 fibroblasts. Hypersensitivity to dNTP synthesis inhibition in POLD1 mutant lines was confirmed using gemcitabine. Our finding is consistent with the notion that reduced dNTP concentration negatively affects the cell growth of hTERT fibroblasts expressing exonuclease and polymerase mutant POLD1.

Sterile α-motif/histidine-aspartate domain-containing protein 1 (SAMHD1) is an intrinsic antiviral restriction factor known to play a vital role in preventing multiple viral infections and in the control of the cellular deoxynucleoside triphosphate (dNTP) pool. Human and mouse SAMHD1 have both been extensively studied; however, our knowledge on porcine SAMHD1 is limited. Here, we report our findings from comprehensive structural and functional studies on porcine SAMHD1. We determined the crystal structure of porcine SAMHD1 and showed that it forms a symmetric tetramer. Moreover, we modified the deoxynucleotide triphosphohydrolase (dNTPase) activity of SAMHD1 by site-directed mutagenesis based on the crystal structure, and obtained an artificial dimeric enzyme possessing high dNTPase activity. Taken together, our results define the mechanism underlying dNTP regulation and provide a deeper understanding of the regulation of porcine SAMHD1 functions. Directed modification of key residues based on the protein structure enhances the activity of the enzyme, which will be beneficial in the search for new antiviral strategies and for future translational applications.

l-Rhamnose is an important monosaccharide both as nutrient source and as building block in prokaryotic glycoproteins and glycolipids. Generation of those composite molecules requires activated precursors being provided e. g. in form of nucleotide sugars such as dTDP-β-l-rhamnose (dTDP-l-Rha). dTDP-l-Rha is synthesized in a conserved 4-step reaction which is canonically catalyzed by the enzymes RmlABCD. An intact pathway is especially important for the fitness of pseudomonads, as dTDP-l-Rha is essential for the activation of the polyproline specific translation elongation factor EF-P in these bacteria. Within the scope of this study, we investigated the dTDP-l-Rha-biosynthesis route of Pseudomonas putida KT2440 with a focus on the last two steps. Bioinformatic analysis in combination with a screening approach revealed that epimerization of dTDP-4-keto-6-deoxy-d-glucose to dTDP-4-keto-6-deoxy-l-mannose is catalyzed by the two paralogous proteins PP_1782 (RmlC1) and PP_0265 (RmlC2), whereas the reduction to the final product is solely mediated by PP_1784 (RmlD). Thus, we also exclude the distinct RmlD homolog PP_0500 and the genetically linked nucleoside diphosphate-sugar epimerase PP_0501 to be involved in dTDP-l-Rha formation, other than suggested by certain databases. Together our analysis contributes to the molecular understanding how this important nucleotide-sugar is synthesized in pseudomonads.

Ribonucleotide reductases (RNRs) catalyze the reduction of ribonucleotides to the corresponding deoxyribonucleotides, the building blocks of DNA. RNRs are specific for either ribonucleoside diphosphates or triphosphates as substrates. As far as is known, oxygen-dependent class I RNRs (NrdAB) all reduce ribonucleoside diphosphates, and oxygen-sensitive class III RNRs (NrdD) are all ribonucleoside triphosphate reducers, whereas the adenosylcobalamin-dependent class II (NrdJ) contains both ribonucleoside diphosphate and triphosphate reducers. However, it is unknown how this specificity is conveyed by the active site of the enzymes and how this feature developed in RNR evolution. By structural comparison of the active sites in different RNRs, we identified the apical loop of the phosphate-binding site as a potential structural determinant of substrate specificity. Grafting two residues from this loop from a diphosphate- to a triphosphate-specific RNR caused a change in preference from ribonucleoside triphosphate to diphosphate substrates in a class II model enzyme, confirming them as the structural determinants of phosphate specificity. The investigation of the phylogenetic distribution of this motif in class II RNRs yielded a likely monophyletic clade with the diphosphate-defining motif. This indicates a single evolutionary-split event early in NrdJ evolution in which diphosphate specificity developed from the earlier triphosphate specificity. For those interesting cases where organisms contain more than one nrdJ gene, we observed a preference for encoding enzymes with diverse phosphate specificities, suggesting that this varying phosphate specificity confers a selective advantage.

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.

Cells require extra amounts of dNTPs to repair DNA after damage. Polyphosphate (polyP) is an evolutionary conserved linear polymer of up to several hundred inorganic phosphate (Pi) residues that is involved in many functions, including Pi storage. In the present article, we report on findings demonstrating that polyP functions as a source of Pi when required to sustain the dNTP increment essential for DNA repair after damage. We show that mutant yeast cells without polyP produce less dNTPs upon DNA damage and that their survival is compromised. In contrast, when polyP levels are ectopically increased, yeast cells become more resistant to DNA damage. More importantly, we show that when polyP is reduced in HEK293 mammalian cell line cells and in human dermal primary fibroblasts (HDFa), these cells become more sensitive to DNA damage, suggesting that the protective role of polyP against DNA damage is evolutionary conserved. In conclusion, we present polyP as a molecule involved in resistance to DNA damage and suggest that polyP may be a putative target for new approaches in cancer treatment or prevention. We would like to thank all the members of our group (E. Bállega, O. Mirallas, M. Ribeiro, A. Sánchez, B. Semper, and R. Carballar) for day-to-day talks, and Marta Pérez for technical assistance. The yeast ppn1Δ, ppn2Δ, ppx1Δ strain is a kind gift from A Mayer (Université de Lausanne). This work was supported by funding from the Spanish Government, with a MINECO grant (Ref: BFU 2013-44189-P) awarded to J. Clotet and a MICINN grant (Ref: BFU 2013-42895-P) awarded to E. Garí. J.M.M. was the recipient of a post-graduate Junior Faculty Fellowship from the UIC and l’Obra Social la Caixa.

The interplay between replication stress and the S phase checkpoint is a key determinant of genome maintenance, and has a major impact on human diseases, notably, tumour initiation and progression. Recent studies have yielded insights into sequence-dependent and sequence-independent sources of endogenous replication stress. These stresses result in nuclease-induced DNA damage, checkpoint activation and genome-wide replication fork slowing. Several hypotheses have been proposed to account for the mechanisms involved in this complex response. Recent results have shown that the slowing of the replication forks most commonly results from DNA precursor starvation. By concomitantly increasing the density of replication initiation, the cell elicits an efficient compensatory strategy to avoid mitotic anomalies and the inheritance of damage over cell generations.

HL60 myeloid leukemia cells are extensively used as a differentiation model. We investigated a variant of HL60 which is resistant to differentiation induction (HL60-R) by standard differentiation inducers such as retinoic acid and dimethylsulfoxide (DMSO). To find an explanation for this resistance, we examined nucleotide (NTP) and deoxynucleotide (dNTP) pools in HL60-R and its parent cell line, sensitive to differentiation, HL60-S. We also explored whether these differences led to a difference in sensitivity to various antimetabolites. Drug sensitivity was measured with the tetrazolium (MTT) assay, while nucleotides were measured with anion-exchange HPLC. HL60-R cells were between 2- and 5-fold resistant to the antimetabolites 5-fluorouracil, Brequinar, hydroxyurea and N-(phosphonacetyl)-L-aspartate (PALA), but more sensitive to aza-2’-deoxycytidine (DAC), cytarabine and thymidine (5- to 10-fold). The NTP pools in both HL60 variants showed a normal pattern with ATP being the highest (2530–2876 pmol/106 cells) and CTP being lowest. However, UTP pools were 2-fold higher in the HL60-S cells (p 6 cells) being highest in HL60-R cells, but dATP was 4-fold lower in HL60-S cells. In HL-60-R, the triple combination retinoic acid, DMSO and DAC increased all NTPs almost 2-fold in contrast to HL60-S. Uridine increased UTP (1.4-fold), CTP (2-fold) and dCTP (1.4.-fold) pools in both cell lines, but thymidine increased only dTTP pools (4- to 7-fold), with a depletion of dCTP. PALA decreased UTP and CTP in both cell lines, but increased ATP (only in HL60-R). Hydroxyurea decreased dNTP especially in HL60-S cells. In conclusion, the pronounced differences in NTP and dNTP pools between HL60-S and HL60-R possibly play a role in the induction of differentiation and drug sensitivity.

Eukaryotic ribonucleotide reductases are iron-dependent enzymes that catalyze the rate-limiting step in the de novo synthesis of deoxyribonucleotides. Multiple mechanisms regulate the activity of ribonucleotide reductases in response to genotoxic stresses and iron deficiency. Upon iron starvation, the Saccharomyces cerevisiae Aft1 transcription factor specifically binds to iron-responsive cis elements within the promoter of a group of genes, known as the iron regulon, activating their transcription. Members of the iron regulon participate in iron acquisition, mobilization and recycling, and trigger a genome-wide metabolic remodeling of iron-dependent pathways. Here, we describe a mechanism that optimizes the activity of yeast ribonucleotide reductase when iron is scarce. We demonstrate that Aft1 and the DNA-binding protein Ixr1 enhance the expression of the gene encoding for its catalytic subunit, RNR1, in response to iron limitation, leading to an increase in both mRNA and protein levels. By mutagenesis of the Aft1-binding sites within RNR1 promoter, we conclude that RNR1 activation by iron depletion is important for Rnr1 protein and deoxyribonucleotide synthesis. Remarkably, Aft1 also activates the expression of IXR1 upon iron scarcity through an iron-responsive element located within its promoter. These results provide a novel mechanism for the direct activation of ribonucleotide reductase function by the iron-regulated Aft1 transcription factor., This work was supported by predoctoral contracts from the Spanish Ministry of Economy, Industry and Competitiveness to LRA and AMR; and by the BIO2017-87828-C2-1-P grant from the Spanish Ministry of Science, Innovation and Universities, and FEDER (Fondo Europeo de Desarrollo Regional) funds to SP.

Deoxyribonucleotide metabolites (dNTPs) are the substrates for DNA synthesis. It has been proposed that their availability influences the progression of the cell cycle during development and pathological situations such as tumor growth. The mechanism has remained unclear for the link between cell cycle and dNTP levels beyond their role as substrates. Here, we review recent studies concerned with the dynamics of dNTP levels in early embryos and the role of DNA replication checkpoint as a sensor of dNTP levels.

Sterile alpha motif and histidine-aspartic acid domain-containing protein 1 (SAMHD1), a dNTP triphosphohydrolase, regulates the levels of cellular dNTPs through their hydrolysis. SAMHD1 protects cells from invading viruses that depend on dNTPs to replicate and is frequently mutated in cancers and Aicardi-Goutieres syndrome, a hereditary autoimmune encephalopathy. We discovered that SAMHD1 localizes at the immunoglobulin (Ig) switch region, and serves as a novel DNA repair regulator of Ig class switch recombination (CSR). Depletion of SAMHD1 impaired not only CSR but also IgH/c-Myc translocation. Consistently, we could inhibit these two processes by elevating the cellular nucleotide pool. A high frequency of nucleotide insertion at the break-point junctions is a notable feature in SAMHD1 deficiency during activation-induced cytidine deaminase-mediated genomic instability. Interestingly, CSR induced by staggered but not blunt, double-stranded DNA breaks was impaired by SAMHD1 depletion, which was accompanied by enhanced nucleotide insertions at recombination junctions. We propose that SAMHD1-mediated dNTP balance regulates dNTP-sensitive DNA end-processing enzyme and promotes CSR and aberrant genomic rearrangements by suppressing the insertional DNA repair pathway.

Morpholinos are six-membered rings that may provide higher conformational rigidity when incorporated into an oligonucleotide (ODN) backbone. Phosphorodiamidate morpholinos are chemically modified ODNs containing morpholinos in place of 2'-deoxyribose moieties throughout their backbone and have garnered much interest in recent years due to their ability to function as highly effective steric blockers in exon skipping therapy. To further explore the biophysical and biological properties of ODNs derived from morpholino nucleosides, we have replaced the 2'-deoxyribonucleotides of phosphodiester DNA with morpholinonucleotides to generate phosphoramidate ODNs. Here, we evaluate the mechanistic pathways observed during the solution-phase synthesis of morpholinonucleoside phosphoramidites, solid-phase synthesis of morpholinonucleotide phosphoramidates of mA, mG, mC and mT (prefix 'm' represents morpholino) and our first attempts directed at the solid-phase synthesis of chimeric DNA-phosphoramidate ODNs, as well as fully modified 22-mer phosphoramidate ODNs.

Polymerase γ catalytic subunit (POLG) gene encodes the enzyme responsible for mitochondrial DNA (mtDNA) synthesis. Mutations affecting POLG are the most prevalent cause of mitochondrial disease because of defective mtDNA replication and lead to a wide spectrum of clinical phenotypes characterized by mtDNA deletions or depletion. Enhancing mitochondrial deoxyribonucleoside triphosphate (dNTP) synthesis effectively rescues mtDNA depletion in different models of defective mtDNA maintenance due to dNTP insufficiency. In this study, we studied mtDNA copy number recovery rates following ethidium bromide-forced depletion in quiescent fibroblasts from patients harboring mutations in different domains of POLG. Whereas control cells spontaneously recovered initial mtDNA levels, POLG-deficient cells experienced a more severe depletion and could not repopulate mtDNA. However, activation of deoxyribonucleoside (dN) salvage by supplementation with dNs plus erythro-9-(2-hydroxy-3-nonyl) adenine (inhibitor of deoxyadenosine degradation) led to increased mitochondrial dNTP pools and promoted mtDNA repopulation in all tested POLG-mutant cells independently of their specific genetic defect. The treatment did not compromise POLG fidelity because no increase in multiple deletions or point mutations was detected. Our study suggests that physiologic dNTP concentration limits the mtDNA replication rate. We thus propose that increasing mitochondrial dNTP availability could be of therapeutic interest for POLG deficiency and other conditions in which mtDNA maintenance is challenged.-Blazquez-Bermejo, C., Carreno-Gago, L., Molina-Granada, D., Aguirre, J., Ramon, J., Torres-Torronteras, J., Cabrera-Perez, R., Martin, M. A., Dominguez-Gonzalez, C., de la Cruz, X., Lombes, A., Garcia-Arumi, E., Marti, R., Camara, Y. Increased dNTP pools rescue mtDNA depletion in human POLG-deficient fibroblasts.

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.

International audience; The 5'-nucleotidase cN-II has been shown to be associated with the sensitivity to nucleoside analogues, the survival of cytarabine treated leukemia patients and to cell proliferation. Due to the lack of relevant cell models for solid tumors, we developed four cell lines with low cN-II expression and characterized them concerning their in vitro sensitivity to cancer drugs and their intracellular nucleotide pools. All four cell models had an important decrease of cN-II expression but did not show modified sensitivity, cell proliferation or nucleotide pools. Our cell models will be important for the study of the role of cN-II in human cancer cells

Viral DNA replication requires deoxyribonucleotide triphosphates (dNTPs). These molecules, which are found at low levels in noncycling cells, are generated either by salvage pathways or through de novo synthesis. Nucleotide synthesis utilizes the activity of a series of nucleotide-biosynthetic enzymes (NBEs) whose expression is repressed in noncycling cells by complexes between the E2F transcription factors and the retinoblastoma (Rb) tumor suppressor. Rb-E2F complexes are dissociated and NBE expression is activated during cell cycle transit by cyclin-dependent kinase (Cdk)-mediated Rb phosphorylation. The DNA virus human cytomegalovirus (HCMV) encodes a viral Cdk (v-Cdk) (the UL97 protein) that phosphorylates Rb, induces the expression of cellular NBEs, and is required for efficient viral DNA synthesis. A long-held hypothesis proposed that viral proteins with Rb-inactivating activities functionally similar to those of UL97 facilitated viral DNA replication in part by inducing the de novo production of dNTPs. However, we found that dNTPs were limiting even in cells infected with wild-type HCMV in which UL97 is expressed and Rb is phosphorylated. Furthermore, we revealed that both de novo and salvage pathway enzymes contribute to viral DNA replication during HCMV infection and that Rb phosphorylation by cellular Cdks does not correct the viral DNA replication defect observed in cells infected with a UL97-deficient virus. We conclude that HCMV can obtain dNTPs in the absence of Rb phosphorylation and that UL97 can contribute to the efficiency of DNA replication in an Rb phosphorylation-independent manner. IMPORTANCE Transforming viral oncoproteins, such as adenovirus E1A and papillomavirus E7, inactivate Rb. The standard hypothesis for how Rb inactivation facilitates infection with these viruses is that it is through an increase in the enzymes required for DNA synthesis, which include nucleotide-biosynthetic enzymes. However, HCMV UL97, which functionally mimics these viral oncoproteins through phosphorylation of Rb, fails to induce the production of nonlimiting amounts of dNTPs. This finding challenges the paradigm of the role of Rb inactivation during DNA virus infection and uncovers the existence of an alternative mechanism by which UL97 contributes to HCMV DNA synthesis. The ineffectiveness of the UL97 inhibitor maribavir in clinical trials might be better explained with a fuller understanding of the role of UL97 during infection. Furthermore, as the nucleoside analog ganciclovir is the current drug of choice for treating HCMV, knowing the provenance of the dNTPs incorporated into viral DNA may help inform antiviral therapeutic regimens.

Ribonucleotide Reductase (RNR) is the sole enzyme that catalyzes the reduction of ribonucleotides into deoxyribonucleotides. Even though RNR is a recognized target for antiproliferative molecules, and the main target of the approved drug hydroxyurea, few new leads targeted to this enzyme have been developed. We have evaluated a recently identified set of RNR inhibitors with respect to inhibition of the human enzyme and cellular toxicity. One compound, NSC73735, is particularly interesting; it is specific for leukemia cells and is the first identified compound that hinders oligomerization of the mammalian large RNR subunit. Similar to hydroxyurea, it caused a disruption of the cell cycle distribution of cultured HL‐60 cells. In contrast to hydroxyurea, the disruption was reversible, indicating higher specificity. NSC73735 thus defines a potential lead candidate for RNR‐targeted anticancer drugs, as well as a chemical probe with better selectivity for RNR inhibition than hydroxyurea.

Alkylating agents can form O(6)-methylguansine (O(6)-MeG). To study the intrinsic kinetic behaviors of bypassing O(6)-MeG, we used the catalytic core of yeast DNA polymerase η (Pol ηcore, residues 1-513), instead of the full-length Pol η, to study their elementary steps, eliminating the effects of the C-terminal C2H2 motif on dNTP incorporation. The misincorporation frequencies were 10(-4) for G and 0.055-0.446 for O(6)-MeG. O(6)-MeG does not affect the extension efficiency. Pol ηcore showed no fast burst phase for any incorporation opposite G or O(6)-MeG. Primer extension was greatly blocked by O(6)-MeG and about 67% dTTP, 31% dCTP and 2% dATP were incorporated opposite O(6)-MeG. This study provides further understanding of the mutation mechanism of alkylated lesion for yeast DNA polymerase η.

In this method, E. coli DNA Pol I binds to a nick or short gap in duplex DNA. The 5′ → 3′ exonuclease activity of Pol I then removes nucleotides from one strand of the DNA, creating a template for the synthesis of DNA by the 5′ → 3′ polymerase activity of Pol I. The simultaneous elimination of nucleotides from the 5′ side and the addition of nucleotides to the 3′ side result in movement of the nick (nick translation) along the DNA, which becomes labeled to high specific activity.

The Mec1 and Rad53 kinases play a central role during acute replication stress in budding yeast. They are also essential for viability in normal growth conditions, but the signal that activates the Mec1-Rad53 pathway in the absence of exogenous insults is currently unknown. Here, we show that this pathway is active at the onset of normal S phase because deoxyribonucleotide triphosphate (dNTP) levels present in G

Deoxyxylonucleic acid (dxNA) is a synthetic polymer that might have potential for heredity and evolution. Because of dxNA's unusual backbone geometry, sequence information stored in it is presumed to be inaccessible to natural nucleic acids or proteins. Despite a large structural similarity with natural nucleotides, incorporation of 2'-deoxyxylonucleotides (dxNTs) through the action of polymerases is limited. We present the identification of a mutant of the DNA polymerase Therminator with increased tolerance to deoxyxylose-induced backbone distortions. Whereas the original polymerase stops after incorporation of two consecutive dxNTs, the mutant is able to catalyse the extension of incorporated dxNTs with 2'-deoxyribonucleotides (dNTs) and the incorporation of up to four dxNTs alternates with dNTs, thereby translocating a highly distorted double helix throughout the entire polymerase. A single His-to-Arg substitution very close to the catalytic site residues is held to be responsible for interaction with the primer phosphate groups and for stabilizing nucleotide sugar-induced distortions during incorporation and translocation. ispartof: CHEMBIOCHEM vol:19 issue:22 pages:2410-2420 ispartof: location:Germany status: published

Quantification of cellular deoxyribonucleoside mono-(dNMP), di-(dNDP), triphosphates (dNTPs) and related nucleoside metabolites are difficult due to their physiochemical properties and widely varying abundance. Involvement of dNTP metabolism in cellular processes including senescence and pathophysiological processes including cancer and viral infection make dNTP metabolism an important bioanalytical target. We modified a previously developed ion pairing reversed phase chromatography-mass spectrometry method for the simultaneous quantification and 13C isotope tracing of dNTP metabolites. dNMPs, dNDPs, and dNTPs were chromatographically resolved to avoid mis-annotation of in-source fragmentation. We used commercially available 13C15N-stable isotope labeled analogs as internal standards and show that this isotope dilution approach improves analytical figures of merit. At sufficiently high mass resolution achievable on an Orbitrap mass analyzer, stable isotope resolved metabolomics allows simultaneous isotope dilution quantification and 13C isotope tracing from major substrates including 13C-glucose. As a proof of principle, we quantified dNMP, dNDP and dNTP pools from multiple cell lines. We also identified isotopologue enrichment from glucose corresponding to ribose from the pentose-phosphate pathway in dNTP metabolites.

PrimPol is the most recently discovered human DNA polymerase/primase and plays an emerging role in nuclear and mitochondrial genomic maintenance. As a member of archaeo-eukaryotic primase (AEP) superfamily enzymes, PrimPol possesses DNA polymerase and primase activities that are important for replication fork progression in vitro and in cellulo. The enzymatic activities of PrimPol are critically dependent on the nucleotidyl-transfer reaction to incorporate deoxyribonucleotides successively; however, our knowledge concerning the kinetic mechanism of the reaction remains incomplete. Using enzyme kinetic analyses and computer simulations, we dissected the mechanism by which PrimPol transfers a nucleotide to a primer-template DNA, which comprises DNA binding, conformational transition, nucleotide binding, phosphoester bond formation, and dissociation steps. We obtained the rate constants of the steps by steady-state and pre-steady-state kinetic analyses and simulations. Our data demonstrate that the rate-limiting step of PrimPol-catalyzed DNA elongation depends on the metal cofactor involved. In the presence of Mn(2+), a conformational transition step from non-productive to productive PrimPol:DNA complexes limits the enzymatic turnover, whereas, in the presence of Mg(2+), the chemical step becomes rate-limiting. As evidenced from our kinetic and simulation data, PrimPol maintains the same kinetic mechanism under either millimolar or physiological micromolar Mn(2+) concentration. Our study revealed the underlying mechanism by which PrimPol catalyzes nucleotide incorporation with two common metal cofactors, and provides a kinetic basis for further understanding the regulatory mechanism of this functionally diverse primase-polymerase.

The purpose of this study was to investigate the inhibitory effect of ZH-1 ((6S,9aS,6aR,9bR)-6-(phenylcarbonyl)-6,6a,9a,9b-tetrahydro-8H-azolidino[3,4-a]b enzo [e]indolizine-7,9-dione) and its potential interaction with gemcitabine in A549 cells. MTT assay showed that the combined use of gemcitabine and ZH-1 presented a significant inhibition effect on A549 cell growth with the cell viability from 82.3 ± 5.6% to 51.0 ± 6.6%. The CI value was 0.60 suggesting a synergistic effect between these two drugs. HPLC-MS/MS data indicated that combined treatment with gemcitabine and ZH-1 induced a significant decrease in deoxyadenosine triphosphate, deoxycytidine triphosphate, deoxyguanosine triphosphate and deoxythymidine triphosphate levels compared with use of gemcitabine alone. Five RNs as well as seven dRNs were considered to be significantly contributive to the discrimination of samples. Furthermore, western blot analysis revealed that the combination treatment caused A549 cell apoptosis via the intrinsic pathway by up-regulating Bax/Bcl-2 ratio, activating caspase-9, caspase-3 and poly-ADP-ribose polymerase, and promoting caspase-7, caspase-9 and poly-ADP-ribose polymerase cleavage. Collectively, the combined treatment with gemcitabine and ZH-1 exerted a strong synergistic action on anticancer activity through growth inhibition, perturbations in ribonucleotides and deoxyribonucleotides and the activation of intrinsic apoptotic signaling pathway.

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

SAMHD1 is a cellular dNTPase that restricts lentiviral infection presumably by lowering cellular dNTP levels to below a critical threshold required for reverse transcription; however, lowering cellular dNTP levels may not be the sole mechanism of restriction. In particular, an exonuclease activity of SAMHD1 was reported to contribute to virus restriction. We further investigated the requirements for SAMHD1 restriction activity in both differentiated U937 and cycling HeLa cells. Using hydroxyurea treatment to lower baseline dNTP levels in HeLa cells, we were able to document efficient SAMHD1 restriction without significant further reduction in dNTP levels by SAMHD1. These results argue against a requirement for additional myeloid-specific host factors for SAMHD1 function but further support the notion that SAMHD1 possesses an additional dNTP-independent function contributing to lentiviral restriction. However, our own experiments failed to associate this presumed additional SAMHD1 antiviral activity with a reported nuclease activity.

The leadzyme refers to a small ribozyme that cleaves a RNA substrate in the presence of Pb(2+). In an optimized form, the enzyme strand contains only two unpaired nucleotides. Most RNA-cleaving DNAzymes are much longer. Two classical Pb(2+)-dependent DNAzymes, 8-17 and GR5, both contain around 15 nucleotides in the enzyme loop. This is also the size of most RNA-cleaving DNAzymes that use other metal ions for their activity. Such large enzyme loops make spectroscopic characterization difficult and so far no high-resolution structural information is available for active DNAzymes. The goal of this work is to search for DNAzymes with smaller enzyme loops. A simple replacement of the ribonucleotides in the leadzyme by deoxyribonucleotides failed to produce an active enzyme. A Pb(2+)-dependent in vitro selection combined with deep sequencing was then performed. After sequence alignment and DNA folding, a new DNAzyme named PbE22 was identified, which contains only 5 nucleotides in the enzyme catalytic loop. The biochemical characteristics of PbE22 were compared with those of the leadzyme and the two classical Pb(2+)-dependent DNAzymes. The rate of PbE22 rises with increase in Pb(2+) concentration, being 1.7 h(-1) in the presence of 100 μM Pb(2+) and reaching 3.5 h(-1) at 500 µM Pb(2+). The log of PbE22 rate rises linearly in a pH-dependent fashion (20 µM Pb(2+)) with a slope of 0.74. In addition, many other abundant sequences in the final library were studied. These sequences are quite varied in length and nucleotide composition, but some contain a few conserved nucleotides consistent with the GR5 structure. Interestingly, some sequences are active with Pb(2+) but none of them were active with even 50 mM Mg(2+), which is reminiscent of the difference between the GR5 and 8-17 DNAzymes.

Gene-silencing strategies based on catalytic nucleic acids have been rapidly developed in the past decades. Ribozymes, antisense oligonucleotides and RNA interference have been actively pursued for years due to their potential application in gene inactivation. Pioneered by Joyce et al., a new class of catalytic nucleic acid composed of deoxyribonucleotides has emerged via an in vitro selection system. The therapeutic potential of these RNA-cleaving DNAzymes have been shown both in vitro and in vivo. Although they rival the activity and stability of synthetic ribozymes, they are limited by inefficient delivery to the intracellular targets. Recent successes in clinical testing of the DNAzymes in cancer patients have revitalized the potential clinical utility of DNAzymes.

Abasic sites (Apurinic/apyrimidinic (AP) sites), produced ∼50,000 times/cell/day, are very blocking and miscoding. To better understand miscoding mechanisms of abasic site for yeast DNA polymerase η, pre-steady-state nucleotide incorporation and LC–MS/MS sequence analysis of extension product were studied using pol ηcore (catalytic core, residues 1–513), which can completely eliminate the potential effects of the C-terminal C2H2 motif of pol η on dNTP incorporation. The extension beyond the abasic site was very inefficient. Compared with incorporation of dCTP opposite G, the incorporation efficiencies opposite abasic site were greatly reduced according to the order of dGTP > dATP >> dCTP and dTTP. Pol ηcore showed no fast burst phase for any incorporation opposite G or abasic site, suggesting that the catalytic step is not faster than the dissociation of polymerase from DNA. LC–MS/MS sequence analysis of extension products showed that 53% products were dGTP misincorporation, 33% were dATP and 14% were -1 frameshift, indicating that Pol ηcore bypasses abasic site by a combined G-rule, A-rule and -1 frameshift deletions. Compared with full-length pol η, pol ηcore relatively reduced the efficiency of incorporation of dCTP opposite G, increased the efficiencies of dNTP incorporation opposite abasic site and the exclusive incorporation of dGTP opposite abasic site, but inhibited the extension beyond abasic site, and increased the priority in extension of A: abasic site relative to G: abasic site. This study provides further understanding in the mutation mechanism of abasic sites for yeast DNA polymerase η.

Regulating gene expression programmes is a central facet of the DNA damage response. The Dun1 kinase protein controls expression of many DNA damage induced genes, including the ribonucleotide reductase genes, which regulate cellular dNTP pools. Using a combination of gene expression profiling and chromatin immunoprecipitation, we demonstrate that in the absence of DNA damage the yeast Rad4-Rad23 nucleotide excision repair complex binds to the promoters of certain DNA damage response genes including DUN1, inhibiting their expression. UV radiation promotes the loss of occupancy of the Rad4-Rad23 complex from the regulatory regions of these genes, enabling their induction and thereby controlling the production of dNTPs. We demonstrate that this regulatory mechanism, which is dependent on the ubiquitination of Rad4 by the GG-NER E3 ligase, promotes UV survival in yeast cells. These results support an unanticipated regulatory mechanism that integrates ubiquitination of NER DNA repair factors with the regulation of the transcriptional response controlling dNTP production and cellular survival after UV damage.

Ribonucleotide reductase (RNR) catalyzes the reduction of ribonucleotides to the corresponding deoxyribonucleotides, which are used as building blocks for DNA replication and repair. This process is tightly regulated via two allosteric sites, the specificity site (s-site) and the overall activity site (a-site). The a-site resides in an N-terminal ATP cone domain that binds dATP or ATP and functions as an on/off switch, whereas the composite s-site binds ATP, dATP, dTTP, or dGTP and determines which substrate to reduce. There are three classes of RNRs, and class I RNRs consist of different combinations of α and β subunits. In eukaryotic and Escherichia coli class I RNRs, dATP inhibits enzyme activity through the formation of inactive α6 and α4β4 complexes, respectively. Here we show that the Pseudomonas aeruginosa class I RNR has a duplicated ATP cone domain and represents a third mechanism of overall activity regulation. Each α polypeptide binds three dATP molecules, and the N-terminal ATP cone is critical for binding two of the dATPs because a truncated protein lacking this cone could only bind dATP to its s-site. ATP activates the enzyme solely by preventing dATP from binding. The dATP-induced inactive form is an α4 complex, which can interact with β2 to form a non-productive α4β2 complex. Other allosteric effectors induce a mixture of α2 and α4 forms, with the former being able to interact with β2 to form active α2β2 complexes. The unique features of the P. aeruginosa RNR are interesting both from evolutionary and drug discovery perspectives.

Cyclins control the activation of cyclin-dependent kinases (CDK), which in turn, control the cell cycle and cell division. Intracellular availability of deoxynucleotides (dNTP) plays a fundamental role in cell cycle progression. SAM domain and HD domain-containing protein 1 (SAMHD1) degrades nucleotide triphosphates and controls the size of the dNTP pool. SAMHD1 activity appears to be controlled by CDK. Here, we show that knockdown of cyclin D3 a partner of CDK6 and E2 a partner of CDK2 had a major impact in SAMHD1 phosphorylation and inactivation and led to decreased dNTP levels and inhibition of HIV-1 at the reverse transcription step in primary human macrophages. The effect of cyclin D3 RNA interference was lost after degradation of SAMHD1 by HIV-2 Vpx, demonstrating the specificity of the mechanism. Cyclin D3 inhibition correlated with decreased activation of CDK2. Our results confirm the fundamental role of the CDK6-cyclin D3 pair in controlling CDK2-dependent SAMHD1 phosphorylation and dNTP pool in primary macrophages.

The Escherichia coli SOS system is a well-established model for the cellular response to DNA damage. Control of SOS depends largely on the RecA protein. When RecA is activated by single-stranded DNA in the presence of a nucleotide triphosphate cofactor, it mediates cleavage of the LexA repressor, leading to expression of the 30(+)-member SOS regulon. RecA activation generally requires the introduction of DNA damage. However, certain recA mutants, like recA730, bypass this requirement and display constitutive SOS expression as well as a spontaneous (SOS) mutator effect. Presently, we investigated the possible interaction between SOS and the cellular deoxynucleoside triphosphate (dNTP) pools. We found that dNTP pool changes caused by deficiencies in the ndk or dcd genes, encoding nucleoside diphosphate kinase and dCTP deaminase, respectively, had a strongly suppressive effect on constitutive SOS expression in recA730 strains. The suppression of the recA730 mutator effect was alleviated in a lexA-deficient background. Overall, the findings suggest a model in which the dNTP alterations in the ndk and dcd strains interfere with the activation of RecA, thereby preventing LexA cleavage and SOS induction.

The absolute and relative pool sizes of deoxyribonucleotides (dRNs) are essential in DNA replication fidelity, DNA damage and repair. We found in this study that although DNA damage induced by methyl methanesulfonate (MMS) seemed similar in cancer (HepG2) and normal (LO2) cells, more extensive alterations in ribonucleotides (RNs) and dRNs pools occurred in HepG2 cells indicating that HepG2 cells were more vigilant to DNA damage. After 10 h repair, RNs pools were still severely perturbed in LO2 cells. Compared to LO2 cells, deoxyribonucleotide triphosphates (dNTPs) pools in HepG2 cells elevated by more folds which could facilitate more efficient DNA repair and improve survival probability following DNA damage, although this should definitely lead to higher mutation rates. DNA repair was more efficient in HepG2 cells at S phase and it partly came to an end while DNA repair was still uncompleted in LO2 cells outside S phase. In conclusion, our results demonstrated that HepG2 and LO2 cells presented many differences in nucleotide metabolism, cell cycle checkpoints and DNA repair pathways in response to DNA damage, which could be potential targets for cancer treatment.

The human POLG gene encodes the catalytic subunit of mitochondrial DNA polymerase gamma (pol gamma). Mutations in pol gamma are associated with a spectrum of disease phenotypes including autosomal dominant and recessive forms of progressive external ophthalmoplegia, spino-cerebellar ataxia and epilepsy, and Alpers-Huttenlocher hepatocerebral poliodystrophy. Multiple deletions, or depletion of mtDNA in affected tissues, are the molecular hallmarks of pol gamma mutations. To shed light on the pathogenic mechanisms leading to these phenotypes, we have introduced in MIP1, the yeast homologue of POLG, two mutations equivalent to the human Y955C and G268A mutations, which are associated with dominant and recessive PEO, respectively. Both mutations induced the generation of petite colonies, carrying either rearranged (rho(-)) or no (rho(0)) mtDNA. Mutations in genes that control the mitochondrial supply of deoxynucleotides (dNTP) affect the mtDNA integrity in both humans and yeast. To test whether the manipulation of the dNTP pool can modify the effects of pol gamma mutations in yeast, we have overexpressed a dNTP checkpoint enzyme, ribonucleotide reductase, RNR1, or deleted its inhibitor, SML1. In both mutant strains, the petite mutability was dramatically reduced. The same result was obtained by exposing the mutant strains to dihydrolipoic acid, an anti-oxidant agent. Therefore, an increase of the mitochondrial dNTP pool and/or a decrease of reactive oxygen species can prevent the mtDNA damage induced by pol gamma mutations in yeast and, possibly, in humans.

The question of whether RNA is more stable or unstable compared to DNA or other nucleic acids has long been a subject of extensive scrutiny and public attention. Recently, thermodynamically stable and degradation-resistant RNA motifs have been utilized in RNA nanotechnology to build desired architectures and integrate multiple functional groups. Here we report the effects of phosphorothioate deoxyribonucleotides (PS-DNA), deoxyribonucleotides (DNA), ribonucleotides (RNA), 2′-F nucleotides (2′-F), and locked nucleic acids (LNA) on the thermal and in vivo stability of the three-way junction (3WJ) of bacteriophage phi29 motor packaging RNA. It was found that the thermal stability gradually increased following the order of PS-DNA/PS-DNA < DNA/DNA < DNA/RNA < RNA/RNA < RNA/2′-F RNA < 2’-F RNA/2′-F RNA < 2′-F RNA/LNA < LNA/LNA. This proposition is supported by studies on strand displacement and the melting of homogeneous and heterogeneous 3WJs. By simply mixing different chemically modified oligonucleotides, the thermal stability of phi29 pRNA 3WJ can be tuned to cover a wide range of melting temperatures from 21.2°C to over 95°C. The 3WJLNA was resistant to boiling temperature denaturation, urea denaturation, and 50% serum degradation. Intravenous injection of fluorescent LNA/2′-F hybrid 3WJs into mice revealed its exceptional in vivo stability and presence in urine. It is thus concluded that incorporation of LNA nucleotides, alone or in combination with 2′-F, into RNA nanoparticles derived from phi29 pRNA 3WJ can extend the half-life of the RNA nanoparticles in vivo and improve their pharmacokinetics profile.