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Ramon Marti - One of the best experts on this subject based on the ideXlab platform.
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limited dctp availability accounts for mitochondrial dna depletion in mitochondrial neurogastrointestinal encephalomyopathy mngie
PLOS Genetics, 2011Co-Authors: Emiliano Gonzalezvioque, Antoni L. Andreu, Javier Torrestorronteras, Ramon MartiAbstract:Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is a severe human disease caused by mutations in TYMP, the gene encoding thymidine phosphorylase (TP). It belongs to a broader group of disorders characterized by a pronounced reduction in mitochondrial DNA (mtDNA) copy number in one or more tissues. In most cases, these disorders are caused by mutations in genes involved in deoxyribonucleoside triphosphate (dNTP) metabolism. It is generally accepted that imbalances in mitochondrial dNTP pools resulting from these mutations interfere with mtDNA replication. Nonetheless, the precise mechanistic details of this effect, in particular, how an excess of a given dNTP (e.g., imbalanced dTTP excess observed in TP deficiency) might lead to mtDNA depletion, remain largely unclear. Using an in organello replication experimental model with isolated murine liver mitochondria, we observed that overloads of dATP, dGTP, or dCTP did not reduce the mtDNA replication rate. In contrast, an excess of dTTP decreased mtDNA synthesis, but this effect was due to secondary dCTP depletion rather than to the dTTP excess in itself. This was confirmed in human cultured cells, demonstrating that our conclusions do not depend on the experimental model. Our results demonstrate that the mtDNA replication rate is unaffected by an excess of any of the 4 separate dNTPs and is limited by the availability of the dNTP present at the lowest concentration. Therefore, the availability of dNTP is the key factor that leads to mtDNA depletion rather than dNTP imbalances. These results provide the first test of the mechanism that accounts for mtDNA depletion in MNGIE and provide evidence that limited dNTP availability is the common cause of mtDNA depletion due to impaired anabolic or catabolic dNTP pathways. Thus, therapy approaches focusing on restoring the deficient substrates should be explored.
Vera Bianchi - One of the best experts on this subject based on the ideXlab platform.
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mitochondrial deoxynucleotide pools in quiescent fibroblasts a possible model for mitochondrial neurogastrointestinal encephalomyopathy mngie
Journal of Biological Chemistry, 2005Co-Authors: Paola Ferraro, Peter Reichard, Giovanna Pontarin, Laura Crocco, Sonia Fabris, Vera BianchiAbstract:Abstract Mitochondrial (mt) DNA depletion syndromes can arise from genetic deficiencies for enzymes of dNTP metabolism, operating either inside or outside mitochondria. MNGIE is caused by the deficiency of cytosolic thymidine phosphorylase that degrades thymidine and deoxyuridine. The extracellular fluid of the patients contains 10–20 μm deoxynucleosides leading to changes in dTTP that may disturb mtDNA replication. In earlier work, we suggested that mt dTTP originates from two distinct pathways: (i) the reduction of ribonucleotides in the cytosol (in cycling cells) and (ii) intra-mt salvage of thymidine (in quiescent cells). In MNGIE and most other mtDNA depletion syndromes, quiescent cells are affected. Here, we demonstrate in quiescent fibroblasts (i) the existence of small mt dNTP pools, each usually 3–4% of the corresponding cytosolic pool; (ii) the rapid metabolic equilibrium between mt and cytosolic pools; and (iii) the intra-mt synthesis and rapid turnover of dTTP in the absence of DNA replication. Between 0.1 and 10 μm extracellular thymidine, intracellular thymidine rapidly approaches the extracellular concentration. We mimic the conditions of MNGIE by maintaining quiescent fibroblasts in 10–40 μm thymidine and/or deoxyuridine. Despite a large increase in intracellular thymidine concentration, cytosolic and mt dTTP increase at most 4-fold, maintaining their concentration for 41 days. Other dNTPs are marginally affected. Deoxyuridine does not increase the normal dNTP pools but gives rise to a small dUTP and a large dUMP pool, both turning over rapidly. We discuss these results in relation to MNGIE.
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molecular analysis of mutations in the hprt gene of v79 hamster fibroblasts effects of imbalances in the dctp dgtp and dttp pools
Journal of Molecular Biology, 1995Co-Authors: Elisabetta Dare, Dag Jenssen, Vera Bianchi, Lihua ZhangAbstract:Abstract dCMP-deaminase-deficient V79/dC hamster cells have highly imbalanced deoxyribonucleoside triphosphate (dNTP) pools, i.e. a 17-fold larger dCTP pool, a slightly reduced dTTP and a very low dGTP pool, compared to dCMP-deaminase-proficient V79/p cells. Nevertheless, the two lines showed the same rates of spontaneous mutation at the hprt and ouabain-resistance loci. Analysis of spontaneous hprt mutations indicated an increase in misincorporation of C in V79/dC cells, although it was not statistically significant. When the dCTP pool was further increased fivefold by incubating V79/dC cells with cytidine, C misincorporation increased to 88%, but the mutation frequency remained unchanged. The dNTP pools of V79/dC cells were also altered by treatment with thymidine, or with thymidine plus deoxycytidine. After incubation with thymidine alone, the dCTP pool all but disappeared, whereas it maintained a normal level in the presence of deoxycytidine. In both cases dTTP rose to nmol amounts, and dGTP accumulated. Incubation with 10 mM thymidine was the only treatment that increased the mutation frequency; T misincorporation then accounted for 94% of the base substitutions. In the presence of deoxycytidine the cells had a dTTP/dCTP ratio of 0.04, but 86% of the base substitutions involved C misincorporation and most probably originated from G misincorporation caused by excess dGTP. Alterations of RNA splicing and hot spots for base substitutions varied with the imbalance, the latter showed “next-nucleotide effects”. Our results suggest that the fidelity of DNA replication in V79 cells is only affected by large changes in the pool and is more sensitive to changes in dGTP than in dCTP or dTTP.
Jinwoo Ahn - One of the best experts on this subject based on the ideXlab platform.
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substrate specificity of samhd1 triphosphohydrolase activity is controlled by deoxyribonucleoside triphosphates and phosphorylation at thr592
Biochemistry, 2016Co-Authors: Sunbok Jang, Xiaohong Zhou, Jinwoo AhnAbstract:The sterile alpha motif (SAM) and histidine-aspartate (HD) domain containing protein 1 (SAMHD1) constitute a triphosphohydrolase that converts deoxyribonucleoside triphosphates (dNTPs) into deoxyribonucleosides and triphosphates. SAMHD1 exists in multiple states. The monomer and apo- or GTP-bound dimer are catalytically inactive. Binding of dNTP at allosteric site 2 (AS2), adjacent to GTP-binding allosteric site 1 (AS1), induces formation of the tetramer, the catalytically active form. We have developed an enzyme kinetic assay, tailored to control specific dNTP binding at each site, allowing us to determine the kinetic binding parameters of individual dNTPs at both the AS2 and catalytic sites for all possible combinations of dNTP binding at both sites. Here, we show that the apparent Km values of dNTPs at AS2 vary in the order of dCTP < dGTP < dATP < dTTP. Interestingly, dCTP binding at AS2 significantly reduces the dCTP hydrolysis rate, which is restored to a rate comparable to that of other dNTPs upon d...
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Substrate Specificity of SAMHD1 Triphosphohydrolase Activity Is Controlled by Deoxyribonucleoside Triphosphates and Phosphorylation at Thr592
2016Co-Authors: Sunbok Jang, Xiaohong Zhou, Jinwoo AhnAbstract:The sterile alpha motif (SAM) and histidine-aspartate (HD) domain containing protein 1 (SAMHD1) constitute a triphosphohydrolase that converts deoxyribonucleoside triphosphates (dNTPs) into deoxyribonucleosides and triphosphates. SAMHD1 exists in multiple states. The monomer and apo- or GTP-bound dimer are catalytically inactive. Binding of dNTP at allosteric site 2 (AS2), adjacent to GTP-binding allosteric site 1 (AS1), induces formation of the tetramer, the catalytically active form. We have developed an enzyme kinetic assay, tailored to control specific dNTP binding at each site, allowing us to determine the kinetic binding parameters of individual dNTPs at both the AS2 and catalytic sites for all possible combinations of dNTP binding at both sites. Here, we show that the apparent Km values of dNTPs at AS2 vary in the order of dCTP < dGTP < dATP < dTTP. Interestingly, dCTP binding at AS2 significantly reduces the dCTP hydrolysis rate, which is restored to a rate comparable to that of other dNTPs upon dGTP, dATP, or dTTP binding at AS2. Strikingly, a phosphomimetic mutant, Thr592Asp SAMHD1 as well as phospho-Thr592, show a significantly altered substrate specificity, with the rate of dCTP hydrolysis being selectively reduced regardless of which dNTP binds at AS2. Furthermore, cyclin A2 binding at the C-terminus of SAMHD1 induces the disassembly of the SAMHD1 tetramer, suggesting an additional layer of SAMHD1 activity modulation by cyclin A2/CDK2 kinase. Together, our results reveal multiple allosteric mechanisms for controlling the rate of dNTP destruction by SAMHD1
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structural basis of allosteric activation of sterile α motif and histidine aspartate domain containing protein 1 samhd1 by nucleoside triphosphates
Journal of Biological Chemistry, 2014Co-Authors: Leonardus M I Koharudin, Maria Delucia, Jennifer Mehrens, Angela M Gronenborn, Jinwoo AhnAbstract:Sterile α motif and histidine-aspartate domain-containing protein 1 (SAMHD1) plays a critical role in inhibiting HIV infection, curtailing the pool of dNTPs available for reverse transcription of the viral genome. Recent structural data suggested a compelling mechanism for the regulation of SAMHD1 enzymatic activity and revealed dGTP-induced association of two inactive dimers into an active tetrameric enzyme. Here, we present the crystal structures of SAMHD1 catalytic core (residues 113–626) tetramers, complexed with mixtures of nucleotides, including dGTP/dATP, dGTP/dCTP, dGTP/dTTP, and dGTP/dUTP. The combined structural and biochemical data provide insight into dNTP promiscuity at the secondary allosteric site and how enzymatic activity is modulated. In addition, we present biochemical analyses of GTP-induced SAMHD1 full-length tetramerization and the structure of SAMHD1 catalytic core tetramer in complex with GTP/dATP, revealing the structural basis of GTP-mediated SAMHD1 activation. Altogether, the data presented here advance our understanding of SAMHD1 function during cellular homeostasis.
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mechanisms of allosteric activation and inhibition of the deoxyribonucleoside triphosphate triphosphohydrolase from enterococcus faecalis
Journal of Biological Chemistry, 2014Co-Authors: Ivan I Vorontsov, Maria Delucia, Jennifer Mehrens, George Minasov, Ludmilla Shuvalova, W F Anderson, Jinwoo AhnAbstract:EF1143 from Enterococcus faecalis, a life-threatening pathogen that is resistant to common antibiotics, is a homo-tetrameric deoxyribonucleoside triphosphate (dNTP) triphosphohydrolase (dNTPase), converting dNTPs into the deoxyribonucleosides and triphosphate. The dNTPase activity of EF1143 is regulated by canonical dNTPs, which simultaneously act as substrates and activity modulators. Previous crystal structures of apo-EF1143 and the protein bound to both dGTP and dATP suggested allosteric regulation of its enzymatic activity by dGTP binding at four identical allosteric sites. However, whether and how other canonical dNTPs regulate the enzyme activity was not defined. Here, we present the crystal structure of EF1143 in complex with dGTP and dTTP. The new structure reveals that the tetrameric EF1143 contains four additional secondary allosteric sites adjacent to the previously identified dGTP-binding primary regulatory sites. Structural and enzyme kinetic studies indicate that dGTP binding to the first allosteric site, with nanomolar affinity, is a prerequisite for substrate docking and hydrolysis. Then, the presence of a particular dNTP in the second site either enhances or inhibits the dNTPase activity of EF1143. Our results provide the first mechanistic insight into dNTP-mediated regulation of dNTPase activity.
John A. Tainer - One of the best experts on this subject based on the ideXlab platform.
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structural basis for recognition and catalysis by the bifunctional dctp deaminase and dUTPase from methanococcus jannaschii
Journal of Molecular Biology, 2003Co-Authors: Joy L Huffman, Hong Li, John A. TainerAbstract:Abstract Potentially mutagenic uracil-containing nucleotide intermediates are generated by deamination of dCTP, either spontaneously or enzymatically as the first step in the conversion of dCTP to dTTP. dUTPases convert dUTP to dUMP, thus avoiding the misincorporation of dUTP into DNA and creating the substrate for the next enzyme in the dTTP synthetic pathway, thymidylate synthase. Although dCTP deaminase and dUTPase activities are usually found in separate but homologous enzymes, the hyperthermophile Methanococcus jannaschii has an enzyme, DCD-DUT, that harbors both dCTP deaminase and dUTP pyrophosphatase activities. DCD-DUT has highest activity on dCTP, followed by dUTP, and dTTP inhibits both the deaminase and pyrophosphatase activities. To help clarify structure–function relationships for DCD-DUT, we have determined the crystal structure of the wild-type DCD-DUT protein in its apo form to 1.42 A and structures of DCD-DUT in complex with dCTP and dUTP to resolutions of 1.77 A and 2.10 A, respectively. To gain insights into substrate interactions, we complemented analyses of the experimentally defined weak density for nucleotides with automated docking experiments using dCTP, dUTP, and dTTP. DCD-DUT is a hexamer, unlike the homologous dUTPases, and its subunits contain several insertions and substitutions different from the dUTPase β barrel core that likely contribute to dCTP specificity and deamination. These first structures of a dCTP deaminase reveal a probable role for an unstructured C-terminal region different from that of the dUTPases and possible mechanisms for both bifunctional enzyme activity and feedback inhibition by dTTP.
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human dUTP pyrophosphatase uracil recognition by a β hairpin and active sites formed by three separate subunits
Structure, 1996Co-Authors: Clifford D Mol, Jonathan M Harris, Evan M Mcintosh, John A. TainerAbstract:Abstract Background The essential enzyme dUTP pyrophosphatase (dUTPase) is exquisitely specific for dUTP and is critical for the fidelity of DNA replication and repair. dUTPase hydrolyzes dUTP to dUMP and pyrophosphate, simultaneously reducing dUTP levels and providing the dUMP for dTTP biosynthesis. A high cellular dTTP: dUTP ratio is essential to avoid uracil incorporation into DNA, which would lead to strand breaks and cell death. We report the first detailed atomic-resolution structure of a eukaryotic dUTPase, human dUTPase, and complexes with the uracil-containing deoxyribonucleotides, dUMP, dUDP and dUTP. Results The crystal structure reveals that each subunit of the dUTPase trimer folds into an eight-stranded jelly-roll β barrel, with the C-terminal β strands interchanged among the subunits. The structure is similar to that of the E. coli enzyme, despite low sequence homology between the two enzymes. The nucleotide complexes reveal a simple and elegant way for a β hairpin to recognize specific nucleic acids: uracil is inserted into a distorted antiparallel β hairpin and hydrogen bonds entirely to main-chain atoms. This interaction mimics DNA base pairing, selecting uracil over cytosine and sterically precluding thymine and ribose binding. Residues from the second subunit interact with the phosphate groups and a glycine-rich C-terminal tail of the third subunit caps the substrate-bound active site, causing total complementary enclosure of substrate. To our knowledge, this is the first documented instance of all three subunits of a trimeric enzyme supplying residues that are critical to enzyme function and catalysis. Conclusions The dUTPase nucleotide-binding sites incorporate some features of other nucleotide-binding proteins and protein kinases, but seem distinct in sequence and architecture. The novel nucleic acid base recognition motif appears ancient; higher order structures, such as the ribosome, may have evolved from a motif of this kind. These uracil–β-hairpin interactions are an obvious way for peptides to become early coenzymes in an RNA world, providing a plausible link to the protein–DNA world. Within the β hairpin, there is a tyrosine corner motif that normally specifies β-arch connections; this tyrosine motif was apparently recruited to discriminate against ribonucleotides, more recently than the evolution of the β hairpin itself.
Peter F Guengerich - One of the best experts on this subject based on the ideXlab platform.
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efficient and high fidelity incorporation of dctp opposite 7 8 dihydro 8 oxodeoxyguanosine by sulfolobus solfataricus dna polymerase dpo4
Journal of Biological Chemistry, 2005Co-Authors: Hong Zang, Adriana Irimia, Karen C Angel, Martin Egli, Lioudmila V Loukachevitch, Jeongyun Choi, Peter F GuengerichAbstract:Abstract DNA polymerases insert dATP opposite the oxidative damage product 7,8-dihydro-8-oxodeoxyguanosine (8-oxoG) instead of dCTP, to the extent of >90% with some polymerases. Steady-state kinetics with the Y-family Sulfolobus solfataricus DNA polymerase IV (Dpo4) showed 90-fold higher incorporation efficiency of dCTP > dATP opposite 8-oxoG and 4-fold higher efficiency of extension beyond an 8-oxoG:C pair than an 8-oxoG:A pair. The catalytic efficiency for these events (with dCTP or C) was similar for G and 8-oxoG templates. Mass spectral analysis of extended DNA primers showed ≥95% incorporation of dCTP > dATP opposite 8-oxoG. Pre-steady-state kinetics showed faster rates of dCTP incorporation opposite 8-oxoG than G. The measured Kd,dCTP was 15-fold lower for an oligonucleotide containing 8-oxoG than with G. Extension beyond an 8-oxoG:C pair was similar to G:C and faster than for an 8-oxoG:A pair, in contrast to other polymerases. The Ea for dCTP insertion opposite 8-oxoG was lower than for opposite G. Crystal structures of Dpo4 complexes with oligonucleotides were solved with C, A, and G nucleoside triphosphates placed opposite 8-oxoG. With ddCTP, dCTP, and dATP the phosphodiester bonds were formed even in the presence of Ca2+. The 8-oxoG:C pair showed classic Watson-Crick geometry; the 8-oxoG:A pair was in the syn:anti configuration, with the A hybridized in a Hoogsteen pair with 8-oxoG. With dGTP placed opposite 8-oxoG, pairing was not to the 8-oxoG but to the 5′ C (and in classic Watson-Crick geometry), consistent with the low frequency of this frameshift event observed in the catalytic assays.
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kinetics of nucleotide incorporation opposite dna bulky guanine n2 adducts by processive bacteriophage t7 dna polymerase exonuclease and hiv 1 reverse transcriptase
Journal of Biological Chemistry, 2005Co-Authors: Hong Zang, Thomas M Harris, Peter F GuengerichAbstract:Six oligonucleotides with carcinogen derivatives bound at the N2 atom of deoxyguanosine were prepared, including adducts derived from butadiene, acrolein, crotonaldehyde, and styrene, and examined for effects on the replicative enzymes bacteriophage DNA polymerase T7- (T7-) and HIV-1 reverse transcriptase for comparison with previous work on smaller DNA adducts. All of these adducts strongly blocked dCTP incorporation opposite the adducts. dATP was preferentially incorporated opposite the acrolein and crotonaldehyde adducts, and dTTP incorporation was preferred at the butadiene- and styrene-derived adducts. Steady-state kinetic analysis indicated that the reduced catalytic efficiency with adducted DNA involved both an increased Km and attenuated kcat. Fluorescence estimates of Kd and pre-steady-state kinetic measurements of koff showed no significantly decreased affinity of T7- with the adducted oligonucleotides or the dNTP. Pre-steady-state kinetics showed no burst phase kinetics for dNTP incorporation with any of the modified oligonucleotides. These results indicate that phosphodiester bond formation or a conformational change of the enzyme.DNA complex is rate-limiting instead of the step involving release of the oligonucleotide. Thio elemental effects for dNTP incorporation were generally relatively small but variable, indicating that the presence of adducts may sometimes make phosphodiester bond formation rate-limiting but not always.
