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1.
Shen R Olcott MC Kim J Rajagopal I Mathews CK 《The Journal of biological chemistry》2004,279(31):32225-32232
In both prokaryotic and eukaryotic organisms, nucleoside diphosphate kinase is a multifunctional protein, with well defined functions in ribo- and deoxyribonucleoside triphosphate biosynthesis and more recently described functions in genetic and metabolic regulation, signal transduction, and DNA repair. This paper concerns two unusual properties of nucleoside diphosphate (NDP) kinase from Escherichia coli: 1) its ability to interact specifically with enzymes encoded by the virulent bacteriophage T4 and 2) its roles in regulating metabolism of the host cell. By means of optical biosensor analysis, fluorescence spectroscopy, immunoprecipitation, and glutathione S-transferase pull-down assays, we have shown that E. coli NDP kinase interacts directly with T4 thymidylate synthase, aerobic ribonucleotide reductase, dCTPase-dUTPase, gene 32 single-strand DNA-binding protein, and deoxycytidylate hydroxymethylase. The interactions with ribonucleotide reductase and with gp32 are enhanced by nucleoside triphosphates, suggesting that the integrity of the T4 dNTP synthetase complex in vivo is influenced by the composition of the nucleotide pool. The other investigations in this work stem from the unexpected finding that E. coli NDP kinase is dispensable for successful T4 phage infection, and they deal with two observations suggesting that the NDP kinase protein plays a genetic role in regulating metabolism of the host cell: 1) the elevation of CTP synthetase activity in an ndk mutant, in which the structural gene for NDP kinase is disrupted, and 2) the apparent ability of NDP kinase to suppress anaerobic growth in a pyruvate kinase-negative E. coli mutant. Our data indicate that the regulatory roles are metabolic, not genetic, in nature. 相似文献
2.
Kim J Shen R Olcott MC Rajagopal I Mathews CK 《The Journal of biological chemistry》2005,280(31):28221-28229
Adenylate kinase, which catalyzes the reversible ATP-dependent phosphorylation of AMP to ADP and dAMP to dADP, can also catalyze the conversion of nucleoside diphosphates to the corresponding triphosphates. Lu and Inouye (Lu, Q., and Inouye, M. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 5720-5725) showed that an Escherichia coli ndk mutant, lacking nucleoside diphosphate kinase, can use adenylate kinase as an alternative source of nucleoside triphosphates. Bacteriophage T4 can reproduce in an Escherichia coli ndk mutant, implying that adenylate kinase can meet a demand for deoxyribonucleoside triphosphates that increases by up to 10-fold as a result of T4 infection. In terms of kinetic linkage and specific protein-protein associations, NDP kinase is an integral component of T4 dNTP synthetase, a multienzyme complex containing phage-coded enzymes, which facilitates the synthesis of dNTPs and their flow into DNA. Here we asked whether, by similar criteria, adenylate kinase of the host cell is also a specific component of the complex. Experiments involving protein affinity chromatography, immunoprecipitation, optical biosensor measurements, and glutathione S-transferase pulldowns demonstrated direct interactions between adenylate kinase and several phage-coded enzymes, as well as E. coli nucleoside diphosphate kinase. These results identify adenylate kinase as a specific component of the complex. The rate of DNA synthesis after infection of an ndk mutant was found to be about 40% of the rate seen in wild-type infection, implying that complementation of the missing NDP kinase function by adenylate kinase is fairly efficient, but that adenylate kinase becomes rate-limiting for DNA synthesis when it is the sole source of dNTPs. 相似文献
3.
DNA precursor imbalances are known to be mutagenic in both eukaryotic and prokaryotic systems. Almost certainly, such mutagenesis involves competition between correctly and incorrectly base-paired precursors at replication sites. Since other factors may be involved, it is important to identify specific mutations induced by specific pool imbalances. Using bacteriophage T4, we have developed a system for such analysis. We prepare double mutants of T4; one mutation affects a phage-coded enzyme of deoxyribonucleoside triphosphate (dNTP) metabolism, while the second is an rII mutation known to revert along a specific pathway. We determine dNTP pools in infection by such a mutant and measure both the spontaneous reversion rate of the rII mutation and, in some cases, the nucleotide sequence at the mutant site. In this paper we analyze mutations induced by a deficiency of T4-encoded deoxycytidylate deaminase. This causes pools of 5-hydroxymethyl-dCTP to expand some 30-fold, while dTTP pools contract. This specifically stimulates AT-to-GC reversion. One of the four AT-to-GC reverters tested, rIIUV215, increases its reversion rate at least 1000-fold under these pool-imbalance conditions, while the other mutants tested show increases of only about 10-fold. Therefore, factors other than dNTP competition, including local DNA sequence environment, must be invoked to fully explain mechanisms of dNTP pool imbalance-induced mutagenesis. We discuss models for this, and we also report unexpected effects of the dCMP deaminase deficiency upon pools of ribonucleoside triphosphates. 相似文献
4.
Adenylate kinase (Adk) that catalyses the synthesis of ADP from ATP and AMP has also been shown to perform an ATP dependent phosphorylation of ribo- and deoxynucleoside diphosphates to their corresponding nucleoside triphosphate; ATP+(d)NDP<-->ADP+(d)NTP. This reaction, suggested to occur by the transfer of the gamma-phosphoryl from ATP to the nucleoside diphosphate, is overall similar to that normally carried out by nucleoside diphosphate kinase (Ndk). Accordingly, Adk was proposed to be responsible for residual Ndk-like activity measured in a mutant strain of Escherichia coli, where the ndk gene was disrupted. We present data supporting a mechanism for the synthesis of nucleoside triphosphates by Adk that unlike the previously suggested mechanism mentioned above are in complete agreement with the current knowledge about the Adk enzyme and its various catalytic properties. We propose that nucleoside triphosphate synthesis occurs by beta-phosphoryl transfer from ADP to any bound nucleoside diphosphate. Our results point to the fact that the proposed Ndk-like mechanism of Adk originated from an erroneous interpretation of data, in that contamination of ATP preparations with AMP and ADP was not taken into account. Our results also address the proposed role of Adk in restoring a normal growth rate of mutant strains of E. coli lacking Ndk. These mutant strains apparently, in spite of a mutator phenotype, are able to synthesise nucleoside triphosphates by alternative pathways to maintain the same growth rate as the wildtype. 相似文献
5.
Temperature-sensitive DNA mutant of Chinese hamster ovary cells with a thermolabile ribonucleotide reductase activity.
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B E Wojcik J J Dermody H L Ozer B Mun C K Mathews 《Molecular and cellular biology》1990,10(11):5688-5699
JB3-B is a Chinese hamster ovary cell mutant previously shown to be temperature sensitive for DNA replication (J. J. Dermody, B. E. Wojcik, H. Du, and H. L. Ozer, Mol. Cell. Biol. 6:4594-4601, 1986). It was chosen for detailed study because of its novel property of inhibiting both polyomavirus and adenovirus DNA synthesis in a temperature-dependent manner. Pulse-labeling studies demonstrated a defect in the rate of adenovirus DNA synthesis. Measurement of deoxyribonucleoside triphosphate (dNTP) pools as a function of time after shift of uninfected cultures from 33 to 39 degrees C revealed that all four dNTP pools declined at similar rates in extracts prepared either from whole cells or from rapidly isolated nuclei. Ribonucleoside triphosphate pools were unaffected by a temperature shift, ruling out the possibility that the mutation affects nucleoside diphosphokinase. However, ribonucleotide reductase activity, as measured in extracts, declined after cell cultures underwent a temperature shift, in parallel with the decline in dNTP pool sizes. Moreover, the activity of cell extracts was thermolabile in vitro, consistent with the model that the JB3-B mutation affects the structural gene for one of the ribonucleotide reductase subunits. The kinetics of dNTP pool size changes after temperature shift are quite distinct from those reported after inhibition of ribonucleotide reductase with hydroxyurea. An indirect effect on ribonucleotide reductase activity in JB3-B has not been excluded since human sequences other than those encoding the enzyme subunits can correct the temperature-sensitive growth defect in the mutant. 相似文献
6.
Ribonucleoside and deoxyribonucleoside triphosphate pools have been measured in Escherichia coli infected with bacteriophage T4 DNA polymerase mutator, wild type, and antimutator alleles during mutagenesis by the base analogue 2-aminopurine. ATP and GTP pools expand significantly during mutagenesis, while CTP and UTP pools contract slightly. The DNA polymerase (gene 43) alleles and an rII lesion perturb normal dNTP pools more than does the presence of 2-aminopurine. We find no evidence that 2-aminopurine induces mutations indirectly by causing an imbalance in normal dNTP pools. Rather, it seems likely that, by forming base mispairs with thymine and with cytosine, 2-aminopurine is involved directly in causing bidirectional A.T in equilibrium G.C transitions. The ratios for 2-aminopurine deoxyribonucleoside triphosphate/dATP pools are 5-8% for tsL56 mutator and 1-5% for tsL141 antimutator and 43+ alleles. We conclude that the significant differences observed in the frequencies of induced transition mutations in the three alleles can be attributed primarily to the properties of the DNA polymerases with their associated 3'-exonuclease activities in controlling the frequency of 2-aminopurine.cystosine base mispairs. 相似文献
7.
Mark A. Bernard Nancy B. Ray Michael C. Olcott Stephen P. Hendricks Christopher K. Mathews 《Journal of bioenergetics and biomembranes》2000,32(3):259-267
This article summarizes research from our laboratory on two aspects of the biochemistry ofnucleoside diphosphate kinase from Escherichia coli—first, its interactions with several T4bacteriophage-coded enzymes, as part of a multienzyme complex for deoxyribonucleosidetriphosphate biosynthesis. We identify some of the specific interactions and discuss whetherthe complex is linked physically or functionally with the T4 DNA replicationmachinery, orreplisome. Second, we discuss phenotypes of an E. coli mutant strain carrying a targeteddeletion of ndk, the structural gene for nucleoside diphosphate kinase. How do bacteria lackingthis essential housekeeping enzyme synthesize nucleoside triphosphates? In view of the specificinteractions of nucleoside diphosphate kinase with T4 enzymes of DNA metabolism, howdoes T4 multiply after infection of this host? Finally, the ndk disruption strain has highlybiased nucleoside triphosphate pools, including elevations of the CTP and dCTP pools of7- and 23-fold, respectively. Accompanied by these biased nucleotide pools is a strong mutatorphenotype. What is the biochemical basis for the pool abnormalities and what are the mutagenicmechanisms? We conclude with brief references to related work in other laboratories. 相似文献
8.
Quantification of the effects on viral DNA synthesis of reverse transcriptase mutations conferring human immunodeficiency virus type 1 resistance to nucleoside analogues
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Bouchonnet F Dam E Mammano F de Soultrait V Henneré G Benech H Clavel F Hance AJ 《Journal of virology》2005,79(2):812-822
9.
Relations between synthesis of deoxyribonucleotides and DNA replication in 3T6 fibroblasts 总被引:4,自引:0,他引:4
In exponentially growing 3T6 cells, the synthesis of deoxythymidine triphosphate (dTTP) is balanced by its utilization for DNA replication, with a turnover of the dTTP pool of around 5 min. We now investigate the effects of two inhibitors of DNA synthesis (aphidicolin and hydroxyurea) on the synthesis and degradation of pyrimidine deoxynucleoside triphosphates (dNTPs). Complete inhibition of DNA replication with aphidicolin did not decrease the turnover of pyrimidine dNTP pools labeled from the corresponding [3H]deoxynucleosides, only partially inhibited the in situ activity of thymidylate synthetase and resulted in excretion into the medium of thymidine derived from breakdown of dTTP synthesized de novo. These data demonstrate continued synthesis of dTTP in the absence of DNA replication. In contrast, hydroxyurea decreased the turnover of pyrimidine dNTP pools 5-50-fold. Hydroxyurea is an inhibitor of ribonucleotide reductase and stops DNA synthesis by depleting cells of purine dNTPs but not pyrimidine dNTPs. Our results suggest that degradation of dNTPs is turned off by an unknown mechanism when de novo synthesis is blocked. 相似文献
10.
Mutant cells lines of 3T6 mouse fibroblasts, resistant to thymidine and deoxyadenosine, have an altered allosteric regulation of the enzyme ribonucleotide reductase (Meuth, M. and Green, H., Cell, 3, 367, 1974). Compared to 3T6, these lines contain larger pools of deoxynucleoside triphosphates, in particular deoxycytidine triphosphate, but show a normal rate of DNA synthesis. Addition of thymidine or deoxyadenosine to 3T6 cells results in large accumulations of the corresponding triphosphates and a dramatic decrease in the dCTP pool, concomitant with inhibition of DNA synthesis. Addition of thymidine to the mutant cell lines also leads to an increase in the dTTP pool but does not result in a depletion of dCTP or inhibition of DNA synthesis. Addition of deoxyadenosine only leads to a small increase of the dATP pool. In general the change in the allosteric regulation of bibonucleotide reductase is reflected in the deoxynucleotide pools. 相似文献
11.
The accumulation of RNA and protein and the kinetics of nucleoside triphosphate and guanosine polyphosphate pools during amino acid starvation and carbon source downshift were investigated in Streptomyces hygroscopicus. RNA accumulation was controlled stringently during both amino acid starvation and carbon source downshift. The pool size of ppGpp increased dramatically under these conditions. However, the intracellular concentrations of nucleoside triphosphates were low and the concentration of guanosine polyphosphates was much lower than in Escherichia coli. The possible significance of this phenomenon in the regulation is discussed. 相似文献
12.
A method for the enzymatic synthesis and purification of [alpha-32P] nucleoside triphosphates. 总被引:1,自引:1,他引:0
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A simplified method is described for the enzymatic synthesis and purification of [alpha-32P]ribo- and deoxyribonucleoside triphosphates. The products are obtained at greater than 97% radiochemical purity with yields of 50--70% (relative to 32Pi) by a two-step elution from DEAE-Sephadex. All reactions are done in one vessel as there is no need for intermediate product purifications. This method is therefore suitable for the synthesis of these radioactive compounds on a relatively large scale. The sequential steps of the method involve first the synthesis of [gamma-32P]ATP and the subsequent phosphorylation of nucleoside 3' monophosphate with T4 polynucleotide kinase to yield nucleoside 3', [5'-32P]diphosphate. Hexokinase is used after the T4 reaction to remove any remaining [gamma-32P]ATP. Nucleoside 3',[5'-32P]diphosphate is treated with nuclease P-1 to produce the nucleoside [5'-32P]monophosphate which is phosphorylated to the [alpha-32P]nucleoside triphosphate with pyruvate kinase and nucleoside monophosphate kinase. Adenosine triphosphate used as the phosphate donor for [alpha-32P]deoxynucleoside triphosphate syntheses is readily removed in a second purification step involving affinity chromatography on boronate-polyacrylamide. [alpha-32P]Ribonucleoside triphosphates can be similarly purified when deoxyadenosine triphosphate is used as the phosphate donor. 相似文献
13.
Shishir K. Das Earl P. Benditt Lawrence A. Loeb 《Biochemical and biophysical research communications》1983,114(2):458-464
In this communication we describe the rapid increase in cellular deoxynucleoside triphosphate (dNTP) concentrations in Chinese Hamster cell line V79 after exposure to known mutagens. With this cell line an expansion of dATP and dTTP pools was detected; changes in dCTP were not large; changes in dGTP were either not significant or too low to quantitate. This situation may reflect the existence of imbalances in dNTP pools at the DNA replication fork. The expansion of dATP and dTTP pools occurred within 2 to 4 hours after exposure of cultured cells to N-methyl-N′-nitro-N-nitrosoguanidine (MNNG). Ultraviolet light (UV), mitomycin C, and cytosine arabinoside also caused similar dNTP pool changes. 相似文献
14.
Alterations of deoxyribonucleoside triphosphate pools in Escherichia coli: effects on deoxyribonucleic acid replication and evidence for compartmentation.
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M L Pato 《Journal of bacteriology》1979,140(2):518-524
Inhibition of ribonucleic acid synthesis in Escherichia coli 15 TAU bar with rifampin or streptolydigin leads to large increases in the sizes of cellular ribonucleoside and deoxyribonucleoside triphosphate pools. Inhibition of protein synthesis leads to increases in the sizes of all nucleoside triphosphate pools except the guanosine triphosphate and deoxyguanosine triphosphate pools; a decrease in the size of the latter pool may be responsible for the slowing of deoxyribonucleic acid replication fork movement observed in this strain in the absence of protein synthesis. Analysis of the kinetics of incorporation of labeled precursors into deoxyribonucleic acid and into cellular pools suggests that functional compartmentation of nucleotide pools exists, allowing the incorporation of exogenously supplied precursors into deoxyribonucleic acid without prior equilibration with the cellular pools. 相似文献
15.
The accuracy of DNA synthesis depends on the accuracy of the polymerase as well as the quality and concentration(s) of the available 5′-deoxynucleoside-triphosphate DNA precursors (dNTPs). The relationships between dNTPs and error rates have been studied in vitro, but only limited insights exist into these correlations during in vivo replication. We have investigated this issue in the bacterium Escherichia coli by analyzing the mutational properties of dcd and ndk strains. These strains, defective in dCTP deaminase and nucleoside diphosphate kinase, respectively, are characterized by both disturbances of dNTP pools and a mutator phenotype. ndk strains have been studied before, but were included in this study, as controversies exist regarding the source of its mutator phenotype. We show that dcd strains suffer from increased intracellular levels of dCTP (4-fold) and reduced levels of dGTP (2-fold), while displaying, as measured using a set of lacZ reversion markers in a mismatch-repair defective (mutL) background, a strong mutator effect for G·C→T·A and A·T→T·A transversions (27- and 42-fold enhancement, respectively). In contrast, ndk strains possess a lowered dATP level (4-fold) and modestly enhanced dCTP level (2-fold), while its mutator effect is specific for just the A·T→T·A transversions. The two strains also display differential mutability for rifampicin-resistant mutants. Overall, our analysis reveals for both strains a satisfactory correlation between dNTP pool alterations and the replication error rates, and also suggests that a minimal explanation for the ndk mutator does not require assumptions beyond the predicted effect of the dNTP pools. 相似文献
16.
We have generated mutator strains of Bacillus anthracis Sterne by using directed gene knockouts to investigate the effect of deleting genes involved in mismatch repair, oxidative repair, and maintaining triphosphate pools. The single-knockout strains are deleted for mutS, mutY, mutM, or ndk. We also made double-knockout strains that are mutS ndk or mutY mutM. We have measured the levels of mutations in the rpoB gene that lead to the Rif(r) phenotype and have examined the mutational specificity. In addition, we examined the mutational specificity of two mutagens, 5-azacytidine and N-methyl-N'-nitro-N-nitroso-guanidine. The mutY and mutM single knockouts are weak mutators by themselves, but the combination of mutY mutM results in very high mutation rates, all due to G:C --> T:A transversions. The situation parallels that seen in Escherichia coli. Also, mutS knockouts are strong mutators and even stronger in the presence of a deletion of ndk. The number of sites in rpoB that can result in the Rif(r) phenotype by single-base substitution is more limited than in certain other bacteria, such as E. coli and Deinococcus radiodurans, although the average mutation rate per mutational site is roughly comparable. Hotspots at sites with virtually identical surrounding sequences are organism specific. 相似文献
17.
Hydroxyurea (HU) causes inhibition of DNA synthesis in regenerating rat liver due to an inhibition of the ribonucleotide reductase. We studied the consequences of a continuous HU infusion for deoxyribonucleoside triphosphate (dNTP) pools in the liver after partial hepatectomy and tried to modify imbalances by application of deoxyribonucleosides in vivo. In normal liver, an intracellular concentration of 0.16, 0.84, 0.33 and 0.27 pmol/micrograms DNA was observed for dATP, dCTP, dGTP and dTTP, respectively. In regenerating liver the dNTP pools show minor changes until 18 h after partial hepatectomy. During and after a continuous HU infusion 14--24 h after partial hepatectomy, the intracellular dNTP pools change considerably. At 19.5 h after partial hepatectomy, 5.5 h after the start of HU infusion, and at 25 h after partial hepatectomy, 1 h after termination of HU infusion, the dTTP pool was more than 10-times, and the dGTP pool about 2-times higher than in controls, while the dATP and dCTP pools remain relatively unchanged. Simultaneous infusion of HU and deoxythymidine (dThd) 14--25 h after partial hepatectomy results in a further increase of the dTTP pool during and after HU infusion. Administration of deoxycytidine (dCyd) leads to a moderate increase of the dCTP pool and a weak decrease of the dTTP pool during HU infusion. The combined application of dCyd and dThd after HU infusion had similar effects on dNTP pools as observed with dThd alone. These results show that intracellular pools of dNTPs in hepatocytes can be altered by exogenous factors in a controlled pattern. This system can be used as a model for studying the implications of induced dNTP pool dysbalances for the initiation of liver carcinogenesis by mutagenic chemicals. 相似文献
18.
Induction of mutations in Drosophila melanogaster gypsy retroelements by modulation of intracellular deoxynucleoside triphosphate pools in vivo
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The retroviral mutation rate is susceptible to a number of variables, including the balance between intracellular deoxynucleoside triphosphate (dNTP) pools. While this follows from tissue culture studies, the issue has never been addressed directly in vivo. To explore this question in a tractable experimental system, we analyzed the impact of thymidine treatment on the synthesis of gypsy retroelement cDNA from Drosophila melanogaster during development through to hatching. The mutation frequency was enhanced approximately 16-fold over the levels seen in the experimental background. Due to the lack of proofreading, these gypsy elements represent hypervariable loci within the Drosophila genome, suggesting that dNTP pool imbalances in vivo are mutagenic. 相似文献
19.
2'-Azidocytidine is a specific inhibitor of DNA synthesis in cultured 3T6 mouse fibroblasts. Earlier work (Akerblom, L., Pontis, E., and Reichard, P. (1982) 257, 6776-6782) indicated that the nucleoside, after phosphorylation, acted by inhibiting both ribonucleotide reduction and DNA strand elongation. We now demonstrate that the effect on strand elongation was due to a contamination of azidocytidine with less than 0.3% of arabinosyl cytosine. Pure azidocytidine inhibits specifically ribonucleotide reductase and its effects on DNA synthesis are secondary to this inhibition. The results with azidocytidine concerning the size and turnover of deoxyribonucleoside triphosphate pools parallel those of hydroxyurea and are distinct from those of arabinosyl cytosine. Together with hydroxyurea, azidocytidine is a useful compound in studies aiming at a specific block of the production of deoxynucleoside triphosphates in intact cells. Comparisons of the effects of azidocytidine and arabinosyl cytosine complement earlier studies with hydroxyurea and aphidicolin concerning inter-relations between dNTP synthesis and DNA replication. 相似文献
20.
Detection of activities that interfere with the enzymatic assay of deoxyribonucleoside 5'-triphosphates 总被引:15,自引:0,他引:15
Several enzymes that interfere with the enzymatic assay of deoxyribonucleoside 5'-triphosphates (dNTP's) are present as contaminants when nucleotides are extracted from HeLa cells with 60% methanol. These activities include a nuclease, nucleoside diphosphokinase, and deoxyribonucleoside monophosphokinases which phosphorylate dAMP, dGMP, and dCMP. Collectively, these enzymes are able to degrade and reutilize the DNA template which is used together with DNA polymerase for dNTP assays. This process introduces large errors when dNTP assays are performed in this manner. Attempts to block the enzymatic conversion of deoxyribonucleoside diphosphates to triphosphates by inhibition of nucleoside diphosphokinase were unsuccessful because of the inability to block completely the kinase activity. Acid extraction of nucleotides also results in the presence of an activity that interferes with the enzymatic dNTP assay. The error introduced by this interfering activity is much smaller than that arising from the enzymes present in methanol extracts. All of these interfering activities are removed when cells are first extracted with 60% methanol and the resulting extract is subsequently treated with perchloric acid. 相似文献