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1.
《DNA Repair》2017
O6-Methylguanine (O6-MeG) is highly mutagenic and is commonly found in DNA exposed to methylating agents, generally leads to G:C to A:T mutagenesis. To study DNA replication encountering O6-MeG by the DNA polymerase (gp90) of P. aeruginosa phage PaP1, we analyzed steady-state and pre-steady-state kinetics of nucleotide incorporation opposite O6-MeG by gp90 exo−. O6-MeG partially inhibited full-length extension by gp90 exo−. O6-MeG greatly reduces dNTP incorporation efficiency, resulting in 67-fold preferential error-prone incorporation of dTTP than dCTP. Gp90 exo− extends beyond T:O6-MeG 2-fold more efficiently than C:O6-MeG. Incorporation of dCTP opposite G and incorporation of dCTP or dTTP opposite O6-MeG show fast burst phases. The pre-steady-state incorporation efficiency (kpol/Kd,dNTP) is decreased in the order of dCTP:G > dTTP:O6-MeG > dCTP:O6-MeG. The presence of O6-MeG at template does not affect the binding affinity of polymerase to DNA but it weakened their binding in the presence of dCTP and Mg2+. Misincorporation of dTTP opposite O6-MeG further weakens the binding affinity of polymerase to DNA. The priority of dTTP incorporation opposite O6-MeG is originated from the fact that dTTP can induce a faster conformational change step and a faster chemical step than dCTP. This study reveals that gp90 bypasses O6-MeG in an error-prone manner and provides further understanding in DNA replication encountering mutagenic alkylation DNA damage for P. aeruginosa phage PaP1. 相似文献
2.
Richard Y.-H. Wang Lan-Hsiang Huang Melanie Ehrlich 《Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression》1982,696(1):31-36
Bacteriophage XP-12-infected Xanthomonas oryzae have been found to be a source of a kinase preparation which converts m5dCMP to m5dCDP and then to m5dCTP using ATP as the phosphate donor. Optimal formation of the triphosphate required the presence of creatine phosphate and creatine kinase. In the presence of dGTP, dTTP and dATP, Escherichia coli DNA polymerase I and T4 DNA polymerase catalyzed the incorporation of m5dCTP into DNA just as efficiently as that of dCTP. Neither dTMP nor dCMP served as substrate for the m5dCMP monophosphate kinase. Analogous preparations from uninfected X. oryzae were unable to phosphorylate m5dCMP. 相似文献
3.
Requirements and optimal conditions have been studied for measurements of dGTP and dCTP in cellular extracts using the copolymer [d(1 ? C)] as primer in a reaction catalysed by the large fragment of DNA polymerase from E. coli. The pool size of dGTP and dCTP in the human lymphocytes in the absence of PHA was found to be about 0.1 and 0.15 pmoles/106 cells, respectively. After treatment with PHA the pool size of both deoxynucleotides increased. The pool size of dCTP reached a maximum after 67 h simultaneously with the peak value of labelled deoxythymidine incorporation into DNA and the variation in these two parameters was very similar. The variation in the dGTP pool, however, was not so distinctly related to deoxythymidine incorporation as in the dCTP pool, since the increase in the dGTP pool was very small from 52–67 h. During transformation the dGTP pool was found to be the smallest pool. The relative cellular content of mono-, di- and triphosphate esters of deoxyadenosine, deoxyguanosine and deoxycytidine was studied. 相似文献
4.
Base-exchange activity was contrasted to the usual phosphatidohydrolase activity of commercial phospholipase D preparation from cabbage. The former activity was assayed by measuring the incorporation of labeled ethanolamine and choline into phospholipids. The latter activity was assayed by measuring the formation of phosphatidic acid with radioactive phosphatidylcholine microdispersion as substrate. The pH optimum for the base-exchange activity was about 9.0, whereas the phosphatidohydrolase activity had a pH optimum around 5.6. The incorporation of ethanolamine and choline into phospholipid was dependent upon the amount of acceptor asolectin microdispersion present. The optimum concentration of Ca2+ in the base-exchange reaction was about 4 mm, whereas the optimum concentration for the phosphatidohydrolase activity was greater than 28 mm. The incorporation of ethanolamine into phospholipid was decreased 50% by heating the enzyme preparation at 50°C for about 10 min, whereas the choline incorporation decreased approximately 20% and the phosphatidohydrolase activity decreased by about 10% under these conditions.Hemicholinium-3 was found to be a noncompetitive inhibitor for the incorporation of both ethanolamine and choline into phospholipid with respective Ki, values of 1.25 × 10?3 and 2.50 × 10?3m. The Km values for ethanolamine and choline in the base-exchange reaction were 1.25 × 10?3 and 2.50 × 10?3m, respectively. The apparent Km for phosphatidylcholine for the phosphatidohydrolase activity was about 1.5 × 10?3m, and there was no inhibition by hemicholinium-3. 相似文献
5.
Joon Ja Kim Richard Abrams James S. Franzen 《Archives of biochemistry and biophysics》1977,178(2):674-675
Ribonucleoside triphosphate reductase from Lactobacillus leichmannii, after reduction by exposure to dithiothreitol, has been alkylated with N-ethylmaleimide. Under conditions where the unreduced enzyme does not incorporate N-ethylmaleimide residues, the reduced enzyme is rapidly alkylated to the extent of one N-ethylmaleimide per molecule of enzyme. Loss of enzyme activity parallels the incorporation of N-ethylmaleimide. The value of the second-order rate constant for the alkylation at 0 °C of the reduced enzyme is influenced by the presence of some of the effectors of the enzyme, e.g., dATP at 200 μm reduces this parameter from 0.61 to 0.33 mm?1 min?1. The addition of coenzyme B12 did not significantly affect the rate of alkylation of the reduced enzyme nor did it change the rate of alkylation of the dATP-reduced enzyme complex. Reduced enzyme, freed of dithiol, was shown to be unable to convert CTP stoichiometrically to dCTP when all of the usual enzyme assay components, except the dithiol, were present, nor did addition of CTP to the otherwise complete mixture decrease the level of N-ethylmaleimide-reactive thiol. However, the subsequent addition of dithiol was found to result in essentially complete reduction of CTP to dCTP. Hence, although reduction of the enzyme is probably required to generate an active form of the enzyme, the reduced enzyme does not appear to be capable of transferring its reducing equivalents stoichiometrically to the substrate to form dCTP from CTP. These results are discussed in terms of the mechanism of action of this enzyme. 相似文献
6.
R S Tobin 《Life sciences》1974,14(5):957-965
Isolated nuclei of Ehrlich ascites tumor cells synthesize DNA in vitro using endogenous template and enzymes. Deoxycytidine nucleoside triphosphate (dCTP) is incorporated into acid-insoluble material to a much greater extent than the other substrates, even in the absence of the other triphosphates. Much of the [3H] dCTP is converted to [3H]CTP, some of which is incorporated into RNA, as evidenced by alkali-lability and density on cesium sulfate gradients. 相似文献
7.
The adenovirus priming protein pTP contributes to the kinetics of initiation of DNA replication
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Adenovirus (Ad) precursor terminal protein (pTP) in a complex with Ad DNA polymerase (pol) serves as a primer for Ad DNA replication. During initiation, pol covalently couples the first dCTP with Ser-580 of pTP. By using an in vitro reconstituted replication system comprised of purified proteins, we demonstrate that the conserved Asp-578 and Asp-582 residues of pTP, located close to Ser-580, are important for the initiation activity of the pTP/pol complex. In particular, the negative charge of Asp-578 is essential for this process. The introduced pTP mutations do not alter the binding capacity to DNA or polymerase, suggesting that the priming mechanism is affected. The Asp-578 or Asp-582 mutations increase the Km for dCTP incorporation, and higher dCTP concentrations or Mn2+ replacing Mg2+ partially relieve the initiation defect. Moreover, the kcat/Km values are reduced as a consequence of the pTP mutations. These observations demonstrate that pTP influences the catalytic activity of pol in initiation. Since both Asp residues are situated close to the pol active site during initiation, they may contribute to correct positioning of the OH group in Ser-580. Our results indicate that specific amino acids of the protein primer influence the ability of Ad5 DNA polymerase to initiate DNA replication. 相似文献
8.
Gerda Tyrsted 《Experimental cell research》1975,91(2)
Requirements and optimal conditions have been studied for measurements of dGTP and dCTP in cellular extracts using the copolymer [d(1 − C)] as primer in a reaction catalysed by the large fragment of DNA polymerase from E. coli. The pool size of dGTP and dCTP in the human lymphocytes in the absence of PHA was found to be about 0.1 and 0.15 pmoles/106 cells, respectively. After treatment with PHA the pool size of both deoxynucleotides increased. The pool size of dCTP reached a maximum after 67 h simultaneously with the peak value of labelled deoxythymidine incorporation into DNA and the variation in these two parameters was very similar. The variation in the dGTP pool, however, was not so distinctly related to deoxythymidine incorporation as in the dCTP pool, since the increase in the dGTP pool was very small from 52–67 h. During transformation the dGTP pool was found to be the smallest pool. The relative cellular content of mono-, di- and triphosphate esters of deoxyadenosine, deoxyguanosine and deoxycytidine was studied. 相似文献
9.
W C Claycomb 《Developmental biology》1977,61(2):245-251
[3H]dTMP incorporation into DNA of nuclei isolated from differentiating cardiac muscle of the rat has been characterized. Nuclei prepared at different times during the terminal phase of differentiation by a procedure not involving a detergent (Triton X-100) wash show a progressively diminished capacity to support in vitro [3H]dTMP incorporation; this diminution parallels the loss of DNA polymerase α from cardiac muscle. The rate of incorporation of [3H]dTMP into DNA of nuclei washed twice with 0.5% Triton X-100 does not correlate with the in vivo DNA synthetic activity. As determined by electron microscopy the Triton X-100 wash removes the outer nuclear membrane; the pellet obtained by centrifuging the Triton X-100 extract of these nuclei consists of circular membrane vesicles. The predominant DNA polymerase activity in these preparations was characterized using pH optimum, N-ethylmaleimide sensitivity, and correlation to in vivo DNA synthetic activity as criteria. DNA polymerase α activity predominated in the non-Triton X-100-extracted nuclei and in the outer nuclear membrane fraction; DNA polymerase β activity was the predominant activity observed in Triton X-100-extracted nuclei. These data emphasize that the procedure which is used to isolate nuclei from proliferating cells can greatly influence the nature of the DNA synthetic activity that is observed in vitro, suggest that DNA polymerase α is associated with the outer nuclear membrane, and add support to the idea that this enzyme is involved in eukaryotic DNA replication. 相似文献
10.
The model carcinogen N-2-acetylaminofluorene covalently binds to the C8 position of guanine to form two adducts, the N-(2′-deoxyguanosine-8-yl)-aminofluorene (G-AF) and the N-2-(2′-deoxyguanosine-8-yl)-acetylaminofluorene (G-AAF). Although they are chemically closely related, their biological effects are strongly different and they are processed by different damage tolerance pathways. G-AF is bypassed by replicative and high-fidelity polymerases, while specialized polymerases ensure synthesis past of G-AAF. We used the DNA polymerase I fragment of a Bacillus stearothermophilus strain as a model for a high-fidelity polymerase to study the kinetics of incorporation of deoxy-CTP (dCTP) opposite a single G-AF. Pre-steady-state kinetic experiments revealed a drastic reduction in dCTP incorporation performed by the G-AF-modified ternary complex. Two populations of these ternary complexes were identified: (i) a minor productive fraction (20%) that readily incorporates dCTP opposite the G-AF adduct with a rate similar to that measured for the adduct-free ternary complexes and (ii) a major fraction of unproductive complexes (80%) that slowly evolve into productive ones. In the light of structural data, we suggest that this slow rate reflects the translocation of the modified base within the active site, from the pre-insertion site into the insertion site. By making this translocation rate limiting, the G-AF lesion reveals a novel kinetic step occurring after dNTP binding and before chemistry. 相似文献
11.
12.
Kimball and Wilson1 reported that the arabinose analogue of cytidine (ara-C) inhibited DNA polymerase in a crude extract prepared from Ehrlich ascites cells. Furth and Cohen2 observed cytosine arabinoside triphosphate (ara-CTP) inhibited DNA polymerase in extracts from either calf thymus or bovine lymphosarcoma tissue, although these investigators3 had already found no effect of ara-CTP on DNA polymerase from Escherichia coli. The inhibition in both of these cases could be substantially reversed by dCTP; but incorporation of the arabinose nucleotide (ara-CMP) into DNA could not be unequivocally demonstrated. Graham and Whitmore4 reported the incorporation of ara-C into DNA in vivo and the inhibition of a DNA polymerase from L cells by ara-CTP. They found that ara-CMP was initially incorporated into small DNA strands but subsequently appeared in long strands. Momparler5 has presented evidence that, in vitro, ara-C incorporation was limited to the 3′-hydroxyl end of DNA chains. Such incorporation might be expected to block further chain elongation but this expectation was not supported by the evidence presented by Graham and Whitmore. 相似文献
13.
Partial purification and properties of CTP:phosphatidic acid cytidylyltransferase from membranes of Escherichia coli.
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The cytosine liponucleotides CDP-diglyceride and dCDP-diglyceride are key intermediates in phospholipid biosynthesis in Escherichia coli (C. R. H. Raetz and E. P. Kennedy, J. Biol. Chem. 248:1098--1105, 1973). The enzyme responsible for their synthesis, CTP:phosphatidic acid cytidylytransferase, was solubilized from the cell envelope by a differential extraction procedure involving the detergent digitonin and was purified about 70-fold (relative to cell-free extracts) in the presence of detergent. In studies of the heat stability of the enzyme, activity decayed slowly at 63 degrees C. Initial velocity kinetic experiments suggested a sequential, rather than ping-pong, reaction mechanism; isotopic exchange reaction studies supported this conclusion and indicated that inorganic pyrophosphate is released before CDP-diglyceride in the reaction sequence. The enzyme utilized both CTP and dCTP as nucleotide substrate for the synthesis of CDP-diglyceride and dCDP-diglyceride, respectively. No distinction was observed between CTP and dCTP utilization in any of the purification, heat stability, and reaction mechanism studies. In addition, CTP and dCTP were competitive substrates for the partially purified enzyme. It therefore appears that a single enzyme catalyzes synthesis of both CDP-diglyceride and dCDP-diglyceride in E. coli. The enzyme also catalyzes a pyrophosphorolysis of CDP-diglyceride, i.e., the reverse of its physiologically important catalysis. 相似文献
14.
The influence of Cu2+ ions (in the form of CuCl2) in the concentration range 10?3 to 10?6 M on the content and biosynthesis of indole glucosinolates glucobrassicin and neoglucobrassicin has been studied on etiolated seedlings of rape (Brassica napus var.arvensis (Lam.) Thell.). Ions Cu2+ acted on the seedlings either chronically from the beginning of the germination or acutely, during 3 to 72 h, on seven days old seedlings. The biosynthesis of both glucosinolates was followed by the incorporation of35S from Na2 35SO4 into them in hypocotyl segments from seven days old intact etiolated seedlings. After the entry of small amounts of Cu2+ ions into the plants, stimulation of the glucosinolates formation occurs, as was found after three h action of Cu2+ ions. After the entry of a greater amount of Cu2+ ions into the plant, harmful effects appear, as was found after chronic two days action or after 24 and 48 hours acute action of Cu2+ ions. Later further stimulation of glucosinolate formation occurs, probably due to enhanced metabolism during reparation processes, as was manifested after chronic action of Cu2+ ions lasting four and eight days. The optimal effect of copper was found mainly in the concentration range 5×10?4 M to 10?5 M. Ions Cu2+ in higher concentration increased the uptake of sulphate ions by hypocotyl segments, and in lower concentrations increased the incorporation of35S from35SO4 2? into the proteins. 相似文献
15.
Rat liver nuclei incubated in vitro catalyze a sustained incorporation of32Pi into polyphosphate. A preliminary estimate indicates a minimal rate of 10 moles of Pi incorporation into polyphosphates/h/mg protein. Polyphosphate is the predominant acid-insoluble product of nuclear phosphorylation; its formation is dependent on the presence of a divalent cation and is catalyzed by a system or systems as yet uncharacterized. 相似文献
16.
Repair of heteroduplex DNA containing an A/G mismatch in a mutL background requires the Escherichia coli mutY gene function. The mutY-dependent in vitro repair of A/G mismatches is accompanied by repair DNA synthesis on the DNA strand bearing mispaired adenines. The size of the mufY-dependent repair tract was measured by the specific incorporation of α-[32P]dCTP into different restriction fragments of the repaired DNA. The repair tract is shorter than 12 nucleotides and longer than 5 nucleotides and is localized to the 3′ side of the mismatched adenine. This repair synthesis is carried out by DNA polymerase I. 相似文献
17.
《Biochimica et Biophysica Acta (BBA)/Lipids and Lipid Metabolism》1990,1042(1):62-69
Rainbow trout leucocytes contain high levels of neutral lipid (about 70% of total lipid on a wt% basis) consisting of mostly triacylglycerol, free sterols and sterol esters (25%, 15% and 52% of neutral lipid, respectively). The phospholipids, separated by thin-layer chromatography, consisted predominantly of phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine, each present at about 30% of the total phospholipid. Radiolabelling of the leucocytes for 1 h with 1 μCi (approx. 6 μM) [1−14C]20:4(n−6), [1−14C]20:5(n−3) or [1−14C]22:6(n−3) each gave similar uptake values (approx. 1 · 105 cpm/107 leucocytes). The incorporation into total phospholipids was highest for 22:6(n−3) and lowest for 20:4(n−6). A higher percentage of radiolabel from [1−14C]22:6(n − 3) was found incorporated into phosphatidylcholine and phosphatidylethanolamine as compared to that from [1−14C]20:4(n − 6) and [1−14C]20:5(n−3), while the reverse situation was found with phosphatidylinositol and phosphatidylserine. The relative rates of incorporation into the different phospholipid classes for all three fatty acids were in the order phosphatidylinositol > sphingomyelin > diphosphatidylglycerol > phosphatidylcholine > phosphatidylethanolamine > phosphatidylserine. Calcium ionophore-challenge did not significantly alter the pattern of phospholipid radiolabel. Ionophore-challenge released large amounts of radiolabel, much of which was recovered after high-performance liquid chromatographic separation as free fatty acid/monohydroxy fatty acids, although only approx. 0.3% was recovered in leukotriene B4 and leukotriene B5 for the [1−14C]20:4(n−6) and [1−14C]20:5(n−3) labelled leucocytes, respectively. Other lipoxygenase products were also radiolabelled and tentatively identified as 20-carboxy-LTB4, 20-hydroxy-LTB4, 6-trans-LTB4, 6-trans-12-epi-LTB4, 6-trans-8-cis-12-epi-LTB4 and the corresponding LTB5 structures. No ‘6-series’ leukotrienes were produced from [1−14C]22:6(n−3), nor was there any evidence for the synthesis of ‘5-series’ leukotrienes via retroconversion of 22:6(n−3) to 20:5(n−3). This latter finding shows that, despite the preponderance of 22:6(n−3) in the membranes of trout leucocytes, this fatty acid is not a substrate for leukotriene generation. 相似文献
18.
Deoxycytidylate deaminase. Properties of the enzyme from cultured kidney cells of baby hamster 总被引:4,自引:3,他引:1
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dCMP deaminase was partially purified from BHK-21/C13 cells grown in culture. The molecular weight of the enzyme was estimated by gel filtration and gradient centrifugation to be 130000 and 115000 respectively. The enzyme had a pH optimum of 8.4. Its activity versus substrate concentration curve was sigmoid, the substrate concentration at half-maximal velocity being 4.4mm. dCTP activated the deaminase maximally at 40μm, gave a hyperbolic curve for activity versus dCMP concentration and a Km value for dCMP of 0.91mm. dCTP activation required the presence of Mg2+ or Mn2+ ions. dTTP inhibited the deaminase maximally at 15μm; the inhibition required the presence of Mg2+ or Mn2+ ions. The enzyme was very heat-labile but could be markedly stabilized by dCTP at 0.125mm and ethylene glycol at 20% (v/v). 相似文献
19.
《Analytical biochemistry》1987,163(2):537-545
Two versions of an approach to identify DNA-protein interactions at sites of DNA replication in HeLa cell nuclei are described. In this procedure, newly replicated DNA chains are first labeled and photosensitized in vitro by the incorporation of [α-32P]dCTP and bromodeoxyuridine triphosphate, respectively. Irradiation with ultraviolet light is then used to covalently crosslink the proteins that are adjacent to the photosensitized and isotopically labeled strands of newly replicated DNA. After the bulk of the DNA is digested with nucleases, the crosslinked proteins—marked by short covalently linked radioactive DNA tags—are fractionated by electrophoresis in sodium dodecyl sulfate-polyacrylamide gels and detected by autoradiography. With this technology, certain proteins have been shown to associate selectively with newly replicated DNA. The method appears adaptable for application to a variety of problems involving DNA-protein association. 相似文献
20.
Effects of Sulfite on Metabolism in Isolated Mesophyll Cells from Papaver somniferum 总被引:3,自引:3,他引:0
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Exposure (30 minutes) of leaf-free mesophyll cells from the C-3 plant, Papaver somniferum, to concentrations of sulfite (SO2 + HSO3− + SO3−) up to 20 millimolar stimulated the rate of CO2 incorporation as much as 30%. The sulfite rapidly affects the metabolism of newly incorporated CO2. Ammonia incorporation into glutamine and subsequent transamination reactions were stimulated during the short term exposure periods while glycolate metabolism apparently was inhibited by bisulfite at two points in the pathway. The results further indicate that glycolate is the major precursor of glycine in these cells. Prolonged periods of exposure (24 hours) to sulfite had somewhat different effects on carbon metabolism: the high concentrations (10 to 20 millimolar) severely inhibited all aspects of cellular metabolism while lower concentrations (1 millimolar) appeared to inhibit ammonia incorporation but stimulated synthesis of sucrose and starch. 相似文献