Effect of ATP analogues on T4 polynucleotide ligase.
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Adenosine Triphosphate↗
Biomedical subjects
K Kleppe
Publications and source records attributed to K Kleppe.
Effect of caffeine on DNA polymerase I from Escherichia coli: studies in vitro and in vivo.
The influence of caffeine on the activity of DNA polymerase I from E. coli was investigated. Caffeine had no effect on the polymerizing activity but did inhibit both 5' leads to 3' and 3' leads to 5' nuclease activities. The highest inhibition was observed with d(A--T)n as substrate: at a concentration of caffeine of 10 mM, inhibition was about 50%. In studies in vivo with 3 isogenic strains of E. coli, carrying different mutations in the DNA polymerase I gene, the effect of caffeine on survival after ultraviolet irradiation was most marked for the wild-type, pol+, followed by those mutants defective in 3' leads to 5', polA1, and 5' leads to 3' nuclease activities, polA107. Caffeine had little influence on survival of the resA1 mutant which lacks both 5' leads to 3' and 3' leads to 5' nuclease activities. These results support the idea that the influence of caffeine on dark repair may be explained in part by its effect on the nuclease activities of DNA polymerase I.
Caffeine↗
Phosphorylation of tRNA by T4 polynucleotide kinase.
The phosphorylation of various intact tRNA species by T4 polynucleotide kinase has been studied. The apparent Michaelis constant was on the average found to be 100 times lower than for some single-stranded DNAs previously studied. (J.R. Lillehaug and K. Kleppe, (1975) Biochemistry, 14, 1221). Conditions which result in complete phosphorylation of different tRNA species have also been established. Studies on equilibrium constants and the reversibility of the reaction revealed that the phosphorylation reaction is not a true equilibrium reaction under the conditions used in this work.
Coliphages↗
Polyamine induced aggregation of DNA.
Polyamine induced aggregation of various DNAs has been studied under conditions usually employed in many enzymatic assays where DNA is one of the substrates. Spermine was by far the most efficient polyamine in causing aggregation followed by spermidine and cadaverine. All double-stranded and naturally occurring single-stranded DNAs were found to aggregate. No aggregation of single-stranded homodeoxypolymers could be detected under the same conditions. The concentration of polyamine at which the aggregation commenced was found to be a linear function of the DNA concentration. The slope of the curves depended on the nature of the polyamine, DNA the concentration of Mg++ and the ionic strength.
Cadaverine↗
Phosphorylation of double-stranded DNAs by T4 polynucleotide kinase.
The phosphorylation by T4 polynucleotide kinase of various double-stranded DNAs containing defined 5'-hydroxyl end group structures has been studied. Particular emphasis was placed on finding conditions that allow complete phosphorylation. The DNAs employed were homodeoxyoligonucleotides annealed on the corresponding homopolymers, DNA duplexes corresponding to parts of the genes for alanine yeast tRNA, and a suppressor tyrosine tRNA from Escherichia coli. The rate of phosphoylation of DNAs with 5'-hydroxyl groups in gaps was approximately ten times slower than for the corresponding single-stranded DNA. At low concentrations of ATP, 1 muM, incomplete phosphorylation was obtained, whereas with higher concentrations of ATP, 30 muM, complete phosphorylation was achieved. In the case of DNAs with 5'-hydroxyl groups at nicks approximately 30% phosphorylation could be detected using 30 muM ATP. A DNA containing protruding 5'-hydroxyl group ends was phosphorylated to completion using the same conditions as for single-stranded DNA, i.e., a ratio between the concentrations of ATP and 5'-hydroxyl groups of 5:1 and a concentration of ATP of approximately 1 muM. For a number of DNAs containing protruding 3'-hydroxyl group ends and one DNA containing even ends incomplete phosphorylation was found under similar conditions. For all these DNAs a plateau level was observed varying from 20 to 45% of complete phosphorylation. At 20 muM and higher ATP concentrations, the phosphorylation was complete also for these DNAs. With low concentrations of ATP a rapid production of inorganic phosphate was noted for all the latter DNAs. The apparent equilibrium constants for the forward and reverse reaction were determined for a number of different DNAs, and these data revealed that the plateau levels of phosphorylation obtained at low concentrations of ATP for DNAs with protruding 3'-hydroxyl group and even ends is not a true equilibrium resulting from the forward and reverse reaction. It is suggested that the plateau levels are due to formation of inactive enzyme-substrate and enzyme-product complexes. For all double-stranded DNAs tested, except DNAs containing protruding 5'-hydroxyl group ends, addition of KCl to the reaction mixture resulted in a drastic decrease in the rate of phosphorylation, as well as in the maximum level phosphorylated. Spermine, on the other hand, had little influence. Both of these agents have previously been shown to activate T4 polynucleotide kinase using single-stranded DNAs as substrates (Lillehaug, J.R., and Kleppe, K. (1975), Biochemistry 14, 1221). The inhibition of phosphorylation of double-stranded DNAs by salt might be the result of stabilization of the 5'-hydroxyl group regions of these DNAs.
Adenosine Triphosphate↗
Total synthesis of the structural gene for the precursor of a tyrosine suppressor transfer RNA from Escherichia coli. 10. Enzymatic joining of chemically synthesized segments to form the DNA duplex corresponding to the nucleotide sequence 86-126.
The polynucleotide ligase-catalyzed joining of the eight chemically synthesized deoxypolynucleotides (segments 19 to 26), comprising the nucleotide sequence 86-126 of the DNA corresponding to the Escherichia coli tyrosine tRNA precursor has been investigated. Joining was studied using various combinations of 3, 4, or larger number of segments at a time. The extent of joining was in general low (0 to 40%) for the three-component as well as for the four-component systems. Joining of the five- and six- component systems was more satisfactory with yields from 25 to about 60%. The three duplexes [IVa] to [IVc]were prepared in single step reactions in yields of about 50% and were characterized. Duplex [IVd] could not be prepared in a single step reaction because of the failure of 5'-phosphorylated segment 26 to join to the rest of the duplex. Using a carefully annealed mixture of segments 24, 25, and phosphorylated segment 26, the joining of the latter to segment 24 could be realized in about 25% yield, much activated intermediate being concurrently present.
Adenosine Triphosphate↗
Total synthesis of the structural gene for the precursor of a tyrosine suppressor transfer RNA from Escherichia coli. 11. Enzymatic joining to form the total DNA duplex.
The DNA duplex corresponding to the entire length (126 nucleotides) of the precursor for an Escherichia coli tyrosine tRNA has been synthesized. Duplex [I] (Sekiya, T., Besmer, P., Takeya, T., and Khorana, H. G.(1976) J. Biol. Chem. 251, 634-641), corresponding to the nucleotide sequence 1-26, containing single-stranded ends and carrying one appropriately labeled 5'-phosphate group, was joined to duplex [II] (Loewen, P. C., Miller, R. C., Panet, A., Sekiya, T., and Khorana, H. G. (1976) J. Biol. Chem. 251, 642-650) (nucleotide sequence 23-66 or 23-60) was phosphorylated with [gamma-33P]ATP at the 5'-OH ends. Duplex [III] (Panet, A., Kleppe, R., Kleppe, K., and Khorana, H. G. (1976) J. Biol. Chem. 251, 651-657) (nucleotide sequence 57-94 (Fig. 2)) was also phosphorylated at 5'-ends with [gamma-33P]ATP and was joined to duplex [IV] (Caruthers, M. H., Kleppe, R., Kleppe, K., and Khorana, H. G. (1976) J. Biol. Chem. 251, 658-666) (nucleotide sequence 90-126) which carried a 33P-labeled phosphate group on nucleotide 90. The joined product, duplex [III + IV] (nucleotide sequence 57-126) was characterized. The latter duplex was joined to the duplex [I + II] to give the total duplex. The latter contains singlestranded ends (nucleotides 1 to 6 and 121 to 126) which can either be "filled in" to produce the completely base-paired duplex or may be used to add the promoter and terminator regions at the appropriate ends.
Base Sequence↗
Total synthesis of the structural gene for the precursor of a tyrosine suppressor transfer RNA from Escherichia coli. 1. General introduction.
With the ultimate objective of the total synthesis of a tRNA gene including its transcriptional signals, an Escherichia coli tyrosine suppressor tRNA gene was chosen. The arguments in favor of this choice are presented. A plan for the total synthesis of the 126-nucleotide-long DNA duplex corresponding to a precursor (Altman S., and Smith, J. D. (1971) Nature New Biol. 233, 35) to the above tRNA is formulated. The plan involves: (a) the chemical synthesis of 26 deoxyribooligonucleotide segments, (b) polynucleotide ligase-catalyzed joining of several segments at a time to form a total of four DNA duplexes with appropriate comlementary single-stranded ends, and (c) the joining of the duplexes to form the entire DNA duplex. Ten accompanying papers describe the experimental realization of this objective.
Base Sequence↗
Total synthesis of the structural gene for the precursor of a tyrosine suppressor transfer RNA from Escherichia coli. 9. Enzymatic joining of chemically synthesized deoxyribopolynucleotide segments corresponding to nucleotide sequence 57-94.
The DNA duplexes representing nucleotide sequences 61-89 and 57-94 have been synthesized, isolated pure, and fully characterized. Synthesis of the duplex with the nucleotide sequence 61-89 involved the DNA ligase-catalyzed joining of chemically synthesized deoxyoligonucleotide segments 14 to 18 shown in Fig. 1A, while for the longer duplex (sequences 57-94) seven deoxyribooligonucleotides (segments 13 to 19, Fig. 1B) were used in one-step enzymatic joining. The joining of the short tetranucleotide (segment 16) to the segment 17 required the presence of the adjacent segment 14, even if the latter did not contain a 5'-phosphate group, to allow its joining to segment 16. However, in the synthesis of both of the DNA duplexes, the yields were comparatively low (30 to 40%) and could not be significantly increased although a variety of conditions was tried. The main cause in both cases evidently was the sluggish joining of segment 14 to 16 and of segment 16 to segment 17. Although the original plan for the total synthesis of this part of the gene for the tRNA precursor involved the DNA duplex consisting of segments 14 to 18, this duplex could not be quantitatively phosphorylated at the two 5'-OH ends for subsequent joining to the adjoining parts of the gene. The DNA duplex consisting of segments 13 to 19, which possesses both terminal 5'-OH groups at protruding single-stranded ends, was readily phosphorylated and used successfully in the total synthesis of the gene as described in an accompanying paper.
Base Sequence↗
Preparations and properties of ribonucleic acid polymerase from Acinetobacter calcoaceticus.
Deoxyribonucleic acid (DNA)-dependent ribonucleic acid (RNA) polymerase (EC 2.7.7.6) from Acinetobacter calcoaceticus was purified to apparent homogeneity and its properties were compared with those of the Escherichia coli B enzyme. The molecular weights of the two native active enzymes as well as their alpha and beta subunits appeared to be similar. No subunit corresponding to that of sigma from E. coli was found, and furthermore no separation between the beta subunits could be detected by gel electrophoresis. A number of different DNAs were transcribed by the enzyme from A. calcoaceticus. Maximal RNA synthesis occurred at pH 8.7, 10 mM Mg2+, or 0.3 mM Mn2+ and at a total ionic strength of 0.1. Higher ionic strengths led to increasing inhibition of transcription and at mu = 0.4 complete inhibition was observed. The mechanism of inhibition of salt was not related to the initiation event as observed with T4 core RNA polymerase (R.Kleppe, 1975). In an attempt to understand the mechanism of inhibition by salt, the effect of ionic strength on the sedimentation properties of the enzyme was investigated. At low ionic strength, enzyme species with sedimentation coefficients, s20,w, of 5.8S, 12.4S, and 19.3S were present. In buffers with higher ionic strengths the relative amounts of the 12.4S species decreased. It is suggested, therefore, that the inhibition of activity at higher salt concentrations is caused by a decrease in concentration of the active enzyme species.
Acinetobacter↗
Envelope-associated folded chromosomes for Escherichia coli: variations under different physiological conditions.
The folded chromosome of Escherichia coli has been investigated under various lysis and physiological conditions. A new gradient system was devised that allows excellent separation between unlysed cells and envelope-associated and envelope-free chromosomes. Isotope incorporation experiments showed that the fraction often called "membrane-bound nucleoids" contains cell wall in addition to nucleic acids, membranes, and proteins. The amount of lysozyme added and the lysozyme digestion time were found to be important when comparing the rate of sedimentation of envelope-associated chromosomes obtained under various physiological conditions. Amino acid-starved cells were found to be much harder to lyse with lysozyme than exponentially grown cells, The difference in sedimentation coefficient of envelope-associated chromosomes described earlier (Ryder and Smith, 1974) was not detected when the latter two types of cells had been given equivalent, but not identical, lysozyme treatment such that detergent-mediated lysis proceeded at the same rate. Analysis of pulse- and uniformly labeled chromosomes from amino acid-starved cultures revealed no preferential labeling of either envelope-associated or -released nucleoids. Nor was there a difference in sedimentation coefficient between uniform and pulse-labeled envelope-associated nucleoids. These results are in disagreement with the models for chromosome replication of Worcel and Burgi (1974) and Ryder and Smith (1974), respectively. Growing cells on carbon sources poorer than glucose demonstrated that the replicating chromosomes sediment faster than the bulk of envelope-associated nucleoids. The slower the growth rate, the greater this difference became. An alternative hypothesis regarding chromosome replication and its association with the cell envelope is presented.
Amino Acids↗
Kinetics and effect of salts and polyamines on T4 polynucleotide ligase.
The kinetics of T4 polynucleotide ligase has been investigated at pH 8,20 degrees C and using the double-stranded DNA substrate (dA)n - [(dT)10]n/10. Double-reciprocal plots of initial rates vs substrate concentrations as well as product inhibition studies have indicated that the enzyme reacts according to a ping-pong mechanism. The overall mechanism was found to be non-processive. The true Km for the DNA substrate was 0.6 muM and that of ATP 100 muM. Several attempts were made to reverse the T4 polynucleotide ligase joining reaction using 32-p-labelled (dA)n - [(DT)40]n/40 as substrate. No breakdown of this DNA could be detected. The joining reaction was inhibited by high concentrations, i.e. above approximately 70mM, of salts such as KCl, NaCl, NH4Cl and CsCl. At a concentration of 200 mM almost 100% inhibition was observed. Polyamines also caused inhibition of the enzyme, the most efficient inhibitor being spermine followed by spermidine. At a concentration of 1 mM spermine, virtually no joining took place. Addition of salts or polyamines resulted in a large increase in the apparent Km for the DNA substrate whereas the apparent Km for ATP remained unchanged. It is suggested that the affinity of the enzyme for the DNA substrate is decreased in the presence of inhibiting agents.
Adenosine Monophosphate↗
Kinetics and specificity of T4 polynucleotide kinase.
The kinetics of T4 polynucleotide kinase has been investigated at pH 8.0 and 37 degrees. Double reciprocal plots of initial rates vs. substrate concentrations as well as product inhibition studies have indicated that the enzyme reacts according to the ordered sequential mechanism shown in eq 2 in the text for phosphorylation of a DNA molecule. Based on this mechanism the rate equation for the overall reaction was deduced and the various kinetic constants estimated. Hill plots indicated little or no interaction between active sites in the enzyme. The apparent Michaelis constants and V-max were determined at a fixed ATP concentration, 66 muM, for a number of different substrates varying in chain length, base composition, and nature of the sugar, and a wide variation was found. For the nucleoside 3'-monophosphates tested both the apparent Michaelis constant and V-max values were from approximately 2 to 5 times larger than for the corresponding oligonucleotide. The following orders were obtained with regard to apparent Michaelis constants and V-max for the nucleoside 3'-monophosphates investigated: Michaelis constant, rGP greater than rUp greater than rCp greater than rAp greater than dTp; V-max, rGp greater than rCp greater than rAp greater than dTp greater than rUp. Somewhat similar results were also obtained with the deoxyoligonucleotides tested.
Binding, Competitive↗
Effect of salts and polyamines on T4 polynucleotide kinase.
The activity of T4 polynucleotide kinase (EC 2.7.1.78) was found to be greatly stimulated by salts, such as NaCl and KCl, and polyamines such as spermine and spermidine. Up to a sixfold increase in initial rates was observed with a variety of different single-stranded DNAs and mono- and oligonucleotides. The optimal concentrations of salts were 0.125 M, corresponding to a total ionic strength of mu equals 0.19. For polyamines the optimal concentrations were found to be at approximately 2 mM. With low enzyme concentration and in the absence of activators complete phosphorylation was not achieved for a number of substrates. In the presence of salts or polyamines or high concentration of enzyme the phosphorylation proceeded to completion. Addition of salt led to an increase in both the apparent V-max and the Michaelis constant for the DNA substrate whereas the Michaelis constant of ATP remained unchanged. Polyamines had a similar influence on the kinetic constants for the DNA substrate whereas a decrease was found for the apparent Michaelis constant for ATP. The overall mechanism in the presence of activators was found to be sequential but probably of a rapid equilibrium random type. Of the inorganic anions tested both P-i and PP-i inhibited the enzyme in a competitive manner with both substrates.
Coliphages↗
Preparation and separation of d(pT)-10-n oligonucleotides.
A series of oligomers having the general formula d(pT)-10-n, n varying from 2 to 20, has been prepared by enzymatic joining of d(pT)-10, annealed on poly dA, employing T-4 polynucleotide ligase. The oligomers could be separated on 8 or 12% polyacrylamide gels. Such oligomers may prove useful as molecular weight markers and initiators for various polymerases.
Coliphages↗
DNA polymerase I from Acinetobacter calcoaceticus: enzymatic properties.
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Acinetobacter↗
Multiple DNA polymerases in Acinetobacter calcoaceticus.
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Adenine Nucleotides↗
Effect of pH on incorporation of ribonucleotides into dna by DNA polymerase I.
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Adenosine Triphosphate↗