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PubMed1975

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↗

PubMed1979

The effect of antibiotics on the T4 polynucleotide ligase catalyzed template dependent polymerization of oligodeoxythymidylates.

The poly(dA) dependent T4 polynucleotide ligase catalyzed polymerization of oligodeoxythymidylates is dependent upon duplex stability. The antibiotics ethidium bromide, netropsin and Hoechst 33258 stabilize the duplex poly(dA) . P(dT)n (n = 6-10) to thermal denaturation. Ethidium bromide to DNA ratio of 1.25 and netropsin or Hoechst 33258 to DNA ratio of 0.1 the Tm of d(pT) 10 . poly (dA) was increased by 10 degrees and 25 degrees C respectively. The T4 polynucleotide ligase activity was not inhibited under these conditions and temperature optimum of joining of d(pT) 10 . poly(dA) was increased 5 degrees to 10 degrees by the binding of the antibiotics. Duplexes containing shorter oligodeoxythymidylates required lower concentrations of the antibiotics netropsin or Hoechst 33258 to show no inhibition of T4 polynucleotide ligase. The temperature optima of joining the duplexes d(pT)6 . POLY(DA) and d(pT) 8 . poly(dA) were increased by 5 degrees C upon binding of the antibiotics. Polyacrylamide gel analysis of the T4 polynucleotide ligase catalyzed joining of the oligodeoxythymidylates showed that the presence of antibiotics affected the product distribution of the polymerized oligomers.

Anti-Bacterial Agents↗

PubMed1978

T4 polynucleotide ligase catalyzed joining on triple-stranded nucleic acids.

dT1O will form triple-stranded complexes with dAn and these complexes can serve as substrate for T4 polynucleotide ligase (EC 6.5.1.1). The rate of phosphodiester formation was found to be approximately the same as for the double-stranded complex and, furthermore, the rate appears to be similar on the two strands in the complex. Joining of dT1O also took place in the presence of the double-stranded complexes dAn.dTn and dAn.rUn. Polyamines increase the rate of joining catalyzed by T4 polynucleotide ligase under certain conditions.

Coliphages↗

PubMed1976

Joining of simian virus 40 DNA molecules at endonuclease R Eco Ri sites by polynucleotide ligase and analysis of the products by agarose gel electrophoresis.

DNA molecules cut with endonuclease R Eco Ri can be joined at Eco Ri cleavage sites by incubation with polynucleotide ligase. In order to define the optimum conditions for this reaction, linear Simian Virus 40 DNA molecules (SV40(Lri)) produced by endonuclease R Eco Ri cleavage of SV40 form i DNA were joined using polynucleotide ligases specified by bacteriophage T4 and Escherichia coli. We have determined that the concentration of the substrate DNA molecules is the most important factor determining the distribution of covalently joined product molecules into a variety of circular and linear monomeric and oligomeric species.

DNA Restriction Enzymes↗

PubMed1978

The activity of mammalian polynucleotide ligase on x-irradiated DNAs.

Selected samples of heterogeneous DNA from calf thymus with similar number-average molecular weight, Mn, and a low incidence of single-strand breaks were exposed in aqueous solutions to a mild X-ray dose of 1500 rads. The irradiation produced on the average about 0.2 bihelical and 2.2 monohelical scissions per DNA molecule of 1708 000 Mn. The percent distribution of the chemical termini released at the radiation nicks of DNA was as follows: 64.0 OH, 9.0 PO4 and 27.0 unknowns at the 3' ends: 3.8 OH, 68.2 PO4 and 28.0 unknowns at the 5' ends. A nuclease-free polynucleotide ligase I purified about 3000-fold over the crude homogenate from calf thymus succeeded in rejoining 50% of the breaks in the X-irradiated DNA. The ability of the enzyme to close radiation nicks in DNA directly was confirmed also by experiments on synthetic poly(dA).poly([3H]dT),poly(dT)-cellulose substrates with an irradiated dT chain at either the 3' or the 5' side of the functional break. The poor discrimination of mammalian ligase versus nicked DNA containing radiation damage is of practical relevance. While rejoining altered nucleotide chains in the helices of DNA, the enzyme might contribute to the fixation of premutational lesions in the genetic material.

Animals↗

PubMed1977

DNA synthesis and degradation in UV-irradiated toluene treated cells of E. coli K12: the role of polynucleotide ligase.

Toluene treated cells have been used to study the processes of DNA synthesis and DNA degradation in ultra-violet irradiated Escherichia coli K12. Synthesis and degradation are both shown to occur extensively if polynucleotide ligase is inhibited, and to occur to a much lesser extent if ligase activity is optimal. Extensive UV-induced DNA synthesis in toluene-treated cells requires ATP for the initial incision step, and DNA polymerase I. Extensive degradation also depends on the early ATP-dependent incision step, and the subsequent degradation shows a partial requirement for ATP. Curtailment of degradation by ligase requires DNA polymerase activity, but is not dependent upon DNA polymerase I. Apparently this process can be carried out with equal facility by either DNA polymerase II or polymerase III. These observations suggest that extensive DNA polymerase I-dependent repair synthesis and extensive DNA degradation are facets of two divergent pathways of excision repair, both of which depend upon the early uvrABC determined ATP-dependent incision step.

DNA Repair↗

PubMed1978

Use of the T4 polynucleotide ligase in the joining of flush-ended DNA segments generated by restriction endonucleases.

Double-stranded DNA segments with completely base-paired ends were obtained by the action of various restriction endonucleases on phage and plasmid DNAs. These segments were joined covalently by the T4 polynucleotide ligase. The joining was monitored by the electron microscopy count of intramolecularly circularized segments. The highest extent of joining, close to 75%, was observed at 15-25 degrees C with the segments resulting from the action of the Bacillus subtilis (strain R) restriction endonuclease Bsu on the DNA of bacteriophage SPPI or of the plasmid pSC 101. The joining of double-stranded termini required about 10 times more enzyme than the short single-stranded termini produced by the Escherichia coli restriction endonuclease EcoRI. A shortened purification of the T4 ligase was found to give an enzyme devoid of interfacing nucleases.

Coliphages↗

PubMed1978

T4 polynucleotide ligase catalyzed joining of short synthetic DNA duplexes at base-paired ends.

The self-complementary octanucleotide dT-A-G-T-A-C-T-A has been synthesized and its sequence confirmed by two-dimensional fingerprinting. Under conditions used for the T4 polynucleotide ligase reaction, this oligonucleotide forms a dimeric duplex which shows a Tm of 18 degrees C. The optimal rate of joining of the 32P-labeled duplex occurs between 12 and 15 degrees C. The rate is highly concentration dependent, as expected for a bimolecular process. Polyacrylamide gel electrophoretic analysis of this reaction shows the presence of products up to 120 nucleotides in length. In a denaturing gel, each product appears as a double band due to the presence of its 5'-adenylylated activated intermediate. Substrates larger than eight base pairs are utilized more rapidly than the eight base pair duplex, indicating that the T4 ligase has a higher affinity for longer substrates. The low level of nicked intermediates suggests that the joining of both strands requires two steps, the rates of which must be similar.

Base Sequence↗

PubMed1976