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Log in to enjoy additional benefits Want to save this information? An account with takarabio. A 34 nt double-stranded oligonucleotide was produced by the annealing of three complementary oligonucleotides as schematically reported at the bottom of the figure. Ligation is revealed, after autoradiography of the dried gel, by the appearance of a 34mer resulting from the joining of oligos a and b.
The position of the adenylated substrate is indicated by an arrowhead. The same double-stranded oligonucleotide shown in Figure 3 was used in band shift assays as described in Materials and Methods.
Lanes 1—6, 0. The position of the retarded band is indicated by an arrow, while free oligo is indicated by an arrowhead. All the mutants were challenged in both nick joining and blunt-end ligation assays. As a substrate in the nick joining reaction we used 32 P-labelled oligo dT 16 annealed to poly dA.
The remaining two mutants were able to carry out nick joining, although to a lower extent than expected from their reduced ability to perform the self-adenylation step. It is worth noting that the ligation assay appeared to amplify the differences already detected by the self-adenylation assay, just as other properties overall protein structure or affinity for the substrate contributed to the final joining activity.
Their inability to carry out blunt-end ligation could correlate with their inability to form a stable complex with the substrate. In order to better characterize this activity we analysed all the mutants in a DNA relaxation assay. This was unexpected where the N-terminal mutant was concerned, since it showed detectable self-adenylation and nick joining activity. Differing from the wild-type protein Fig. Instead, after either prolonged incubation not shown or in the presence of high levels of protein Fig.
The nicked DNA obtained as a final product was a good substrate for wild-type DNA ligase, indicating that ligatable ends were produced Fig. In this paper we report a functional characterization of T4 DNA ligase carried out by means of His-tagged versions of the wild-type and of four mutated enzymes Table 1. In the last, Lys in the active site, known to be covalently bound to AMP during the first step of the reaction 22 , was substituted by a leucine. Several interesting conclusions emerge from this analysis.
First, T4 DNA ligase seems to differ from the T7 enzyme, recently characterized at the structural level 12 , with regard to the DNA binding properties. The failure of the T4 ligase mutants to form a stable complex with DNA appears to correlate with their inability to perform blunt-end ligation. Altogether, these results suggest that the mode of binding of the enzyme to DNA plays a central role in the joining reaction.
DNA relaxation activity of the different His-tagged proteins. Lane 1, substrate; lanes 2 and 3, 0. Lane 1, supercoiled pUC In addition we have investigated whether enzyme adenylation could influence the stability of the ligase-DNA complex. For this analysis we used a band shift assay in which a double-stranded oligonucleotide containing a ligatable nick on one strand was used as substrate. On the basis of these observations we propose a three step model to explain the mode of action of DNA ligase.
During this phase the nucleotide binding pocket is probably still occupied by the AMP molecule, although no longer covalently bound to the K residue. This model offers a new mechanistic viewpoint to explain blunt-end ligation.
This complex, the real intermediate of the reaction, would survive long enough to allow contact with another DNA fragment and hence completion of ligation. On the basis of this we predict that low levels of ATP would increase the rate of blunt-end ligation. In agreement with our model, the efficiency of blunt-end ligation is significantly increased at an ATP concentration of 2. Rossi and A. Montecucco, unpublished observation.
Results of the functional characterization of the His-tagged versions of wild-type T4 DNA ligase and of four mutated enzymes. A three step model of the T4 DNA ligase activity. Taking into account these similarities and on the basis of the data presented here, a new role for DNA ligase during both DNA replication and DNA repair can be proposed. This model could be extrapolated to the related eukaryotic DNA ligases. This is a significant difference from the wild-type enzyme, which produces relaxed covalently close circular DNA molecules in a topoisomerase-like fashion 5.
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Sign In or Create an Account. Sign In. Advanced Search. Search Menu. Article Navigation. Close mobile search navigation Article Navigation. Volume Article Contents Abstract. Materials and Methods. Functional characterization of the T4 DNA ligase: a new insight into the mechanism of action. Rossella Rossi , Rossella Rossi. Nucleic Acids Research, 19 24 , At what temperature is T4 DNA ligase most active? Why do ligation reactions using T4 DNA ligase fail?
How does DNA ligase work? Gently mix the reaction and centrifuge briefly. Incubate the reactions. Heat-inactivate the ligation reactions. Why is heat inactivating T4 DNA ligase necessary? Ligation reactions fail due to these following possible reasons: 1. The presence of ligase inhibitors in the reactions To prevent contaminations of ligase inhibitors in the reactions, such as high salts or EDTA, make sure you purify your DNA insert and plasmid.
To find out how much insert DNA to add in a ligation reaction if you want the ratio to be , use the formula below: 3. ATP concentration in the buffer is too low If the ATP concentration in the ligation reaction is too low, the ligation will fail. The inactive enzyme The ligation reaction might also fail due to the inactive enzyme. Set up also a negative control with no addition of ligase. If your ligase is still active, you should observe a distinct band with a higher molecular weight for the reaction with the ligase compared to the one without the ligase.
T References Johnson, A. Related Articles. This particul Molecular Cloning: A Detailed Introduction. Molecular cloning is a primary procedure in contemporary biosciences.
This involves introducing a sp Since the early s, restriction enzymes have become an important part of cloning and many other a In molecular biology, agarose gel electrophoresis is a common method used for many applications, suc
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