Ligation can be described as the process of joining two nucleic acid fragments catalyzed by an enzyme. It finds its application in molecular cloning and recombinant DNA molecules. Formation of phosphodiester bonds between the 3′-hydroxyl of one DNA terminus with the 5′-phosphoryl of another. Ligation of RNA can be done similarly.Generally, a co-factor is generally involved in the reaction. ATP or NAD+ may act as co-factor.
First, DNA ligase reacts with ATP or NAD+ to form a ligase-AMP. This intermediate with the AMP is linked to the ε-amino group of lysine via a phosphoamide bond.The lysine group is present in the active site of the ligase. Transfer of adenylyl group the phosphate group happens in this step. The phosphate group is present at the 5′ end of a DNA chain. This step ends in the formation a DNA-adenylate complex. Finally, 3′-hydroxyl at the end of a DNA strand attaches to the activated 5′-phosphoryl group of another DNA molecule forms by a nucleophilic reaction. This step ends in a phosphodiester bond between the two DNA ends.
If there is any break in any strands of the double-stranded DNA it is efficiently repaired by the ligase. However, the complication of ligation arises due to the presence of two separate DNA ends.For ligase reaction to start the two ends should come together. When in ligation of sticky-ends or cohesive ends, the ligase enzyme effectively repairs two nicks of the DNA. But in the blunt end, ligation depends on the property on the two nicks coming together i.e. random collision. This makes the efficiency of the process lower of the two.
Factors affecting ligation
Any factor which can affect an enzyme-mediated reaction has an impact on ligase reaction. The factors include the following
- Reactant and enzyme concentration.
- The temperature at which the reaction happens.
- Period of incubation i.e., reaction time.
Additionally, the reactants in a ligase reaction are two different DNA molecules. These molecules should bond together to form a complete final product. During the product formation, there is a wide type of interaction in molecular level that takes place. They can be intermolecular and intramolecular. On top, there is a requirement of an annealing reaction to give a finished product.
The overall reaction of new phosphodiester bond formation happens in three steps:
- Enzyme adenylation
- Adenylyl transfer to DNA
- Nick sealing
Mg(2+) acts as a cofactor for this catalysis reaction. The efficiency of the reaction is directly proportional to the concentration of Mg(2+). In cases when the cofactor as the limiting factor, the nicking reaction is the rate-limiting reaction.therefore at high concentration of Mg(2+) the ligation efficiency is high. If the concentration of Mg(2+) is limited, the nick- sealing is the rate-limiting reaction of the process, The intermediate product adenylylated DNA accumulates in the solution until the availability of the cofactor.
The concentration of the reactants always affects the rate of the reaction. It limits the reaction in both cases of intermolecular or intra-molecular reaction. Simply, ligation is the attachment of DNA ends, but the reaction constitutes DNA fragments which itself has two fragments, in case the two DNA molecules ends is not compatible with each other, there is a high chance that the DNA can circularize to join to its own ends. If the concentration of DNA is high, the chance of one end of a DNA molecule meeting the end of another DNA, thereby forming an intermolecular ligation. In case of lower concentration of DNA, the chance of a DNA molecule meeting its own end increases, thereby increasing the chance of intramolecular interaction. So as a general rule the total DNA concentration should be less than 10 μg/ml.
When considering the overall setup of the reaction. The concentration of the DNA should be proportional to other factors. It is contributed by various factors like DNA fragments, their length, as well as buffer conditions are also factors. All these factors can affect whether intermolecular or intramolecular reactions are favored.
Condensing agents can also increase the concentration of DNA artificially. Condensing agents such as cobalt hexamine and biogenic polyamines such as spermidine, also crowding agents such as polyethylene glycol can be used to effectively increases the concentration of enzymes. Adding these additives can produce a more intermolecular reaction. They form linear concatemers which are suitable for transformation of plasmid DNA. This process is undesirable for plasmid ligation. Therefore for plasmid digestion, PEG is preferable because it promotes both intramolecular and intermolecular reaction.
The ligase concentration is directly proportional to the rate of ligation. As the efficiency of Blunt-end ligation is always much less efficient than sticky end ligation. Therefore a higher concentration of ligase should be used for blunt-end ligations. High DNA ligase concentration may be used in conjunction with PEG for a faster ligation.
In selecting the temperature of a ligation reaction two functions should be considered. Primarily the optimum activity temperature of the DNA ligase enzyme which is 37°C, secondly the melting temperature (Tm) of the DNA ends to be ligated. The base composition of the DNA overhand and the length determines the melting temperature. The base composition increases the Tm because of the hydrogen bonds. Since G-C base pair contains three hydrogen bonds and A-T base pairs contain two hydrogen bonds, the melting point increases with the high number of G-C bonds. The efficiency of a ligation reaction the ends of the DNA strand should be stably annealed. Generally, in ligation experiments, the Tm is much lower than 37°C. Different restriction enzymes can produce different ends so the optimal temperature can vary widely based on this property of restriction process.
Ligation can be affected by the ionic strength of the buffer. Type of the cations present can also influence the ligation reaction. For instance, if the amount of Na+is excess, will cause the DNA to become more rigid. This increases the likelihood of intermolecular ligation. High concentration of monovalent cation (>200 mM) almost completely inhibits ligation. The standard buffer used for ligation is to be designed to minimize ionic effects.
The methodology and materials required for a DNA-joining reaction are discussed below. The DNA- joining reaction is catalyzed by the Escherichia coli DNA ligase. Ammonium ions played an important factor the rate of ligase reaction.
- coli DNA ligase.
T4 polynucleotide ligase,
- coli alkaline phosphatase purified to remove endonuclease.
- coli DNA polymerase I and the “large” proteolytic fragment of this enzyme
Calf thymus terminal deoxynucleotidyl transferase.
Nucleotides and Substrates
Unlabeled deoxyribonucleoside triphosphates, NMN, and DPN.
ddTMP (Dideoxythymidine mono phosphate) and converted to the triphosphate (ddTTP).[4-3H]Nicotinamide-DPN (50 mCi per mmole). [g-32P]ATP (6 to 14 Ci per mmole). [a-32P]ddTTP was synthesized. [32P]AMP. [32P]DPN.
d(pA)5100 and d(pA)6900.[3H]d(pA-pT)n
The triethylammonium salt of [32P]AMP-morpholidate (0.39 pmole, 4 Ci per mmole) was condensed with the tributylammonium salt of d(pT)4 (0.16 pmole of oligomer) in 0.3 ml of anhydrous pyridine for 84 hours at 40o. The reaction mixture was taken to dryness, dissolved in 1.5 ml of 0.05 M Tris-HCl (pH 8.0), and treated with 90 mg of E. coli alkaline phosphatase for 2 hours at 37o. The A[32P]p-p(dT)1-d(pT)3 was then isolated by chromatography on a DEAE-Sephadex A-25 column (12 cm x 0.5 cm2, bicarbonate cycle equilibrated with H20) ; elution was with a 100-ml linear gradient of triethylammonium bicarbonate (pH 7.8, 0 to 1.2 M) at a flow rate of 7 ml per hour. The molar extinction coefficient of the adenylylated oligomer at 260 nm was 56,000 and the over-all yield relative to the starting d(pT)d was 40%. The A[32P]p-p(dT)1-d(pT)3 was extended to a number average chain length of 240 thymidylate residues using calf thymus terminal deoxynucleotidyltransferase. except that the concentration of dTTP was 3.1 mM, the enzyme concentration was 180 pg per ml, and incubation was at, 35” for 70 min. The reaction was terminated by adding EDTA to 10 mM and heating to 70” for 10 min. After removal of the denatured protein by centrifugation, the adenylylated polymer was purified by filtration through a Sephadex G-50 column (41 cm x 0.74 cm2) equilibrated with 0.01 M Tris-HCl (pH 8.0), 0.5 mM EDTA. Less than 1% of the 32P in the isolated polymer was sensitive to alkaline phosphatase. For purposes of comparison with the A[32P]p-p(dT)1-d(pT)240 [5’-32P]d(pT)240 was also prepared. (dT) 1-d(pT)3 was extended to a number average chain length of 240 thymidylate residues under exactly the same conditions as described above for the synthesis of the adenylated d(pT)240. The isolated polymer was then labeled with 32P at the 5’-terminus using [32P]ATP and polynucleotide kinase without prior phosphatase treatment.[5’-32P]d(pT)160 and (dT)1-d(pT)160 were prepared by methods very similar to those used for the synthesis of the d(pT)240 oligomers. (dT)1-d(pT)160 was terminated with a ddTMP residue by incubation with ddTTP and calf thymus terminal deoxynucleotidyltransferase. The essentially complete addition of dideoxythymidylate to the termini was demonstrated in two ways. First, parallel reactions using [32P]ddTTP showed that 0.94 mole of ddTMP was added per mole of the (dT)1-d(pT)160 oligomer. Secondly, when the terminated oligomer was annealed to a lo-fold excess of d(pA) 5100, the initial rate of polymerization of dTTP with the large fragment of E. coli DNA polymerase I (in the presence of an excess of enzyme over 3’ termini) was only 3% of t.hat observed with the oligomer lacking a dideoxythymidine terminus, thus indicating that at least 97% of the 3’ termini had received a ddTMP residue. d(pT)160- dd(pT)1 was prepared from (dT)1-d(pT)160-dd(pT)1, using polynucleotide kinase and unlabeled ATP. To verify that the 5’ termini had been phosphorylated, it was shown that they could not be further phosphorylated by [32 P]ATP in the presence of polynucleotide kinase unless they were pretreated with alkaline phosphatase.
- All reactions involving the E. coli DNA ligase were conducted at 30” in a buffer composed of 0.015 M Tris-HCl (pH 8.0), 4 mM MgClz, 1 mM EDTA, 100 pg per ml of BSA (this composition takes into account the contribution by the enzyme diluent).
- Substrates and monovalent cations were present at the indicated concentrations.
- Ammonium salts were diluted from stock solutions neutralized to pH 7.5 with NH4OH, CH3NH3Cl and CH3CH2NH3Cl were prepared by neutralization of methylamine and ethylamine with HCl. KCl, NaCl, and NH&l were Baker Reagent grade. RbCl and CsCl were optical grade.
- DNA-joining reactions using [3H]d(pA-pT)n or d(pA)n .d(pT)n were carried out as described previously except that the buffer composition was slightly changed as indicated above.
- In reactions employing d(pA)n.d(pT)n, the d(pA)n, and d(pT)n, were present at equimolar nucleotide concentrations.
- Formation of the product was monitored by adsorption of a portion of the reaction mixture to 1.5.cm squares of DEAE-paper which were washed as described previously.
- Experiments on the reversible formation of ligase-AMP in the absence of DNA were conducted in siliconized glass tubes.
- The formation of ligase-[32P]AMP from [32P]DPN was followed by acid precipitation.
- The release of [32P]AMP from isolated ligase-[32P]AMP by NMN or nicked DNA was determined by conversion of the 32P to an acid soluble form.
- Samples (0.4 ml) of the incubation mixture were added to 0.15 ml of ice-cold 0.5 M Tris base, 50 mM EDTA, 5 mg per ml of BSA to terminate the reaction.
- The protein was precipitated by adding 0.1 ml of cold 50% trichloroacetic acid.
- After 5 min at 0o, the precipitate was removed by centrifugation at 30,000 x g for 5 min.
- The radioactivity in 0.4 ml of the supernatant fluid was determined using a toluene based scintillation fluid containing Triton X-100.
- The rate of the ligase-catalyzed exchange between DPN and NMN was determined by measuring the exchange of 3H from [4-3H]nicotinamide-DPN into a pool of unlabeled NMN at 30o.
- The reaction was terminated by spotting 10-ml samples on top of 2 ml of 0.2 M EDTA on Whatman No. 1 paper.
- The DPN and NMN were separated by descending chromatography in 1 M ammonium acetate (pH 5.0), ethanol (3 :7, v/v) for 16 hours.
- The DPN and NMN spots were cut out and the fraction of 3H in the NMN determined.
- DNA-adenylate formation was measured with d(pA)5100 d(pT)160-dd(pT)1 as substrate.
- At the conclusion of the reaction, 0.1 ml of phenol saturated with 0.1 M Tris-HCl, (pH 7.4), 0.1 M NaCl was added and the mixture extracted for 2 min at 4o.
- Portions (60ml) of the aqueous phase were spotted on DEAE-paper squares which were then washed.
- Control incubations included reactions without ligase, reactions without DNA, and reactions in which the d(pT)100.dd(pT)1 was replaced by (dT)1-d(pT)160-dd(pT)1.
- The 32P retained on DEAE-paper was essentially the same for the various controls and typically represented < 10% of the incorporation observed in complete reaction mixtures.
- The release of AMP from DNA-adenylate was examined in 0.2-ml reaction mixtures containing d(pA)5100. A [32P]p-p(dT)1-d(pT)240.
- Reactions containing the indicated amounts of substrate and enzyme were incubated for 15 min at 30o and the release of 32P from the polymer determined by acid precipitation or by thin layer chromatography on PEI-cellulose.
- In the former case, the reaction was terminated by adding 0.1 ml of 5.8 mM calf thymus DNA and 0.5 ml of cold 10% trichloroacetic acid.
- After 10 min at Oo, the precipitate was removed by centrifugation at 25,000 x g for 10 min and the acid-soluble 32P determined.
- In the latter case, the reaction was terminated by adding 10ml of 0.2 M EDTA and streaking out 100ml of the mixture along a 2-cm line on a PEI sheet (2 cm from the bottom).
- The PEI sheet was developed by ascending chromatography using step elution with LiCl as follows: 2 min with 0.2 M LiCl, then 6 min with 1 M LiCl, and finally with 1.6 M LiCl until the solvent front had migrated 15 cm from the origin.
- The chromatogram was cut into 1-cm strips and the 32P at the origin (unreacted DNA-adenylate), in AMP, and in DPN was determined.
- Double reciprocal plots were fitted by a weighted regression analysis. End group labeling and determination of number average polynucleotide chain lengths were performed.
- Radioactivity was determined by liquid scintillation counting in a Spectrometer.
- A spectrophotometer was used for all optical measurements.
- Unless indicated otherwise, polynucleotide concentrations are expressed as nucleotide equivalents.
- ddTMP, 2’,3’-dideoxythymidine 5’-monophosphate.
- Oligo- and polynucleotides:
- d(pT)240, oligomer of thymidylic acid with 5’-phosphoryl, 3’-hydroxyl termini with a number average chain length of 240 nucleotides;
- (dT)1-d(pT)240, the same oligonucleotide with a 5’-hydroxyl terminus;
- d(pT)160- dd(pT)1, an oligonucleotide with one dideoxythymidylate residue at the 3’-terminus ;
- Ap-pd (T)1-d (pT)240, an oligonucleotide in which the 5’.phosphoryl terminus is linked by a pyrophosphate bond to adenosine 5’-monophosphate;
- d(pA)5100, a polymer of deoxyadenylate with a number average chain length of 5100 nucleotides;
- d(pA-pT)480, the alternating copolymer of deoxyadenylate and deoxythymidylate with a number average chain length of 480 nucleotides.