DNA ligases close nicks in the phosphodiester backbone of DNA. Biologically, DNA ligases are essential for the joining of Okazaki fragments during replication, and for completing short-patch DNA synthesis occurring in DNA repair process. There are two classes of DNA ligases. The first uses NAD+ as a cofactor and only found in bacteria. The second uses ATP as a cofactor and found in eukaryotes, viruses and bacteriophages. The smallest known ATP-dependent DNA ligase is the one from the bacteriophage T7 (at 41KdA). Eukaryotic DNA ligases may be much larger (human DNA ligase I is > 100KDA) but they all appear to share some common sequences and probably structural motifs.
The reaction occurs in three stages in all DNA ligases:
1. Formation of a covalent enzyme-AMP intermediate linked to a lysine side-chain in the enzyme.
2. Transfer of the AMP nucleotide to the 5’ phosphate of the nicked DNA strand.
3. Attack on the AMP-DNA bond by the 3’-OH of the nicked DNA sealing the phosphate backbone and resealing AMP.
The following figure illustrates the three reaction stages:
DNA Ligase, T4
The catalytic activity of the enzyme requires the presence of ATP and Mg++. DNAs that lack the required phosphate residues can be rendered capable of ligation by phosphorylation with T4 polynucleotide kinase. The enzyme also catalyzes an addition reaction of phosphate between pyrophosphate and ATP. The ligation and the repair catalyzed reactions of T4 DNA ligases are illustrated in the following:
1- Ligation of DNA with complementary cohesive termini
2- Repair reaction
Characteristic of the Enzyme from E. coli lysogenic NM989
Bacteriophage T4 DNA ligase is a single polypeptide with a M.W of 68,000 Dalton. The maximal activity pH range is 7.5-8.0. The enzyme exhibits 40% of its activity at pH 6.9 and 65% at pH 8.3. The presence of Mg++ ion is required and the optimal concentration is 10mM. Sulfhydryl reagents (DTT, 2-mercapteothanol) are required as well. Concentrations of NaCl that exceeds 200mM are inhibited. “For intermolecular ligation, especially when the substrate DNAs consist of large DNA molecules PEG (concentrations of 1 % - 10%) appears to stimulate the enzymatic activity”. The optimal incubation temperature for T4 DNA ligase is 16C. When very high efficiency ligation is desired (e.g. making libraries) this temperature is highly recommended. However, ligase is active at a broad range of temperatures. For routine purposes such as subcloning, convenience often dictates incubating time and temperature-ligations performed at 4C overnight or at room temperature for 30 minutes to a couple of hours usually work well.
T4 DNA ligase is mainly used in joining DNAZ molecules with compatible cohesive termini, or blunt ended double stranded DNA to one another or to synthetic linkers. The reaction involving blunt ended DNA is much slower. However, the rate of ligation can be accelerated by the addition of 150-200 mM NaCl and low concentration of PEG. DNAs that lack the 5’phospahate termini, which is essential for the ligase reaction, have to be phosphoralated prior to ligation. The use of T4 polynucleotide kinase and ATP achieves phosphoralation. “The joining of DNA fragments with protruding 5' termini that are not compatible (for instance, restriction digestion of DNA with Xba I and Hind III) can be accomplished by partial filling of the recessed 3' termini in controlled reactions using the Klenow fragment of E. coli DNA polymerase I.
The following figure illustrates the ligation reaction pathway for DNA ligase and it’s interaction with the DNA strands during the process:
T7 DNA ligase Structure
The T7 DNA ligase structure consists of two distinct domains with the ATP binding site formed by the larger N-terminal domain. In the complex with ATP, the adenine ring is buried in a pocket in the enzyme with the alpha-amino group on the ATP close to the side-chain of lysine 34, with which it forms a covalent bond during the first stage of the reaction. The conformation of the ATP suggests that, in the complex with AMP-DNA, the adenine residue would be ‘flipped-out’ of the DNA duplex, preventing it from interfering with base-pairing of the DNA in the region of the nick-site.
The structure of T7 DNA ligase is shown in the following diagram:
1. Biochemistry C22/C29—DNA Replication Enzymes. http://www.biochem.ucl.ac.uk/bsm/xtal/teach/repl/ligase.html
2. DNA ligase, T4. http://www.worthington-biochem.com/manual/D/DNAT4L.html
3. Nucleic Acid Structure IV. http://www.orst.edu/instruction/bb492/fignumbers/Fig24-21.html
4. DNA ligasehttp://arbl.cvmbs.colostate.edu/hbooks/genetics/biotech/enzymes/ligation.html