2-4.1 Ligases:

• DNA ligase catalyses the formation of phosphodiester bond between two deoxynucleotide residues of two DNA strands.
  
  
• DNA ligase enzyme requires a free hydroxyl group at the 3´-end of one DNA chain and a phosphate group at the 5´-end of the other and requires energy in the process.
  
  
• E.coli and other bacterial DNA ligase utilizes NAD⁺ as energy donor, whereas in T4 bacteriophage, T4 DNA ligase uses ATP as cofactor.
  
  
• The role of DNA ligase is to seal nicks in the backbone of double-stranded DNA after lagging strand formation to join the okazaki fragments.
  
  
• This joining process is essential for the normal synthesis of DNA and for repairing damaged DNA. It has been exploited by genetic engineers to join DNA chains to form recombinant DNA molecules. Usually single stranded break are repaired using the complimentary strand as the template but sometimes double stranded breaks can also be repaired with the help of DNA ligase IV.
  
  
• The most widely used DNA ligase is isolated from T4 bacteriophage. T4 DNA ligase needs ATP as a cofactor. The enzyme from E. coli uses cofactor NAD. Except this, the catalysis mechanism is somewhat similar for both the ligases. The role of cofactor is splitting and forming an enzyme-AMP complex which further aids in formation of phosphodiester bonds between hydroxyl and phosphate groups by exposing them.

 

2-4.1.1 Mechanism of Action of DNA Ligases:

• ATP, or NAD⁺, reacts with the ligase enzyme to form a covalent enzyme–AMP complex in which the AMP is linked to ε -amino group of a lysine residue in the active site of the enzyme through a phosphoamide bond.
  
  •  The AMP moiety activates the phosphate group at the 5´-end of the DNA molecule to be joined. It is called as the donor.
  
  •  The final step is a nucleophilic attack by the 3´-hydroxyl group on this activated phosphorus atom which acts as the acceptor. A phosphodiester bond is formed and AMP is released.
  
  • The reaction is driven by the hydrolysis of the pyrophosphate released during the formation of the enzyme–adenylate complex. Two high-energy phosphate bonds are spent in forming a phosphodiester bond in the DNA backbone with ATP serving as energy source.