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What is the enzymatic mechanism of ligase?
Posted July 22, 2020
Answer
DNA ligase is used to join two DNA strands by forming two covalent phosphodiester bonds between 3’ hydroxyl ends of one nucleotide (acceptor) and the 5’ phosphate end of another (donor). This process proceeds in 4 general steps:
- Reorganization of activity site such as nicks in DNA segments or Okazaki fragments.
- Adenylyation of a lysine residue, i.e., addition of AMP, in the active center of the ligase. One ATP molecule is consumed and pyrophosphate is released.
- Transfer of the AMP to the 5’ phosphate of the donor nucleotide, forming a pyrophosphate bond.
- Formation of a phosphodiester bond by attaching the AMP-phosphate bond with the 3’ hydroxyl of the acceptor nucleotide. AMP molecule is released.
Additional resources
Helixyte™ Green *10,000X Aqueous PCR Solution*
6-ROX glycine *25 uM fluorescence reference solution for PCR reactions*Perdew, G. H., Heuvel, J. P. V., & Peters, J. M. (2008). Regulation of gene expression. Springer Science & Business Media.Lodish, H., Berk, A., Kaiser, C. A., Krieger, M., Scott, M. P., Bretscher, A., ... & Matsudaira, P. (2008). Molecular cell biology. Macmillan.