# Ligase

Ligase catalyses a covalent bond between the 5' phosphate of one piece of DNA and the 3' hydroxyl of another piece of DNA.
example: Ligation of an EcoRI restriction site

Bacteria have developed ligase to repair brakes in their DNA, so ligase occurs naturally in bacteria. Bacteria use $NAD^+$ as energy source, eucaryotes instead use ATP and have four different types of ligases

• DNA ligase I: connects Okazaki fragments of the lagging strand in DNA replication, and can also seal some repair and recombination fragments
• DNA ligase II: an alternatively spliced form of DNA ligase III that is only expressed in non-dividing cells.
• DNA ligase III: works with protein XRCC1, which is a DNA repair protein. DNA ligase III is a primary agent in sealing base excision-repairs and recombination fragments.
• DNA ligase IV: works with protein XRCC4, which is another DNA repair protein. DNA ligase IV is also important in sealing base excision-repairs and recombination fragments, especially during development

The following steps summarize an ligation mechanism:

1. In the adenylation step, ATP binds ligase and cleaves away two inorganic phosphates
2. The resulting AMP that binds ligase is transfered to 5' Phosephate of the DNA, which is thereby activated
3. Displacement of the AMP from the phosphate leads to a covalent bond between the phosphate and the hydroxyl and a sealed DNA
Illustration of the ligation mechanism. Notice that step 2. has to be done on both phosphate groups, at each 5' overhang, otherwise only one bond is catalysed