Part:BBa_K525720

DNA ligase from Escherichia coli (LigA)

NAD⁺-dependent DNA ligase from E. coli in Freiburg BioBrick Assembly Standard (RFC 25)

Usage and Biology

Bacterial DNA ligases are important enzymes for DNA repair and replication. They use NAD⁺ as a cofactor to bind hydrolysed AMP to the active site lysine under release of NMN. AMP is used to catalyse the formation of a phosphodiester bond in nicked DNA by transferring AMP to the 5'-phosphate group. This results in an attack of the nick 3'-OH on the 5'-phosphoanhydride linkage and release of free AMP:

During the reaction mechanism the DNA ligases functional domains undergo conformational changes promoting intra- and intermolecular interactions. The domains are highly conserved throughout the bacterial kingdom and categorized in the following way:

• Domain 1: Ia, Ib (NTase)
  
• Domain 2: Oligomer-binding (OB) beta-barrel
  
• Domain 3: Zinc (Zn)-finger, helix-hairpin-helix motif (HhH)
  
• Domain 4: BRCT domain
  

The NAD⁺-dependent DNA ligase from E. coli (LigA) can be applied to biochemical studies ranging from cloning procedures and enzymatic assays to biomedical investigations. For instance, the purified enzyme can contribute to in vitro experiments dealing with antibiotic drug design because bacterial DNA ligases are essential for fundamental life-sustaining processes and differentiate from eukaryotic DNA ligases due to their specifity for NAD⁺ as a cofactor.

Characterization

Detailed information about the characteristics can be found for the PT7 promoter, RBS and His-tag equipped form of LigA BBa_K525710.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 1422
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 4
  Illegal AgeI site found at 2020
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 1636