ATP-dependent DNA ligase from bacteriophage T7: Difference between revisions

ATP-dependent DNA ligase from bacteriophage T7 (Caudovirales Podoviridae) is used to catalyze a phosphodiester bond between single-strand nicks in double-stranded DNA.  This occurs in replication (connecting okazaki fragments)<ref name="Berg">Berg, Jeremy M, Stryer, Lubert, Tymoczko, John L. ''Biochemistry.'' Sixth edition. New York: W.H. Freeman and Company, 2007: 796. Print.</ref>, DNA repair (excision repair), and recombination.  DNA ligases require either ATP (eukaryotes and viruses) or NAD+ (prokaryotes) as a cofactor.  All ligases require a divalent cation for function.  Bacteriophage T7 DNA ligase uses Magnesium in vivo.  A range of pH 7.2-7.7 is ideal for enzymatic activity.  T7 ligase has a molecular weight of 41 kDa<ref>PMID: 8653795</ref>.

ATP-dependent DNA ligase from bacteriophage T7 is monomeric, forming a tertiary structure consisting of two domains (domain 1 and domain 2).  Domain 1 (residues 2:240) contains the ATP binding site.  Domain 1 is composed of six alpha helices which surround three antiparallel Beta sheets.  Domain 2 (residues 241:349) is composed of an antiparallel Beta sheet and an alpha helix.  A groove is formed between the two domains;  this groove allows ATP to bind with domain 1.  The ribose ring of ATP forms hydrogen bonds with the side chains of <scene name='56/567310/Arg_39_arg_55_glu_93/1'>Arg-39, Arg-55, and Glu-93</scene>.  <scene name='56/567310/Lys232_lys238_lys34/2'>Lys-232, Lys-238, and Lys-34</scene> (the catalytic residue) form hydrogen bonds with the three phosphoryl groups of ATP.  The 6-amino group of the adenine ring creates hydrogen bonds with the main-chain carbonyl of Ile-33 and the side chain of Glu-32.  This could account for the use of ATP rather than GTP.  While consisting of 359 residues, residues 121-127, 307-316, and 350-359 are not easily deciphered from the crystalline structure, and are therefore left out of the diagram.  Domain 1 contains the N terminus, while domain 2 contains the C terminus.  Multiple N and C terminii are shown in the diagram due to the missing residues.

 

ATP-dependent DNA ligase from bacteriophage T7 amends a fractured DNA strand through esterification of a 5'- phosphoryl to a 3'- hydroxyl group.  This mechanism occurs with the aid of ATP in several steps.  First, the ligase is activated through a <scene name='56/567310/Amp_complex/1'>phosphoramidate bond with a lysine residue</scene> in the active site (Lys 34).  A pyrophosphate leaves and the enzyme-AMP complex is formed.  Next, the AMP is transferred to the 5' phosphate group at the nick in the DNA.  Finally, T7 ligase creates the phosphodiester bond between the 5' -phosphoryl and the 3' – hydroxyl group, with AMP being freed.  All ATP-dependent DNA ligases contain a conserved amino acid sequence of KxDGxR.  This includes the lysine residue which binds the ATP in the groove between the two domains.