ATP-Dependent DNA Ligase (Bacteriophage T7)

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ATP-DEPENDENT DNA LIGASE FROM BACTERIOPHAGE T7

Overview (General Function)

ATP-dependent DNA ligase from bacteriophage T7 (Caudovirales Podoviridae[1]) is used to catalyze a phosphodiester bond between single-strand nicks in double-stranded DNA. This occurs in replication (connecting okazaki fragments)[2], DNA repair (excision repair), and recombination. DNA ligases require either ATP (eukaryotes and viruses) or NAD+ (prokaryotes) as a cofactor[3]. 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[4].



Structure

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[4]. 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 Arg-39, Arg-55, and Glu-93. Lys-232, Lys-238, and Lys-34 (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[4]. 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.

main-chain carbonyl of Ile-33 and the side chain of Glu-32


Catalytic function with DNA

ATP-dependent DNA ligase from bacteriophage T7 amends a fractured DNA strand through esterification of a 5'- phosphoryl to a 3'- hydroxyl group[3]. This mechanism occurs with the aid of ATP in several steps. First, the ligase is activated through a phosphoramidate bond with a lysine residue 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[4]. All ATP-dependent DNA ligases contain a conserved amino acid sequence of KxDGxR[3]. This includes the lysine residue which binds the ATP in the groove between the two domains.


Structure of Bacteriophage T7 DNA Ligase(PDB entry 1a0i)

Drag the structure with the mouse to rotate


References

  1. ↑ McGrath S, van Sinderen D. Bacteriophage: Genetics and Molecular Biology. Norfolk: Caister Academic Press, 2007. Print.
  2. ↑ Berg, Jeremy M, Stryer, Lubert, Tymoczko, John L. Biochemistry. Sixth edition. New York: W.H. Freeman and Company, 2007: 796. Print.
  3. ↑ 3.0 3.1 3.2 Doherty AJ, Ashford SR, Subramanya HS, Wigley DB. Bacteriophage T7 DNA ligase. Overexpression, purification, crystallization, and characterization. J Biol Chem. 1996 May 10;271(19):11083-9. PMID:8626651
  4. ↑ 4.0 4.1 4.2 4.3 Subramanya HS, Doherty AJ, Ashford SR, Wigley DB. Crystal structure of an ATP-dependent DNA ligase from bacteriophage T7. Cell. 1996 May 17;85(4):607-15. PMID:8653795

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