DNA ligase (NAD+)

 

DNA ligases catalyse the formation of phosphodiester bonds at single-strand breaks in double stranded DNA and are required in DNA replication, repair and recombination. DNA ligases are also used extensively for in vitro DNA manipulation and cloning techniques. Eukaryotic, viral and achaebacteria encoded enzyme al require ATP, where as NAD+ requiring DNA ligases are found exclusively in eubacteria.

 

Reference Protein and Structure

Sequence
Q9ZHI0 UniProt (6.5.1.2) IPR001679 (Sequence Homologues) (PDB Homologues)
Biological species
Thermus filiformis (Bacteria) Uniprot
PDB
1dgs - CRYSTAL STRUCTURE OF NAD+-DEPENDENT DNA LIGASE FROM T. FILIFORMIS (2.9 Å) PDBe PDBsum 1dgs
Catalytic CATH Domains
3.30.1490.70 CATHdb 3.30.470.30 CATHdb (see all for 1dgs)
Cofactors
Magnesium(2+) (1)
Click To Show Structure

Enzyme Reaction (EC:6.5.1.2)

DNA polyanion
CHEBI:83828ChEBI
+
DNA polyanion
CHEBI:83828ChEBI
+
NAD(1-)
CHEBI:57540ChEBI
→
DNA polyanion
CHEBI:83828ChEBI
+
NMN(-)
CHEBI:14649ChEBI
+
adenosine 5'-monophosphate(2-)
CHEBI:456215ChEBI
Alternative enzyme names: DNA joinase, DNA ligase, DNA repair enzyme, DNA-joining enzyme, Deoxyribonucleate ligase, Deoxyribonucleic acid joinase, Deoxyribonucleic acid ligase, Deoxyribonucleic joinase, Deoxyribonucleic ligase, Deoxyribonucleic repair enzyme, Deoxyribonucleic-joining enzyme, Polydeoxyribonucleotide synthase (NAD(+)), Polynucleotide ligase, Polynucleotide ligase (nicotinamide adenine dinucleotide), Polynucleotide synthetase, Polynucleotide synthetase (nicotinamide adenine dinucleotide), Polynucleotide ligase (NAD+), Polydeoxyribonucleotide synthase (NAD+), Polynucleotide ligase (NAD(+)), Poly(deoxyribonucleotide):poly(deoxyribonucleotide) ligase (AMP-forming, NMN-forming),

Enzyme Mechanism

Introduction

The overall mechanism involves three reversible steps. The nicked DNA substrate is formed by annealing two short oligonucleotides to a longer complementary oligonucleotide. First, a covalently adenylated enzyme intermediate is formed by the transfer of the adenylate group of NAD+ to the E-amino group of Lys116. Second, the adenylate moiety is transferred from the enzyme to the 5'-terminal phosphate on one of the oligomers (oligomer A). Finally, a phosphodiester bond is formed by a nucleophilic attack of the 3'-hydroxyl terminus of the other oligomer (oligomer B) on the activated 5'-phosphoryl group of oligomer A in an Sn2 like mechanism with a pentavalent phosphorous transition state. Two Mg(2+) binding sites have been identified within the catalytic site (although the metals are not present in the pdb 1dgs) . The positive charge stabilises the developing negative charge of the pentavalent transition state and also the charged reaction intermediates.

Catalytic Residues Roles

UniProt PDB* (1dgs)
Lys118 Lys116A Acts as a nucleophile and forms a covalent attachment to the adenylate group of NAD+. covalent catalysis
Cys409, Cys412, Cys425, Cys430 Cys406A, Cys409A, Cys422A, Cys427A Forms the catalytic Mg(II) binding site. metal ligand
Glu116, Asp120, Tyr226, Lys315 Glu114A, Asp118A, Tyr221A, Lys312A Stabilises intermediate. electrostatic stabiliser
*PDB label guide - RESx(y)B(C) - RES: Residue Name; x: Residue ID in PDB file; y: Residue ID in PDB sequence if different from PDB file; B: PDB Chain; C: Biological Assembly Chain if different from PDB. If label is "Not Found" it means this residue is not found in the reference PDB.

Chemical Components

References

  1. Doherty AJ et al. (2000), Nucleic Acids Res, 28, 4051-4058. Structural and mechanistic conservation in DNA ligases. DOI:10.1093/nar/28.21.4051. PMID:11058099.
  2. Luo J et al. (1996), Nucleic Acids Res, 24, 3079-3085. Identification of essential residues in Thermus thermophilus DNA ligase. DOI:10.1093/nar/24.15.3079. PMID:8760897.

Step 1.

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Catalytic Residues Roles

Residue Roles
Lys116A covalent catalysis
Cys406A metal ligand
Cys409A metal ligand
Cys422A metal ligand
Cys427A metal ligand
Glu114A electrostatic stabiliser
Tyr221A electrostatic stabiliser
Asp118A electrostatic stabiliser
Lys312A electrostatic stabiliser

Chemical Components

Step 1.

Contributors

Gemma L. Holliday, Alex Gutteridge, James W. Murray, Craig Porter