DNA ligase (ATP)

 

DNA ligase is an essential enzyme required for the repair, replication and recombination of DNA. In all organisms except the prokaryotes it is dependent on the hydrolysis of ATP to AMP and pyrophosphate.

 

Reference Protein and Structure

Sequence
P00969 UniProt (6.5.1.1) IPR016306 (Sequence Homologues) (PDB Homologues)
Biological species
Enterobacteria phage T7 (Virus) Uniprot
PDB
1a0i - ATP-DEPENDENT DNA LIGASE FROM BACTERIOPHAGE T7 COMPLEX WITH ATP (2.6 Å) PDBe PDBsum 1a0i
Catalytic CATH Domains
3.30.1490.70 CATHdb (see all for 1a0i)
Cofactors
Magnesium(2+) (1) Metal MACiE
Click To Show Structure

Enzyme Reaction (EC:6.5.1.1)

ATP(4-)
CHEBI:30616ChEBI
+
dAMP 5'-end residue
CHEBI:53099ChEBI
+
dAMP 3'-end residue
CHEBI:53113ChEBI
→
diphosphate(4-)
CHEBI:18361ChEBI
+
adenosine 5'-monophosphate(2-)
CHEBI:456215ChEBI
+
poly(deoxyadenylic acid)
CHEBI:73276ChEBI
Alternative enzyme names: DNA joinase, DNA ligase, DNA repair enzyme, DNA-joining enzyme, Deoxyribonucleate ligase, Deoxyribonucleic acid joinase, Deoxyribonucleic acid ligase, Deoxyribonucleic acid repair enzyme, Deoxyribonucleic acid-joining enzyme, Deoxyribonucleic joinase, Deoxyribonucleic ligase, Deoxyribonucleic repair enzyme, Deoxyribonucleic-joining enzyme, Polydeoxyribonucleotide synthase (ATP), Polynucleotide ligase, Sealase, Polynucleotide ligase (ATP), Poly(deoxyribonucleotide):poly(deoxyribonucleotide) ligase (AMP-forming),

Enzyme Mechanism

Introduction

Lys34 acts as a nucleophile and attacks the alpha-phosphate of ATP in a substitution reaction, liberating pyrophosphate and forming an intermediate covalently bound to the enzyme. The phosphate group of the first strand of DNA acts as a nucleophile and attacks the phosphate bound to Lys34 in a substitution reaction, liberating Lys34 and forming a DNA-AMP complex. The AMP phosphate deprotonates the hydroxyl group of the second DNA molecule, which then acts as a nucleophile and attacks the DNA phosphate of the DNA-AMP complex in a substitution reaction, liberating AMP and the ligated DNA.

Catalytic Residues Roles

UniProt PDB* (1a0i)
Lys238 Lys238(237)A Stabilises transition states through reaction. May also position the pyrophosphate leaving group. hydrogen bond donor, steric role, electrostatic stabiliser
Lys34 Lys34(33)A Lys34 is the nucleophile in the first step of the reaction and is covalently attached to AMP until the final step. hydrogen bond donor, nucleophile, polar interaction, nucleofuge
Lys240 Lys240(239)A Stabilises transition states through reaction. hydrogen bond donor, 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

bimolecular nucleophilic substitution, overall reactant used, enzyme-substrate complex formation, intermediate formation, overall product formed, enzyme-substrate complex cleavage, proton transfer, intermediate terminated, native state of enzyme regenerated

References

  1. Odell M et al. (2000), Mol Cell, 6, 1183-1193. Crystal Structure of Eukaryotic DNA Ligase–Adenylate Illuminates the Mechanism of Nick Sensing and Strand Joining. DOI:10.1016/s1097-2765(00)00115-5. PMID:11106756.
  2. Sriskanda V et al. (2002), J Biol Chem, 277, 9661-9667. Role of Nucleotidyl Transferase Motif V in Strand Joining byChlorella Virus DNA Ligase. DOI:10.1074/jbc.m110613200. PMID:11751916.
  3. Cherepanov AV et al. (2002), J Biol Chem, 277, 1695-1704. Kinetic mechanism of the Mg2+-dependent nucleotidyl transfer catalyzed by T4 DNA and RNA ligases. DOI:10.1074/jbc.M109616200. PMID:11687591.
  4. Doherty AJ et al. (2000), J Mol Biol, 296, 43-56. Nick recognition by DNA ligases. DOI:10.1006/jmbi.1999.3423. PMID:10656817.
  5. Subramanya HS et al. (1996), Cell, 85, 607-615. Crystal Structure of an ATP-Dependent DNA Ligase from Bacteriophage T7. DOI:10.1016/s0092-8674(00)81260-x. PMID:8653795.
  6. Tomkinson AE et al. (1991), Proc Natl Acad Sci U S A, 88, 400-404. Location of the active site for enzyme-adenylate formation in DNA ligases. DOI:10.1073/pnas.88.2.400. PMID:1988940.
  7. Lehman IR (1974), Science, 186, 790-797. DNA Ligase: Structure, Mechanism, and Function. DOI:10.1126/science.186.4166.790. PMID:4377758.

Step 1. Lys34 acts as a nucleophile and attacks the alpha-phosphate of ATP in a substitution reaction, liberating pyrophosphate and forming an intermediate covalently bound to the enzyme. Mg(II), Lys238 and Lys240 stabilise the intermediates formed, Lys238 positions the pyrophosphate leaving group, allowing for the in-line mechanism to occur.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Lys34(33)A polar interaction, hydrogen bond donor
Lys238(237)A hydrogen bond donor, steric role, electrostatic stabiliser
Lys240(239)A hydrogen bond donor, electrostatic stabiliser
Lys34(33)A nucleophile

Chemical Components

ingold: bimolecular nucleophilic substitution, overall reactant used, enzyme-substrate complex formation, intermediate formation, overall product formed

Step 2. The phosphate group of the first strand of DNA acts as a nucleophile and attacks the phosphate bound to Lys34 in a substitution reaction, liberating Lys34 and forming a DNA-AMP complex. Mg(II), Lys238 and Lys240 stabilise the intermediates formed.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Lys238(237)A hydrogen bond donor, electrostatic stabiliser
Lys240(239)A hydrogen bond donor, electrostatic stabiliser
Lys34(33)A nucleofuge

Chemical Components

ingold: bimolecular nucleophilic substitution, overall reactant used, enzyme-substrate complex cleavage, intermediate formation

Step 3. The AMP phosphate deprotonates the hydroxyl group of the second DNA molecule, which then acts as a nucleophile and attacks the DNA phosphate of the DNA-AMP complex in a substitution reaction, liberating AMP and the ligated DNA. Mg(II), Lys238 and Lys240 stabilise the intermediates formed.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Lys34(33)A polar interaction
Lys238(237)A hydrogen bond donor, electrostatic stabiliser
Lys240(239)A hydrogen bond donor, electrostatic stabiliser

Chemical Components

ingold: bimolecular nucleophilic substitution, proton transfer, overall reactant used, intermediate terminated, overall product formed, native state of enzyme regenerated
Download: Image, Marvin File

Step 1. Lys34 acts as a nucleophile and attacks the alpha-phosphate of ATP in a substitution reaction, liberating pyrophosphate and forming an intermediate covalently bound to the enzyme. Mg(II), Lys238 and Lys240 stabilise the intermediates formed, Lys238 positions the pyrophosphate leaving group, allowing for the in-line mechanism to occur.

Step 2. The phosphate group of the first strand of DNA acts as a nucleophile and attacks the phosphate bound to Lys34 in a substitution reaction, liberating Lys34 and forming a DNA-AMP complex. Mg(II), Lys238 and Lys240 stabilise the intermediates formed.

Step 3. The AMP phosphate deprotonates the hydroxyl group of the second DNA molecule, which then acts as a nucleophile and attacks the DNA phosphate of the DNA-AMP complex in a substitution reaction, liberating AMP and the ligated DNA. Mg(II), Lys238 and Lys240 stabilise the intermediates formed.

Products.

Contributors

Gemma L. Holliday, Daniel E. Almonacid, Emma LR Compton, Craig Porter, Charity Hornby