- Home - Introduction - Projects - People - Publications - Vacancies -

Unlike all other known organisms, eubacteria use NAD in place of ATP to drive the DNA ligation reaction catalysed by DNA ligase. Like the ATP-dependent ligases of other organisms the NAD-dependent enzyme is required for DNA replication and repair. The enzyme catalyses the sealing of nicks between a 3'-hydroxyl and 5'-phosphate group in double stranded DNA. Therefore, it is essential for the survival of the organism and effective inhibition would result in the death of the bacterium. However, before such inhibitors can be developed, it will be necessary to discover more about how the enzyme works.

All DNA ligases, regardless of their energy source, proceed through an enzyme-AMP covalent intermediate. Adenylation occurs as the first stage of the reaction and involves a lysine residue in the active site of the enzyme. This lysine forms part of a sequence motif, KxDG, which is common to all DNA ligases. Indeed, this is the only sequence similarity shared by the ATP- and NAD-dependent ligases. The second stage of the reaction involves the transfer of the AMP group to the 5'-phosphate of the nick. The nick is then sealed in the final stage of the reaction with the elimination of AMP.

The structures of an ATP-dependent DNA ligase and of the structurally and mechanistically related mRNA capping enzyme have been determined in this laboratory. Both these enzymes consist of two domains. The larger, N-terminal, domain contains the active site lysine and is the site of self-adenylation (self-guanylation in the case of mRNA capping enzyme). There is evidence from the structures of the capping enzyme that both domains are required to effect efficient self-guanylation and there is biochemical evidence that the situation is similar in ATP-dependent DNA ligases.

We began our work with no structural information on the NAD-dependent ligases. However, we had recently determined the sequence of the enzyme from the moderate thermophile Bacillus stearothermophilus.

Limited proteolysis studies

Limited proteolysis with thermolysin resulted in two fragments (36kDa and 30kDa) which were resistant to further digestion. This suggests that NAD-dependent DNA ligases consist of at least two structural domains. N-terminal amino acid sequencing and mass spectrometry enabled the identification of the starting and finishing points of the fragments.

To permit further studies of these fragments, equivalent proteins were expressed in E. coli.

Ligation activity

When each fragment was tested for ligation activity, the large fragment showed vastly reduced (at least three orders of magnitude) DNA ligation activity compared to the intact protein. Addition of the small fragment to the large, did not restore ligation activity.

Adenylation activity

When the adenylation activity of each fragment was tested, it was found that the large fragment is adenylated by NAD at the same rate and to the same extent as the intact enzyme. Addition of the small fragment (which cannot, itself, be adenylated since it lacks the active site lysine residue) does not alter the rate of adenylation of the large fragment. This suggests that the large, N-terminal domain is a fully competant self-adenylation module - a situation which contrasts with the ATP-dependent T7 ligase where the C-terminal domain stimulates the self-adenylation activity of the N-terminal domain.

DNA binding

Analysis of the DNA binding activity of the intact ligase and its fragments showed that while the small fragment and the intact enzyme are able to bind nicked, duplex DNA, the large fragment has virtually no affinity for DNA. Approximately the same amount of enzyme was required to produce the same magnitude of shift with both the small fragment and the intact enzyme, suggesting that DNA binding activity is located almost entirely in the C-terminal third of the enzyme. This is also different to the situation in the T7 ligase, where both domains contribute to DNA binding.

Zinc ion binding

Atomic absorption spectroscopy showed the presence of approximately 1 mole of zinc(II) ions per mole of enzyme. A similar amount was detected in the small fragment, but none in the large. The role and exact binding site of the Zn(II) ion remain to be discovered.

Bacterial DNA ligases: Modular proteins

We were unable to detect any interaction between the small and the large fragments. The small fragment does not influence the adenylation rate or ligase activty of the large. The large fragment does not influence the DNA binding activity of the small. Therefore we conclude that the protein consists of two, biochemically independent domains. This is in contrast to the ATP-dependent T7 ligase where there is a greater degree of co-operation between the domains.

Structure of the Large Domain

The structure of the large domain shows striking similarity of fold to the N-terminal domain of T7 ligase. This is particularly surprising considering the lack of sequence similarity between the two enzymes.

The similarity of fold between the ATP- and NAD-dependent DNA ligases is particularly strong around the ATP binding site. This enables us to predict where NAD might bind in B. stearothermophilus ligase.

More about this structure

Reference: Timson DJ & Wigley DB Journal of Molecular Biology 285 73-83.