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DNA ligases are vital enzymes required for important cellular processes such as DNA replication, repair of damaged DNA and recombination. The enzyme mediates the formation of phosphodiester bonds between adjacent 3'-OH and 5'-phosphate termini, thereby joining the nicks in double stranded DNA. Ligases can be classified into two groups depending on their requirement for ATP or NAD+ as the cofactor. All eukaryotic and virally encoded enzymes are ATP-dependent, whereas most prokaryotic enzymes require NAD⁺ for their activity.

ATP-dependent DNA ligase
DNA ligase from bacteriophage T7 is a monomer with a molecular weight of 41 kDa. Crystal structures of the apo form of this enzyme and its complex with ATP have been determined at 2.7 Å and 2.6 Å respectively.

The structure consists of two distinct domains, a larger N-terminal domain and a C-terminal domain. The ATP-binding site is situated in the N-terminal domain in a pocket beneath one of the beta-sheets. This pocket is lined by a number of motifs that are conserved across a wide family of nucleotidyltransferases. The structure of the C-terminal domain is remarkably similar to the oligonucleotide binding fold, observed in a number of proteins. The DNA-binding site is proposed to be in a groove running between the two domains.

The structure has provided the basis for biochemical experiments. We have made the two domains separately and looked at their biochemical properties. The larger N-terminal domain is an active ligase but with greatly reduced activity. Both domains are able to bind to DNA. The N-terminal domain binds both single and double stranded DNA, while the smaller C-terminal domain is only able to bind double stranded DNA despite having a fold that is similar to single strand DNA binding proteins. The affinity for nicked DNA comes from a combination of these two DNA-binding affinities at the active site. Interestingly, the N-terminal domain is poor at the adenylation reaction, but this activity is stimulated greatly by addition of the C-terminal domain. Furthermore, we can demonstrate an association of the domains on gel filtration. These data suggest that a conformational change occurs during the adenylation reaction. This would be similar to that observed directly in our crystal structure of an mRNA capping enzyme, a group of enzymes that are closely related to ligases.

NAD-dependent DNA ligase
We also work on an NAD-dependent DNA ligase from B.stearothermophilus. The enzyme has been overexpressed and biochemical studies are in progress. Limited proteolysis has been used to investigate the domain structure of the enzyme. There are two domains, with the larger N-terminal domain retaining full self adenylation activity and the smaller C-terminal domain having all of the DNA-binding activity of the full length enzyme. The situation is therefore different to that we found in the ATP-dependent enzyme, where both domains contribute to the activities of the enzyme. Structural studies are underway with these domains. The structure of the large N-terminal domain has been solved and work is in progress to determine the structure of the small domain.