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The structure of T7 DNA ligase clearly reveals two structural domains - a larger, N-terminal domain containing the active site lysine residue and a smaller C-terminal domain. The N-terminal domain runs from amino acid residue 1 to 240 and the C-terminal domain from 241 to 359.

The two domains have been expressed in E. coli and their properties investigated.

Adenylation Activity

The N-terminal domain (Domain 1) retains the ability to self-adenylate in the presence of ATP. However, this activity is much reduced compared to the intact protein. Addition of the C-terminal domain (Domain 2) stimulates the activity of Domain 1. This suggests that both domains are required for full self-adenylation activity. This is in contrast to the NAD-dependent ligase where the N-terminal domain is sufficient for full adenylation activity.

Ligation Activity

Domain 1 can act as a DNA ligase, albeit at a reduced (10-100 fold) activity compared to the intact enzyme. Unlike adenylation activity, ligation by Domain 1 is not stimulated by Domain 2.

DNA Binding Activity

DNA binding is shared between the two domains. The larger domain is able to bind both single and double stranded DNA, while the smaller fragment is only able to bind double-stranded DNA. This suggests that the specificity of ATP-dependent DNA ligases for nick sites in DNA is produced by a combination of these two different DNA binding activities in the intact enzyme.

Domain 1 and Domain 2 can be shown to interact.

Gel filtration experiments show that Domain 1 interacts with Domain 2. Taken together with the adenylation data this suggests that the two domains interact in order to bring about adenylation. We postulate that the situation could be similar to the structurally and mechanistically similar mRNA capping enzymes where Domain 2 moves closer to Domain 1, partially closing the inter-domain cleft and pushing the tri-phosphate tail on the nucleotide into a conformation that is correctly positioned for attack by the acitve site lysine residue.

Reference: Doherty AJ & Wigley DB (1999) Functional domains of an ATP-dependent DNA ligase. Journal of Molecular Biology, 285, 63-71.