DNA ligases catalyse joining of DNA ends, a critical step in the replication, repair and recombination of DNA. As the first step in ligation, all DNA ligases form a covalent enzyme-adenylate intermediate, using either NAD⁺ or ATP as the donor of the adenylate group (1). The uniqueness of NAD⁺-dependent DNA ligases to eubacteria makes them an attractive target for novel antibiotics.

High-resolution structures have been obtained for full length NAD⁺-dependent DNA ligases (LigA) from Thermus filiformis (2) and for the N-terminal adenylation domain of LigA from Bacillus stearothermophilus (3). Within the four distinct domains of LigA are several well-characterised protein folds, including a zinc-finger (Zn), an oligomer-binding (OB) β-barrel, a helix-hairpin-helix motif (HhH) and a BRCT motif. A model for the end-joining action of LigA suggests two regions of the enzyme interact independently with DNA (2). To obtain experimental evidence for the molecular details of DNA binding to this enzyme, we have initiated structural analysis of LigA from Escherichia coli.

The C-terminus of all essential NAD⁺-dependent DNA ligases contains a BRCT domain, which mediates macromolecular interactions in other proteins. Using in vitro gel-shift assays, we show that the BRCT domain from E. coli LigA promotes formation of a stable complex between LigA and DNA. The BRCT domain is not essential for DNA end-joining activity in vitro or in vivo, though it does improve the efficiency of end-joining in vitro.

This study confirms two aspects of the predictions from the X-ray crystallographic studies of T. filiformis LigA. Firstly, we show that the BRCT domain represents the ?non-catalytic? DNA binding activity of the enzyme (or at least a critical part of it). Secondly, since deletion of the BRCT domain does not completely abolish ligation activity, other parts of LigA must be able to bind to DNA. This latter point confirms that multiple independent DNA binding activities must reside within the protein. Biophysical chemistry techniques are being used to elucidate the modes of interaction between DNA and LigA.

Acknowledgments

The research was supported by grants from the BBSRC (UK) and the University of East Anglia.