STRUCTURE-FUNCTION RELATIONSHIPS IN DNA LIGASE FROM THE BACTERIOPHAGE T7. Stephen R. Ashford, Aidan J. Doherty, Hosahalli S. Subramanya and Dale B. Wigley, Laboratory of Molecular Biophysics, University of Oxford, South Parks Road, Oxford, Ox1 3QU, UK

DNA ligases are ubiquitous enzymes, necessary for DNA replication and some forms of repair. The enzymes can easily be divided into two groups, and although they all catalyze essentially the same reaction - the sealing of nicks in the DNA backbone via an adenylated enzyme intermediate - they require different co-factors, and possess quite different primary sequence. The eukaryotic and viral DNA ligases require ATP, whereas the bacterial enzymes utilise NAD⁺. All ATP-dependent DNA ligases share a common core structure - as shown by sequence alignments, and therefore the T7 enzyme (one of the smallest in this class) was selected for structural and biochemical analysis.

The enzyme is a monomer of 41 kDa, and the crystal structure at 2.6Å resolution has been solved, with and without ATP bound. The enzyme consists of a larger N terminal domain (residues 1 to 240) containing the co-factor binding pocket and site of adenylated enzyme intermediate formation, and a C terminal domain (240 to 349), which is remarkably similar to the oligonucleotide binding fold (OB fold) found in many proteins including staphylococcal nuclease, and bacterial cold shock protein. Further biochemical study of these two domains, in conjunction with the structure, lead to a number of conclusions about the DNA binding and specificity of the enzyme.