title: Functional domains of an NAD+-dependent DNA ligase.
abstract: Limited proteolysis of the NAD+-dependent DNA ligase from Bacillus stearothermophilus with thermolysin results in two fragments which were resistant to further proteolysis. These fragments were characterised by N-terminal protein sequencing and electrospray mass spectrometry. The larger, N-terminal fragment consists of the first 318 residues and the smaller, C-terminal fragment begins at residue 397 and runs to the C terminus. Both fragments were over-expressed in Escherichia coli and purified to homogeneity from this source. The large fragment retains the full self-adenylation activity of the intact enzyme, has minimal DNA binding activity and vastly reduced ligation activity. The small fragment lacks adenylation activity but binds to nicked DNA with a similar affinity to that of the intact enzyme. It is unable to stimulate the ligation activity of the large fragment. Atomic absorption spectroscopy showed that the intact protein and the small fragment bind a zinc ion but the large fragment does not. No evidence of any interaction between the two fragments could be obtained. Thus, we conclude that NAD+-dependent DNA ligases consist of at least two discrete functional domains: an N-terminal domain which is responsible for cofactor binding and self adenylation, and a C-terminal DNA-binding domain which contains a zinc binding site.
authors.affiliation: Sir William Dunn School of Pathology, The University of Oxford, South Parks Road, Oxford, OX1 3RE, UK.
authors.forename: D J
authors.initials: DJ
authors.lastname: Timson
authors.forename: D B
authors.initials: DB
authors.lastname: Wigley
mesh_terms: D000225:Adenine; D001665:Binding Sites; D002413:Cations, Divalent; D004247:DNA / Q000378:metabolism; D011088:DNA Ligases / Q000378:metabolism*; D001411:Geobacillus stearothermophilus / Q000201:enzymology*; D009243:NAD / Q000378:metabolism; D015032:Zinc
chemical_list: D002413:Cations, Divalent; D009243:NAD; D004247:DNA; D011088:DNA Ligases; D015032:Zinc; D000225:Adenine