title: Characterization of proteolytic fragments of bacteriophage T7 DNA ligase.
abstract: Treatment of T7 DNA ligase with a range of proteases generates two major fragments which are resistant to further digestion. These fragments, of molecular weight 16 and 26 kDa, are derived from the N- and C-termini of the protein, respectively. The presence of ATP or a non-hydrolysable analogue, ADPNP, during limited proteolysis greatly reduces the level of digestion. The N-terminal 16 kDa region of the intact T7 ligase is labelled selectively in the presence of [alpha-32P]ATP, confirming that it contains the active site lysine residue. In common with the intact enzyme, the C-terminal portion of the protein retains the ability to band shift DNA fragments of various lengths, implicating it in DNA binding. It can also inhibit ligation by the intact protein, apparently by competing for target sites on DNA. We conclude that the N-terminal region, which contains the putative active site lysine, plays a role in the transfer of AMP from the enzyme-adenylate complex to the 5'phosphate at the nick site, while the C-terminal 26 kDa fragment appears to position the enzyme at the target site on DNA.
authors.affiliation: Laboratory of Molecular Biophysics, University of Oxford, UK.
authors.forename: A J
authors.initials: AJ
authors.lastname: Doherty
authors.forename: S R
authors.initials: SR
authors.lastname: Ashford
authors.forename: D B
authors.initials: DB
authors.lastname: Wigley
mesh_terms: D000249:Adenosine Monophosphate / Q000378:metabolism; D017123:Bacteriophage T7 / Q000201:enzymology*; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism; D017931:DNA Primers; D010450:Endopeptidases / Q000378:metabolism; D008025:Ligases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008239:Lysine / Q000378:metabolism; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000378:metabolism*; D011487:Protein Conformation
chemical_list: D017931:DNA Primers; D010446:Peptide Fragments; D000249:Adenosine Monophosphate; D004247:DNA; D010450:Endopeptidases; D008025:Ligases; D008239:Lysine