title: Functional characterization of the T4 DNA ligase: a new insight into the mechanism of action.
abstract: ATP-dependent DNA ligases are essential enzymes in both DNA replication and DNA repair processes. Here we report a functional characterization of the T4 DNA ligase. One N-terminal and two C-terminal deletion mutants were expressed in Escherichia coli as histidine- tagged proteins. An additional mutant bore a substitution of Lys159 in the active site that abolished ATP binding. All the proteins were tested in biochemical assays for ATP-dependent self-adenylation, DNA binding, nick joining, blunt-end ligation and AMP- dependent DNA relaxation. From this analysis we conclude that binding to DNA is mediated by sequences at both protein ends and plays a key role in the reaction. The enzyme establishes two different complexes with DNA: (i) a transient complex (T.complex) involving the adenylated enzyme; (ii) a stable complex (S.complex) requiring the deadenylated T4 DNA ligase. The formation of an S. complex seems to be relevant during both blunt-end ligation and DNA relaxation. Moreover the inactive His-K159L substitution mutant, although unable to self-adenylate, still possesses AMP-dependent DNA nicking activity.
authors.affiliation: Istituto di Genetica Biochimica ed Evoluzionistica CNR, Università di Pavia, via Abbiategrasso 207, 27100 Pavia, Italy.
authors.forename: R
authors.initials: R
authors.lastname: Rossi
authors.forename: A
authors.initials: A
authors.lastname: Montecucco
authors.forename: G
authors.initials: G
authors.lastname: Ciarrocchi
authors.forename: G
authors.initials: G
authors.lastname: Biamonti
mesh_terms: D000249:Adenosine Monophosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D000906:Antibodies / Q000276:immunology; D001483:Base Sequence; D004247:DNA / Q000378:metabolism; D011088:DNA Ligases / Q000276:immunology / Q000378:metabolism*; D004260:DNA Repair; D004591:Electrophoresis, Polyacrylamide Gel; D008956:Models, Chemical; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation
chemical_list: D000906:Antibodies; D000249:Adenosine Monophosphate; D000255:Adenosine Triphosphate; D004247:DNA; D011088:DNA Ligases