Nucleic Acids Research, Vol 25, Issue 11 2106-2113, Copyright © 1997 by Oxford University Press
R Rossi, A Montecucco, G Ciarrocchi and G Biamonti
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.
ARTICLES
Functional characterization of the T4 DNA ligase: a new insight into the mechanism of action
Istituto di Genetica Biochimica ed Evoluzionistica CNR, Universita di Pavia, via Abbiategrasso 207, 27100 Pavia, Italy.
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