Functional characterization of the T4 DNA ligase: A new insight into the mechanism of action

被引:84
作者
Rossi, R
Montecucco, A
Ciarrocchi, G
Biamonti, G
机构
[1] UNIV PAVIA, IST GENET BIOCHIM & EVOLUZIONIST, CNR, I-27100 PAVIA, ITALY
[2] UNIV PAVIA, DIPARTIMENTO GENET & MICROBIOL A BUZZATI TRAVERS, I-27100 PAVIA, ITALY
关键词
D O I
10.1093/nar/25.11.2106
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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.
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页码:2106 / 2113
页数:8
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