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PMID: 9016655 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't

Probing the role of the ATP-operated clamp in the strand-passage reaction of DNA gyrase.

Nucleic acids research ·Vol. 24 ·No. 24 ·1996-12-15 ·Pages 4868-73

Tingey AP, Maxwell A

Abstract

The high-resolution structure of the 43 kDa N-terminal fragment of the DNA gyrase B protein shows a large cavity within the protein dimer. The approximate size of this cavity is 20 A, suggesting it could accommodate a DNA helix. Computer-modelling studies of this cavity suggest that it contains a constriction, reducing the width to approximately 13 A, principally caused by the side chain of Arg286. We have used site-directed mutagenesis to alter this residue to Gln. Gyrase bearing this mutation shows virtually no supercoiling activity and near-normal relaxation and DNA cleavage activities. The mutated protein has ATPase activity which cannot be stimulated by DNA. These data support the proposed role of the 43 kDa domain as an ATP-operated clamp which binds DNA during the supercoiling cycle. The lack of DNA-dependent ATPase of the mutant may indicate that binding of DNA within the clamp is a prerequisite for stimulation of the ATPase activity.

MeSH Terms
Adenosine Triphosphatases/metabolism Adenosine Triphosphate/metabolism Cloning, Molecular DNA Topoisomerases, Type I/metabolism DNA Topoisomerases, Type II/genetics,metabolism Molecular Probes Mutagenesis, Site-Directed
Chemicals
Molecular Probes Adenosine Triphosphate Adenosine Triphosphatases DNA Topoisomerases, Type I DNA Topoisomerases, Type II
Authors & Affiliations
2 authors, click to expand affiliations / ORCID
Tingey A P
Department of Biochemistry, University of Leicester, UK.
Maxwell A
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Article Info
Journal
Nucleic acids research
Abbr.
Nucleic Acids Res
ISSN
0305-1048
DOI
10.1093/nar/24.24.4868
Published
1996-12-15
Pages
4868-73
Language
English
Region
England
NLM ID
0411011
PMCID
PMC146357
Subset
IM
External Links
PubMed source DOI: 10.1093/nar/24.24.4868 PMC Full Text View journal details
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