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Nucleic Acids Res. 2011 Jun;39(11):4808-17. doi: 10.1093/nar/gkr018. Epub 2011 Feb 7.

Use of divalent metal ions in the DNA cleavage reaction of topoisomerase IV.

Author information

1
Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, TN 37232-0146, USA.

Abstract

It has long been known that type II topoisomerases require divalent metal ions in order to cleave DNA. Kinetic, mutagenesis and structural studies indicate that the eukaryotic enzymes utilize a novel variant of the canonical two-metal-ion mechanism to promote DNA scission. However, the role of metal ions in the cleavage reaction mediated by bacterial type II enzymes has been controversial. Therefore, to resolve this critical issue, this study characterized the DNA cleavage reaction of Escherichia coli topoisomerase IV. We utilized a series of divalent metal ions with varying thiophilicities in conjunction with oligonucleotides that replaced bridging and non-bridging oxygen atoms at (and near) the scissile bond with sulfur atoms. DNA scission was enhanced when thiophilic metal ions were used with substrates that contained bridging sulfur atoms. In addition, the metal-ion dependence of DNA cleavage was sigmoidal in nature, and rates and levels of DNA cleavage increased when metal ion mixtures were used in reactions. Based on these findings, we propose that topoisomerase IV cleaves DNA using a two-metal-ion mechanism in which one of the metal ions makes a critical interaction with the 3'-bridging atom of the scissile phosphate and facilitates DNA scission by the bacterial type II enzyme.

PMID:
21300644
PMCID:
PMC3113566
DOI:
10.1093/nar/gkr018
[Indexed for MEDLINE]
Free PMC Article
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