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

Pitts SL, Liou GF, Mitchenall LA, Burgin AB, Maxwell A, Neuman KC, Osheroff N - Nucleic Acids Res. (2011)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: 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.

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Cleavage of oligonucleotide substrates by topoisomerase IV in the presence of different divalent metal ions. The central portion of the DNA cleavage substrate is depicted above the graphs. The asterisk denotes the 5′-end-labeled strand and the scissile bonds are indicated by arrows. All modifications are on the bottom strand. Results for the wild-type oligonucleotide (WT, top left panel), and substrates containing a 3′-bridging phosphorothiolate at the scissile (−1/+1) bond (SPO, top right), a non-bridging phosphorothioate at the scissile bond (−1/+1 NB, bottom left) and a non-bridging phosphorothioate at the −2/−1 position (−2/−1 NB, bottom right) are shown. DNA cleavage was carried out in the presence of 10 mM Mg2+ (closed squares), Mn2+ (open circles) or Ca2+ (closed circles). Error bars represent the standard deviation of three independent experiments.
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Figure 3: Cleavage of oligonucleotide substrates by topoisomerase IV in the presence of different divalent metal ions. The central portion of the DNA cleavage substrate is depicted above the graphs. The asterisk denotes the 5′-end-labeled strand and the scissile bonds are indicated by arrows. All modifications are on the bottom strand. Results for the wild-type oligonucleotide (WT, top left panel), and substrates containing a 3′-bridging phosphorothiolate at the scissile (−1/+1) bond (SPO, top right), a non-bridging phosphorothioate at the scissile bond (−1/+1 NB, bottom left) and a non-bridging phosphorothioate at the −2/−1 position (−2/−1 NB, bottom right) are shown. DNA cleavage was carried out in the presence of 10 mM Mg2+ (closed squares), Mn2+ (open circles) or Ca2+ (closed circles). Error bars represent the standard deviation of three independent experiments.

Mentions: Results with the wild-type oligonucleotide (Figure 3, top left panel) were similar to those obtained with plasmid substrate, except that higher concentrations of metal ions were required to yield optimal levels of DNA scission (data not shown; see Figure 6). Once again, Ca2+ supported (by far) the highest levels of topoisomerase IV-mediated DNA cleavage. The only difference between results with the wild-type oligonucleotide and plasmid substrates was the fact that Mn2+ consistently supported higher levels of cleavage with the oligonucleotide than did Mg2+.Figure 3.


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

Pitts SL, Liou GF, Mitchenall LA, Burgin AB, Maxwell A, Neuman KC, Osheroff N - Nucleic Acids Res. (2011)

Cleavage of oligonucleotide substrates by topoisomerase IV in the presence of different divalent metal ions. The central portion of the DNA cleavage substrate is depicted above the graphs. The asterisk denotes the 5′-end-labeled strand and the scissile bonds are indicated by arrows. All modifications are on the bottom strand. Results for the wild-type oligonucleotide (WT, top left panel), and substrates containing a 3′-bridging phosphorothiolate at the scissile (−1/+1) bond (SPO, top right), a non-bridging phosphorothioate at the scissile bond (−1/+1 NB, bottom left) and a non-bridging phosphorothioate at the −2/−1 position (−2/−1 NB, bottom right) are shown. DNA cleavage was carried out in the presence of 10 mM Mg2+ (closed squares), Mn2+ (open circles) or Ca2+ (closed circles). Error bars represent the standard deviation of three independent experiments.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3113566&req=5

Figure 3: Cleavage of oligonucleotide substrates by topoisomerase IV in the presence of different divalent metal ions. The central portion of the DNA cleavage substrate is depicted above the graphs. The asterisk denotes the 5′-end-labeled strand and the scissile bonds are indicated by arrows. All modifications are on the bottom strand. Results for the wild-type oligonucleotide (WT, top left panel), and substrates containing a 3′-bridging phosphorothiolate at the scissile (−1/+1) bond (SPO, top right), a non-bridging phosphorothioate at the scissile bond (−1/+1 NB, bottom left) and a non-bridging phosphorothioate at the −2/−1 position (−2/−1 NB, bottom right) are shown. DNA cleavage was carried out in the presence of 10 mM Mg2+ (closed squares), Mn2+ (open circles) or Ca2+ (closed circles). Error bars represent the standard deviation of three independent experiments.
Mentions: Results with the wild-type oligonucleotide (Figure 3, top left panel) were similar to those obtained with plasmid substrate, except that higher concentrations of metal ions were required to yield optimal levels of DNA scission (data not shown; see Figure 6). Once again, Ca2+ supported (by far) the highest levels of topoisomerase IV-mediated DNA cleavage. The only difference between results with the wild-type oligonucleotide and plasmid substrates was the fact that Mn2+ consistently supported higher levels of cleavage with the oligonucleotide than did Mg2+.Figure 3.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: 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.

Show MeSH
Related in: MedlinePlus