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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 the phosphorothiolate (SPO) oligonucleotide substrate in the simultaneous presence of two different divalent metal ions. Time courses were carried out in the presence of 10 mM Ca2+ alone (closed circles), 100 µM Mn2+ alone (open circles) or a mixture of 10 mM Ca2+ and 100 µM Mn2+ (open squares). The arithmetic sum of the cleavage with Ca2+ or Mn2+ alone is represented by the dotted line. Error bars represent the standard deviation of three independent experiments.
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Figure 9: Cleavage of the phosphorothiolate (SPO) oligonucleotide substrate in the simultaneous presence of two different divalent metal ions. Time courses were carried out in the presence of 10 mM Ca2+ alone (closed circles), 100 µM Mn2+ alone (open circles) or a mixture of 10 mM Ca2+ and 100 µM Mn2+ (open squares). The arithmetic sum of the cleavage with Ca2+ or Mn2+ alone is represented by the dotted line. Error bars represent the standard deviation of three independent experiments.

Mentions: Even more dramatic results were observed when the oligonucleotide that contained a bridging phosphorothiolate was utilized as the substrate. These experiments employed Ca2+, which supports only low levels of cleavage of the sulfur-containing substrate, and Mn2+, which supports the highest levels of topoisomerase IV-mediated scission of this substrate. The first experiment used 10 mM Ca2+ and 100 µM Mn2+, which (as above) represents the concentration that fills only metal ion site A (Figure 6). As seen in Figure 9, the initial rate of cleavage was ∼200-fold faster and the final level of cleavage increased ∼20-fold in reactions that contained both metal ions as compared with the arithmetic sum of cleavage observed in reactions that contained only one of the two individual metal ions. Once again, similar results were observed in reactions that combined 10 mM Ca2+ and 1–100 µM Mn2+ (Figure 10).Figure 9.


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 the phosphorothiolate (SPO) oligonucleotide substrate in the simultaneous presence of two different divalent metal ions. Time courses were carried out in the presence of 10 mM Ca2+ alone (closed circles), 100 µM Mn2+ alone (open circles) or a mixture of 10 mM Ca2+ and 100 µM Mn2+ (open squares). The arithmetic sum of the cleavage with Ca2+ or Mn2+ alone is represented by the dotted line. 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 9: Cleavage of the phosphorothiolate (SPO) oligonucleotide substrate in the simultaneous presence of two different divalent metal ions. Time courses were carried out in the presence of 10 mM Ca2+ alone (closed circles), 100 µM Mn2+ alone (open circles) or a mixture of 10 mM Ca2+ and 100 µM Mn2+ (open squares). The arithmetic sum of the cleavage with Ca2+ or Mn2+ alone is represented by the dotted line. Error bars represent the standard deviation of three independent experiments.
Mentions: Even more dramatic results were observed when the oligonucleotide that contained a bridging phosphorothiolate was utilized as the substrate. These experiments employed Ca2+, which supports only low levels of cleavage of the sulfur-containing substrate, and Mn2+, which supports the highest levels of topoisomerase IV-mediated scission of this substrate. The first experiment used 10 mM Ca2+ and 100 µM Mn2+, which (as above) represents the concentration that fills only metal ion site A (Figure 6). As seen in Figure 9, the initial rate of cleavage was ∼200-fold faster and the final level of cleavage increased ∼20-fold in reactions that contained both metal ions as compared with the arithmetic sum of cleavage observed in reactions that contained only one of the two individual metal ions. Once again, similar results were observed in reactions that combined 10 mM Ca2+ and 1–100 µM Mn2+ (Figure 10).Figure 9.

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