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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 plasmid pBR322 by E. coli topoisomerase IV in the presence of different divalent metal ions. The effects of cation concentration on DNA cleavage (10-min reactions) are shown in the left panel. Mg2+ (closed squares), Mn2+ (open circles) or Ca2+ (closed circles) were titrated from 0.1 to 10 mM. Time courses for DNA cleavage in the presence of 2.5 mM divalent metal ions are shown in the right panel (Mg2+, closed squares; Mn2+, open circles; Ca2+, closed circles). Time courses in the absence of metal ion (open squares) and at the optimal concentration for Mn2+ (0.35 mM Mn2+, open triangles) also are shown. Error bars represent the standard deviation of three independent experiments.
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Figure 2: Cleavage of plasmid pBR322 by E. coli topoisomerase IV in the presence of different divalent metal ions. The effects of cation concentration on DNA cleavage (10-min reactions) are shown in the left panel. Mg2+ (closed squares), Mn2+ (open circles) or Ca2+ (closed circles) were titrated from 0.1 to 10 mM. Time courses for DNA cleavage in the presence of 2.5 mM divalent metal ions are shown in the right panel (Mg2+, closed squares; Mn2+, open circles; Ca2+, closed circles). Time courses in the absence of metal ion (open squares) and at the optimal concentration for Mn2+ (0.35 mM Mn2+, open triangles) also are shown. Error bars represent the standard deviation of three independent experiments.

Mentions: Escherichia coli topoisomerase IV, like other type II topoisomerases, requires a divalent metal ion to support DNA cleavage and overall catalytic activity (Figure 2) (40). Although the physiological metal ion appears to be magnesium, the bacterial enzyme can utilize a variety of divalent cations for the DNA cleavage reaction in vitro (40,41). Figure 2 shows the effects of metal ion concentration on topoisomerase IV-mediated DNA cleavage (left panel) as well as time courses for cleavage at constant metal ion concentrations (right panel). When negatively supercoiled plasmid was used as the substrate, Ca2+ supported the highest levels of DNA scission over a broad range of metal ion concentrations (left panel). Mg2+ and Mn2+ both supported DNA cleavage to a lesser extent (2- to 4-fold lower than Ca2+), but the optimal concentration of Mn2+ (∼0.35 mM) was nearly an order of magnitude lower than that of Mg2+ (∼2.5 mM).Figure 2.


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 plasmid pBR322 by E. coli topoisomerase IV in the presence of different divalent metal ions. The effects of cation concentration on DNA cleavage (10-min reactions) are shown in the left panel. Mg2+ (closed squares), Mn2+ (open circles) or Ca2+ (closed circles) were titrated from 0.1 to 10 mM. Time courses for DNA cleavage in the presence of 2.5 mM divalent metal ions are shown in the right panel (Mg2+, closed squares; Mn2+, open circles; Ca2+, closed circles). Time courses in the absence of metal ion (open squares) and at the optimal concentration for Mn2+ (0.35 mM Mn2+, open triangles) also are shown. Error bars represent the standard deviation of three independent experiments.
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Related In: Results  -  Collection

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Figure 2: Cleavage of plasmid pBR322 by E. coli topoisomerase IV in the presence of different divalent metal ions. The effects of cation concentration on DNA cleavage (10-min reactions) are shown in the left panel. Mg2+ (closed squares), Mn2+ (open circles) or Ca2+ (closed circles) were titrated from 0.1 to 10 mM. Time courses for DNA cleavage in the presence of 2.5 mM divalent metal ions are shown in the right panel (Mg2+, closed squares; Mn2+, open circles; Ca2+, closed circles). Time courses in the absence of metal ion (open squares) and at the optimal concentration for Mn2+ (0.35 mM Mn2+, open triangles) also are shown. Error bars represent the standard deviation of three independent experiments.
Mentions: Escherichia coli topoisomerase IV, like other type II topoisomerases, requires a divalent metal ion to support DNA cleavage and overall catalytic activity (Figure 2) (40). Although the physiological metal ion appears to be magnesium, the bacterial enzyme can utilize a variety of divalent cations for the DNA cleavage reaction in vitro (40,41). Figure 2 shows the effects of metal ion concentration on topoisomerase IV-mediated DNA cleavage (left panel) as well as time courses for cleavage at constant metal ion concentrations (right panel). When negatively supercoiled plasmid was used as the substrate, Ca2+ supported the highest levels of DNA scission over a broad range of metal ion concentrations (left panel). Mg2+ and Mn2+ both supported DNA cleavage to a lesser extent (2- to 4-fold lower than Ca2+), but the optimal concentration of Mn2+ (∼0.35 mM) was nearly an order of magnitude lower than that of Mg2+ (∼2.5 mM).Figure 2.

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