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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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Metal ion concentration dependence for topoisomerase IV-mediated DNA cleavage of linearized pBR322 plasmid. A 4330-bp fragment of pBR322 was singly end-labeled and employed as the cleavage substrate. Reaction products were resolved by acrylamide gel electrophoresis. DNA cleavage in the presence of varying concentrations of Mg2+ (0, 0.25, 0.5, 0.65, 0.85, 1.0, 1.5, 2.0 and 2.5 mM), Mn2+ (0, 0.025, 0.050, 0.075, 0.100, 0.125, 0.150, 0.175 and 0.200 mM) or Ca2+ (0, 0.05, 0.15, 0.25, 0.40, 0.50, 0.75 and 1.00 mM) (left, center or right, respectively) is shown. A DNA standard (DNA) also is shown. The gel is representative of three independent experiments.
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Figure 4: Metal ion concentration dependence for topoisomerase IV-mediated DNA cleavage of linearized pBR322 plasmid. A 4330-bp fragment of pBR322 was singly end-labeled and employed as the cleavage substrate. Reaction products were resolved by acrylamide gel electrophoresis. DNA cleavage in the presence of varying concentrations of Mg2+ (0, 0.25, 0.5, 0.65, 0.85, 1.0, 1.5, 2.0 and 2.5 mM), Mn2+ (0, 0.025, 0.050, 0.075, 0.100, 0.125, 0.150, 0.175 and 0.200 mM) or Ca2+ (0, 0.05, 0.15, 0.25, 0.40, 0.50, 0.75 and 1.00 mM) (left, center or right, respectively) is shown. A DNA standard (DNA) also is shown. The gel is representative of three independent experiments.

Mentions: Metal ion concentration dependence for topoisomerase IV-mediated DNA cleavage of linearized pBR322 plasmid. Data from Figure 4 and additional experiments were quantified. Results for Mg2+(left), Mn2+(middle) and Ca2+ (right) are shown. All values were quantified relative to the most abundant cleavage product (generated at 0.75 mM Ca2+), which was set to 100. Error bars represent the standard deviation of three independent experiments.


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)

Metal ion concentration dependence for topoisomerase IV-mediated DNA cleavage of linearized pBR322 plasmid. A 4330-bp fragment of pBR322 was singly end-labeled and employed as the cleavage substrate. Reaction products were resolved by acrylamide gel electrophoresis. DNA cleavage in the presence of varying concentrations of Mg2+ (0, 0.25, 0.5, 0.65, 0.85, 1.0, 1.5, 2.0 and 2.5 mM), Mn2+ (0, 0.025, 0.050, 0.075, 0.100, 0.125, 0.150, 0.175 and 0.200 mM) or Ca2+ (0, 0.05, 0.15, 0.25, 0.40, 0.50, 0.75 and 1.00 mM) (left, center or right, respectively) is shown. A DNA standard (DNA) also is shown. The gel is representative of three independent experiments.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 4: Metal ion concentration dependence for topoisomerase IV-mediated DNA cleavage of linearized pBR322 plasmid. A 4330-bp fragment of pBR322 was singly end-labeled and employed as the cleavage substrate. Reaction products were resolved by acrylamide gel electrophoresis. DNA cleavage in the presence of varying concentrations of Mg2+ (0, 0.25, 0.5, 0.65, 0.85, 1.0, 1.5, 2.0 and 2.5 mM), Mn2+ (0, 0.025, 0.050, 0.075, 0.100, 0.125, 0.150, 0.175 and 0.200 mM) or Ca2+ (0, 0.05, 0.15, 0.25, 0.40, 0.50, 0.75 and 1.00 mM) (left, center or right, respectively) is shown. A DNA standard (DNA) also is shown. The gel is representative of three independent experiments.
Mentions: Metal ion concentration dependence for topoisomerase IV-mediated DNA cleavage of linearized pBR322 plasmid. Data from Figure 4 and additional experiments were quantified. Results for Mg2+(left), Mn2+(middle) and Ca2+ (right) are shown. All values were quantified relative to the most abundant cleavage product (generated at 0.75 mM Ca2+), which was set to 100. Error bars represent the standard deviation of three independent experiments.

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