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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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Related in: MedlinePlus

Cleavage of the wild-type (WT) oligonucleotide substrate in the simultaneous presence of two different divalent metal ions. Time courses were carried out in the presence of 10 mM Mg2+ alone (closed squares), 1 mM Ca2+ alone (closed circles) or a mixture of 10 mM Mg2+ and 1 mM Ca2+ (open squares). The arithmetic sum of the cleavage with Mg2+ or Ca2+ alone is represented by the dotted line. Error bars represent the standard deviation of three independent experiments.
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Figure 7: Cleavage of the wild-type (WT) oligonucleotide substrate in the simultaneous presence of two different divalent metal ions. Time courses were carried out in the presence of 10 mM Mg2+ alone (closed squares), 1 mM Ca2+ alone (closed circles) or a mixture of 10 mM Mg2+ and 1 mM Ca2+ (open squares). The arithmetic sum of the cleavage with Mg2+ or Ca2+ alone is represented by the dotted line. Error bars represent the standard deviation of three independent experiments.

Mentions: As a second approach to determine the number of metal ions required to support the DNA cleavage reaction of bacterial type II topoisomerases, metal ion mixing experiments were performed (19,24,26). Initial studies utilized the wild-type oligonucleotide substrate and mixtures of Mg2+ and Ca2+. This metal ion pair was used because Mg2+ supports DNA cleavage of the wild-type substrate poorly, even at high concentrations, while Ca2+ efficiently supports high levels of scission. The first time courses for DNA cleavage were generated in the presence of either 10 mM Mg2+, a saturating level, or 1 mM Ca2+, a concentration that saturates the site for metal ion A without appreciably filling the site for metal ion B (Figure 6). As seen in Figure 7, similar low levels of cleavage were generated when either one of the metal ions was included in assays. However, when both metal ions were present in reactions, rates of cleavage increased several-fold above levels predicted from the simple arithmetic sum of scission observed in the presence of the individual metal ions. Furthermore, a large enhancement in the rate and level of DNA scission (compared with calculated sums) was observed when 10 mM Mg2+ was combined with a range of Ca2+ concentrations (0.1–1 mM) that partially filled or saturated the high affinity metal ion site (Figure 8).Figure 7.


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 wild-type (WT) oligonucleotide substrate in the simultaneous presence of two different divalent metal ions. Time courses were carried out in the presence of 10 mM Mg2+ alone (closed squares), 1 mM Ca2+ alone (closed circles) or a mixture of 10 mM Mg2+ and 1 mM Ca2+ (open squares). The arithmetic sum of the cleavage with Mg2+ or Ca2+ 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 7: Cleavage of the wild-type (WT) oligonucleotide substrate in the simultaneous presence of two different divalent metal ions. Time courses were carried out in the presence of 10 mM Mg2+ alone (closed squares), 1 mM Ca2+ alone (closed circles) or a mixture of 10 mM Mg2+ and 1 mM Ca2+ (open squares). The arithmetic sum of the cleavage with Mg2+ or Ca2+ alone is represented by the dotted line. Error bars represent the standard deviation of three independent experiments.
Mentions: As a second approach to determine the number of metal ions required to support the DNA cleavage reaction of bacterial type II topoisomerases, metal ion mixing experiments were performed (19,24,26). Initial studies utilized the wild-type oligonucleotide substrate and mixtures of Mg2+ and Ca2+. This metal ion pair was used because Mg2+ supports DNA cleavage of the wild-type substrate poorly, even at high concentrations, while Ca2+ efficiently supports high levels of scission. The first time courses for DNA cleavage were generated in the presence of either 10 mM Mg2+, a saturating level, or 1 mM Ca2+, a concentration that saturates the site for metal ion A without appreciably filling the site for metal ion B (Figure 6). As seen in Figure 7, similar low levels of cleavage were generated when either one of the metal ions was included in assays. However, when both metal ions were present in reactions, rates of cleavage increased several-fold above levels predicted from the simple arithmetic sum of scission observed in the presence of the individual metal ions. Furthermore, a large enhancement in the rate and level of DNA scission (compared with calculated sums) was observed when 10 mM Mg2+ was combined with a range of Ca2+ concentrations (0.1–1 mM) that partially filled or saturated the high affinity metal ion site (Figure 8).Figure 7.

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