The use of divalent metal ions by type II topoisomerases

Deweese, Joseph E.; Osheroff, Neil

doi:10.1039/c003759a

Cited by 70 publications

(80 citation statements)

References 141 publications

(545 reference statements)

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“…With this context in mind, the FRET jumps specifically observed only in cleavable sequences in Fig The requirement of Mg 2þ ions for DNA bending is reminiscent of the critical role of divalent ions in G-segment cleavage (32). This requirement raises a question as to whether the bending conformation observed in the previous experiments represents a precleavage complex or a product of the cleavage reaction.…”

Section: Resultsmentioning

confidence: 84%

“…S13). Collectively, these results illustrate that the conserved acidic residues in the TOPRIM region (E461, D541, and D543), which are responsible for binding the active site metal ions (30,32) DNA, per se, is not sufficient for DNA cleavage, but a more detailed coordination of protein, DNA, and divalent ions is required.…”

Section: Resultsmentioning

confidence: 95%

“…In bulk DNA cleavage assays, the cleavage reaction of hTopoIIα was greatly hampered by these mutations (32).…”

Section: Resultsmentioning

confidence: 99%

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DNA cleavage and opening reactions of human topoisomerase IIα are regulated via Mg ²⁺ -mediated dynamic bending of gate-DNA

Lee

Jung²,

Heo³

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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Topoisomerase II resolves intrinsic topological problems of doublestranded DNA. As part of its essential cellular functions, the enzyme generates DNA breaks, but the regulation of this potentially dangerous process is not well understood. Here we report singlemolecule fluorescence experiments that reveal a previously uncharacterized sequence of events during DNA cleavage by topoisomerase II: nonspecific DNA binding, sequence-specific DNA bending, and stochastic cleavage of DNA. We have identified unexpected structural roles of Mg 2þ ions coordinated in the TOPRIM (topoisomerase-primase) domain in inducing cleavage-competent DNA bending. A break at one scissile bond dramatically stabilized DNA bending, explaining how two scission events in opposing strands can be coordinated to achieve a high probability of double-stranded cleavage. Clamping of the protein N-gate greatly enhanced the rate and degree of DNA bending, resulting in a significant stimulation of the DNA cleavage and opening reactions. Our data strongly suggest that the accurate cleavage of DNA by topoisomerase II is regulated through a tight coordination with DNA bending.he double helical nature of DNA imposes intrinsic topological problems during replication, repair, and transcription (1-3). Additionally, the topological state of the genetic material needs to be tightly regulated in order to promote proper biochemical interactions between DNA and a variety of proteins (1-4). Topoisomerases are enzymes that resolve topological problems within the double helix by repeated cycles of DNA cleavage and ligation (1-3, 5, 6).As a subclass of the topoisomerase family, type II topoisomerases are found in all organisms from bacteria to human, and even in some viruses (1-3, 5, 6). The essential roles of type II topoisomerases in cell metabolism, differences between bacterial and human homologues, and hyperactivation of these enzymes in cancer cells have been utilized for clinical treatments of bacterial infections and numerous cancers (3, 7-9).Extensive studies for more than twenty years have established that type II topoisomerases use a "two gate" mechanism for DNA strand passage (3, 10, 11), in which a DNA duplex (the transport or T-segment) is transported through an enzyme-mediated transient opening in a separate DNA duplex (the gate or G-segment). The directionality of strand passage is the N-terminal gate of the enzyme to the C-terminal gate. As a result of the double-stranded DNA passage mechanism, each catalytic event changes the linking number of DNA by two. The transport of the T-segment through the G-segment is thought to be initiated by the N-gate clamping motion induced by the binding of ATP to the enzyme (3,6,10,12).Although the double-stranded DNA breaks generated by type II topoisomerases are essential for the cellular functions of these enzymes, it is a dangerous process in which an aberrant operation can damage chromosomal integrity. In fact, widely prescribed anticancer and antibacterial drugs initiate cell death by increasing the cellular conce...

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Section: Resultsmentioning

confidence: 84%

Section: Resultsmentioning

confidence: 95%

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DNA cleavage and opening reactions of human topoisomerase IIα are regulated via Mg ²⁺ -mediated dynamic bending of gate-DNA

Lee

Jung²,

Heo³

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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“…35). A similar phenomenon likely occurs with several antitumor agents and human type II topoisomerases (36). Because cleaved complex formation can be reversed in several ways and assayed as resealing of plasmid DNA, exceptionally strong drug-enzyme interactions can be readily observed.…”

Section: Discussionmentioning

confidence: 90%

Fluoroquinolone-Gyrase-DNA Complexes

Mustaev

Malik

Zhao

et al. 2014

Journal of Biological Chemistry

143

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Background: X-ray crystal structures of fluoroquinolone-gyrase-DNA complexes reveal a single drug-binding mode. Results: A ciprofloxacin derivative with a chloroacetyl moiety at the C-7 end cross-linked with cysteine substitutions in both GyrA and GyrB that were 17 Å apart. Conclusion: Cleaved complexes containing gyrase have two fluoroquinolone-binding modes. Significance: The additional drug-binding mode provides new ways to investigate inhibitor-topoisomerase interactions.

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“…Interestingly, the Mg 2+ to Ca 2+ substitution effects are typically also similarly inhibitory 76 for two-metal ion catalysis as well as for the one-metal ion catalysis in dUTPase [21][22] and in several ATPases [77][78][79][80][81] and kinases 78,[82][83] with some notable exceptions. [84][85] This key metal ion often coordinates the cleaved phosphate. This helps the catalysis by lowering the pK a of the phosphate group.…”

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confidence: 99%

Mutations Decouple Proton Transfer from Phosphate Cleavage in the dUTPase Catalytic Reaction

et al. 2015

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Most enzymes present a catalytic mechanism where one or more proton transfer events occur coupled tightly together with the enzymatic chemical reaction. We show here that inactivating mutations decouple this proton transfer step from the phosphate cleavage reaction in dUTPase. Homotrimeric dUTPase enzymes catalyze the hydrolysis of dUTP to dUMP and pyrophosphate, using largely similar structural and functional groups as most AAA+ enzymes. dUTPases typically use a single Mg 2+ ion as a cofactor in the active site that is formed by direct protein-protein contacts including all three protomers. Here we focus on the C-terminal arm structural motif, which has sequence and functional similarities to P-loop motifs, and is required for catalysis. In this work, we have studied the functional roles of the C-terminal arm in ligand binding and catalysis by using QM/MM (quantum mechanics/molecular mechanics) calculations in conjunction with site-directed mutagenesis experiments. We also present a new method to assess the metal ion coordination symmetry during the catalytic reaction. Using this new implementation, we identified that the coordination symmetry follows a consistent pattern in the three systems studied, reaching the most symmetrical state near the transition states. We found that the phosphate cleavage proceeds with a concerted bimolecular (A N D N ) mechanism with a loose dissociative transition state, and that it is coupled with a proton transfer step involving a unanimously conserved Asp residue. We show that the main mechanistic effect of the lack of the C-terminal arm is to decouple the phosphate cleavage from the subsequent proton transfer step, resulting in a highbarrier altered reaction pathway.

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The use of divalent metal ions by type II topoisomerases

Cited by 70 publications

References 141 publications

DNA cleavage and opening reactions of human topoisomerase IIα are regulated via Mg ²⁺ -mediated dynamic bending of gate-DNA

DNA cleavage and opening reactions of human topoisomerase IIα are regulated via Mg ²⁺ -mediated dynamic bending of gate-DNA

Fluoroquinolone-Gyrase-DNA Complexes

Mutations Decouple Proton Transfer from Phosphate Cleavage in the dUTPase Catalytic Reaction

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The use of divalent metal ions by type II topoisomerases

Cited by 70 publications

References 141 publications

DNA cleavage and opening reactions of human topoisomerase IIα are regulated via Mg 2+ -mediated dynamic bending of gate-DNA

DNA cleavage and opening reactions of human topoisomerase IIα are regulated via Mg 2+ -mediated dynamic bending of gate-DNA

Fluoroquinolone-Gyrase-DNA Complexes

Mutations Decouple Proton Transfer from Phosphate Cleavage in the dUTPase Catalytic Reaction

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Product

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DNA cleavage and opening reactions of human topoisomerase IIα are regulated via Mg ²⁺ -mediated dynamic bending of gate-DNA

DNA cleavage and opening reactions of human topoisomerase IIα are regulated via Mg ²⁺ -mediated dynamic bending of gate-DNA