Hydrolysis of ATP at Only One GyrB Subunit Is Sufficient to Promote Supercoiling by DNA Gyrase

Kampranis, Sotirios C.; Maxwell, Anthony

doi:10.1074/jbc.273.41.26305

Cited by 29 publications

(20 citation statements)

References 19 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The heterodimer also relaxed supercoiled DNA at about one-tenth the rate of the wt enzyme (not shown). These results are similar to what was previously seen for the analogous gyrase B mutant (23).…”

supporting

confidence: 91%

Topoisomerase II drives DNA transport by hydrolyzing one ATP

Baird

Harkins

Morris

et al. 1999

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

127

128

View full text Add to dashboard Cite

DNA topoisomerase II is a homodimeric molecular machine that couples ATP usage to the transport of one DNA segment through a transient break in another segment. In the presence of a nonhydrolyzable ATP analog, the enzyme is known to promote a single turnover of DNA transport. Current models for the enzyme's mechanism based on this result have hydrolysis of two ATPs as the last step, used only to reset the enzyme for another round of reaction. Using rapid-quench techniques, topoisomerase II recently was shown to hydrolyze its two bound ATPs in a strictly sequential manner. This result is incongruous with the models based on the nonhydrolyzable ATP analog data. Here we present evidence that hydrolysis of one ATP by topoisomerase II precedes, and accelerates, DNA transport. These results indicate that important features of this enzyme's mechanism previously have been overlooked because of the reliance on nonhydrolyzable analogs for studying a single reaction turnover. A model for the mechanism of topoisomerase II is presented to show how hydrolysis of one ATP could drive DNA transport.T ype II DNA topoisomerases are ubiquitous enzymes essential for the unlinking of intertwined chromosomes, chromosomal condensation͞decondensation, and manipulation of DNA supercoiling (1, 2). These enzymes catalyze the ATP-dependent transport of one segment of DNA through a transient, enzymemediated break in a second DNA segment (for recent reviews, see refs. 3 and 4). The eukaryotic topoisomerase II enzymes are homodimers, with a monomer molecular mass of Ϸ160-180 kDa, making them relatively simple macromolecular machines for studying the coupling of ATP usage to complex protein movements. The N-terminal half of the enzyme is homologous to Escherichia coli gyrase B and contains the ATPase active site; the C-terminal half is homologous to gyrase A and contains the active site tyrosine for DNA cleavage and the primary dimerization interface. In addition to being mechanistically fascinating, the prokaryotic and eukaryotic members of this enzyme family are the targets of numerous antibiotic and anticancer drugs, respectively (5-7).The mechanism of topoisomerase II is known to involve several steps and associated protein conformational changes (reviewed in ref. 4). The enzyme binds a segment of DNA, the gate or G segment. Both strands of this DNA segment are cleaved and religated by a pair of active site tyrosines, one in each monomer of the dimer. The cleavage reaction results in a four-base staggered break in the DNA, with an enzyme monomer covalently attached to each 5Ј phosphate. Upon binding ATP, the amino-terminal ATPase domains of the enzyme dimerize, capturing a second segment of DNA, the transport or T segment, within the enzyme clamp. The ends of the cleaved G segment are separated, and the T segment is transported through the opening. The gate in the G segment is closed. The T segment is transported out of the topoisomerase, most likely through the C-terminal dimerization interface. The amino terminal dimerization interface re...

show abstract

supporting

confidence: 91%

Topoisomerase II drives DNA transport by hydrolyzing one ATP

Baird

Harkins

Morris

et al. 1999

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

127

128

View full text Add to dashboard Cite

show abstract

“…GyrB dimerization is a prerequisite for ATP hydrolysis to occur (12), although we have previously shown that the rate of ATP hydrolysis already increases with the fraction of gyrase with a narrowed N-gate (8). Gyrase contains two ATP-binding sites, but the ATP hydrolysis rate shows a linear dependence on the concentration of K ϩ , suggesting that the presence of one K ϩ ion is sufficient for nucleotide-induced GyrB dimerization and N-gate closure, in line with the observation that ATP bound to one of the subunits is sufficient to trigger GyrB dimerization and strand passage (13,26). (If two K ϩ ions were required, a dependence on the square of the K ϩ concentration would be expected.)…”

Section: Discussionsupporting

confidence: 69%

Potassium Ions Are Required for Nucleotide-induced Closure of Gyrase N-gate

Gubaev

Klostermeier

2012

Journal of Biological Chemistry

View full text Add to dashboard Cite

Background: ATP-dependent closure of the N-gate is a key step for negative DNA supercoiling by gyrase. Results: Potassium ions are required for efficient nucleotide-induced N-gate closure and DNA supercoiling but are dispensable for DNA relaxation. Conclusion: Potassium ions help coordinate the nucleotide cycle to gate movements of gyrase. Significance: Coordinated conformational changes are crucial for the function of ATP-driven molecular machines in general.DNA gyrase catalyzes ATP-dependent negative supercoiling of DNA by a strand passage mechanism that requires coordinated opening and closing of three protein interfaces, the N-, DNA-, and C-gates. ATP binding to the GyrB subunits of gyrase causes dimerization and N-gate closure. The closure of the N-gate is a key step in the gyrase catalytic cycle, as it captures the DNA segment to be transported and poises gyrase toward strand passage. We show here that K ؉ ions are required for DNA supercoiling but are dispensable for ATP-independent DNA relaxation. Although DNA binding, distortion, wrapping, and DNA-induced narrowing of the N-gate occur in the absence of K ؉ , nucleotide-induced N-gate closure depends on their presence. Our results provide evidence that K ؉ ions relay small conformational changes in the nucleotide-binding pocket to the formation of a tight dimer interface at the N-gate by connecting regions from both GyrB monomers and suggest an important role for K ؉ in synchronization of N-gate closure and DNA-gate opening.Gyrase is a DNA topoisomerase that introduces negative supercoils into plasmid DNA in an ATP-dependent reaction (1) and plays an important role in DNA replication, transcription, and recombination (2). The active unit of gyrase is composed of two GyrA and two GyrB subunits that assemble into an A 2 B 2 heterotetramer. Gyrase forms two cavities, delimited by three protein interfaces termed the N-, DNA-, and C-gates (see Fig. 1A). During ATP-dependent negative supercoiling, these gates open and close in a coordinated manner to allow for strand passage toward negative DNA supercoiling (3-8). The gyrase catalytic cycle begins when a gate DNA (G-segment) is bound at the DNA-gate and distorted (7). Interaction of DNA flanking the G-segment with the C-terminal domains (CTDs) 2 causes the CTDs to move upward and sideways (9), and complete wrapping of DNA leads to narrowing of the N-gate formed by the GyrB subunits (8). The N-gate acts as an ATPdependent clamp (10): ATP binding to the ATPase domains of the GyrB subunits induces GyrB dimerization and N-gate closure (8,(11)(12)(13), leading to the trapping of the transport DNA (T-segment). N-gate closure and DNA-gate opening appear to be coupled, with possible contributions from the T-segment (10). After passage of the T-segment through the gap in the G-segment, the G-segment is religated, the T-segment leaves the enzyme through the C-gate formed by GyrA (14,15), and the N-gate reopens (8, 12). Our recent results provide evidence for a bidirectional communication between the N-and DNAgates of g...

show abstract

“…B. subtilis GyrA is a stable dimer, even at picomolar concentrations (13), whereas isolated GyrB is monomeric (21). To prevent formation of incomplete complexes (22,23) or complex dissociation during smFRET experiments that address conformational changes of the N-gate formed by the GyrB subunits, we fused the coding regions for GyrB and GyrA, such that the two subunits are connected via a linker of the sequence G-A-P (Fig. 1A).…”

Section: Resultsmentioning

confidence: 99%

DNA-induced narrowing of the gyrase N-gate coordinates T-segment capture and strand passage

Gubaev

Klostermeier

2011

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

147

View full text Add to dashboard Cite

DNA gyrase introduces negative supercoils into DNA in an ATPdependent reaction. DNA supercoiling is catalyzed by a strandpassage mechanism, in which a T-segment of DNA is passed through the gap in a transiently cleaved G-segment. Strand passage requires the coordinated closing and opening of three protein interfaces in gyrase, the N-gate, DNA-gate, and C-gate. We show here that DNA binding to the DNA-gate of gyrase and wrapping of DNA around the C-terminal domains of GyrA induces a narrowing of the N-gate. This half-closed state prepares capture of a T-segment in the upper cavity of gyrase. Subsequent N-gate closure upon binding of ATP then poises the reaction toward strand passage. The N-gate reopens after ATP hydrolysis, allowing for further catalytic cycles. DNA binding, cleavage, and wrapping and N-gate narrowing are intimately linked events that coordinate conformational changes at the DNA and the N-gate.gyrase dynamics | nucleotide-induced conformational changes | topoisomerase mechanism | DNA wrapping | negative supercoiling

show abstract

Hydrolysis of ATP at Only One GyrB Subunit Is Sufficient to Promote Supercoiling by DNA Gyrase

Cited by 29 publications

References 19 publications

Topoisomerase II drives DNA transport by hydrolyzing one ATP

Topoisomerase II drives DNA transport by hydrolyzing one ATP

Potassium Ions Are Required for Nucleotide-induced Closure of Gyrase N-gate

DNA-induced narrowing of the gyrase N-gate coordinates T-segment capture and strand passage

Contact Info

Product

Resources

About