Coupling ATP hydrolysis to DNA strand passage in type IIA DNA topoisomerases

2005 ◽  
Vol 33 (6) ◽  
pp. 1460-1464 ◽  
Author(s):  
A. Maxwell ◽  
L. Costenaro ◽  
S. Mitelheiser ◽  
A.D. Bates
Keyword(s):  
Atp Hydrolysis ◽  
Dna Gyrase ◽  
Energy Coupling ◽  
Dna Topoisomerases ◽  
Structural Work ◽  
Structural Details ◽  
Negative Supercoiling ◽  
Topo Ii ◽  
Dna Strand ◽  
Strand Passage

Type IIA topos (topoisomerases) catalyse topological conversions of DNA through the passage of one double strand through a transient break in another. In the case of the archetypal enzyme, DNA gyrase, it has always been apparent that the enzyme couples the free energy of ATP hydrolysis to the introduction of negative supercoiling, and the structural details of this process are now becoming clearer. The homologous type IIA enzymes such as topo IV and eukaryotic topo II also require ATP and it has more recently been shown that the energy of hydrolysis is coupled to a reduction of supercoiling or catenation (linking) beyond equilibrium. The mechanism behind this effect is less clear. We review the energy coupling process in both classes of enzyme and describe recent mechanistic and structural work on gyrase that addresses the mechanism of energy coupling.

DNA supercoiling and relaxation by ATP-dependent DNA topoisomerases

1992 ◽  
Vol 336 (1276) ◽  
pp. 83-91 ◽  
Keyword(s):  
Topoisomerase Ii ◽  
Atp Hydrolysis ◽  
Energy Coupling ◽  
Dna Supercoiling ◽  
Dna Topoisomerases ◽  
Functional Conservation ◽  
Negative Supercoiling ◽  
Topo Ii ◽  
Type Ii Dna Topoisomerases

Bacterial DNA gyrase and the eukaryotic type II DNA topoisomerases are ATPases that catalyse the introduction or removal of DNA supercoils and the formation and resolution of DNA knots and catenanes. Gyrase is unique in using ATP to drive the energetically unfavourable negative supercoiling of DNA, an example of mechanochemical coupling: in contrast, eukaryotic topoisomerase II relaxes DNA in an ATP-requiring reaction. In each case, the enzyme-DNA complex acts as a ‘gate’ mediating the passage of a DNA segment through a transient enzyme-bridged double-strand DNA break. We are using a variety of genetic and enzymic approaches to probe the nature of these complexes and their mechanism of action. Recent studies will be described focusing on the role of DNA wrapping on the A 2 B 2 gyrase complex, subunit activities uncovered by using ATP analogues and the coumarin and quinolone inhibitors, and the identification and functions of discrete subunit domains. Homology between gyrase subunits and the A 2 homodimer of eukaryotic topo II suggests functional conservation between these proteins. The role of ATP hydrolysis by these topoisomerases will be discussed in regard to other energy coupling systems.

Coupling ATP hydrolysis to DNA strand passage in type IIA DNA topoisomerases

2005 ◽  
Vol 33 (6) ◽  
pp. 1460 ◽  
Author(s):  
L. Costenaro ◽  
A. Maxwell ◽  
S. Mitelheiser ◽  
A.D. Bates
Keyword(s):  
Atp Hydrolysis ◽  
Dna Topoisomerases ◽  
Dna Strand ◽  
Strand Passage

The role of ATP in the reactions of type II DNA topoisomerases

2010 ◽  
Vol 38 (2) ◽  
pp. 438-442 ◽  
Author(s):  
Andrew D. Bates ◽  
Anthony Maxwell
Keyword(s):  
Atp Hydrolysis ◽  
Dna Gyrase ◽  
Dna Topology ◽  
Type Ii ◽  
Free Energies ◽  
Dna Topoisomerases ◽  
Type Ii Dna Topoisomerases ◽  
Topology Simplification ◽  
Hydrolysis Of

Type II DNA topoisomerases catalyse changes in DNA topology in reactions coupled to the hydrolysis of ATP. In the case of DNA gyrase, which can introduce supercoils into DNA, the requirement for free energy is clear. However, the non-supercoiling type II enzymes carry out reactions that are apparently energetically favourable, so their requirement for ATP hydrolysis is not so obvious. It has been shown that many of these enzymes (the type IIA family) can simplify the topology of their DNA substrates to a level beyond that expected at equilibrium. Although this seems to explain their usage of ATP, we show that the free energies involved in topology simplification are very small (<0.2% of that available from ATP) and we argue that topology simplification may simply be an evolutionary relic.

scholarly journals CryoEM structures of open dimers of Gyrase A in complex with DNA illuminate mechanism of strand passage

2018 ◽  
Author(s):  
Katarzyna M. Soczek ◽  
Tim Grant ◽  
Peter B. Rosenthal ◽  
Alfonso Mondragon
Keyword(s):  
Conformational Changes ◽  
Dna Cleavage ◽  
Atp Hydrolysis ◽  
Unique Type ◽  
Dna Molecule ◽  
Gyrase A ◽  
A Subunit ◽  
Important Intermediate ◽  
Dna Strand ◽  
Strand Passage

AbstractGyrase is a unique type IIA topoisomerase that uses ATP hydrolysis to maintain the negatively supercoiled state of bacterial DNA. In order to perform its function, gyrase undergoes a sequence of conformational changes that consist of concerted gate openings, DNA cleavage, and DNA strand passage events. Structures where the transported DNA molecule (T-segment) is trapped by the A subunit have not been observed. Here we present the cryoEM structures of two oligomeric complexes of open gyrase A dimers and DNA. The protein subunits in these complexes were solved to 4 Å and 5.16 Å resolution. One of the complexes traps a linear DNA molecule, a putative T-segment, which interacts with the open gyrase A dimers in two states, representing steps either prior to or after passage through the DNA-gate. The structures locate the T-segment in important intermediate conformations of the catalytic cycle and provide insights into gyrase-DNA interactions and mechanism.

scholarly journals The pentapeptide-repeat protein, MfpA, interacts with mycobacterial DNA gyrase as a DNA T-segment mimic

2021 ◽  
Vol 118 (11) ◽  
pp. e2016705118
Author(s):  
Lipeng Feng ◽  
Julia E. A. Mundy ◽  
Clare E. M. Stevenson ◽  
Lesley A. Mitchenall ◽  
David M. Lawson ◽  
...  
Keyword(s):  
Molecular Mechanism ◽  
Dna Cleavage ◽  
Atp Hydrolysis ◽  
Mutational Analysis ◽  
Dna Gyrase ◽  
Dna Breaks ◽  
Repeat Protein ◽  
X Ray Crystallography ◽  
Gyrase A ◽  
Negative Supercoiling

DNA gyrase, a type II topoisomerase, introduces negative supercoils into DNA using ATP hydrolysis. The highly effective gyrase-targeted drugs, fluoroquinolones (FQs), interrupt gyrase by stabilizing a DNA-cleavage complex, a transient intermediate in the supercoiling cycle, leading to double-stranded DNA breaks. MfpA, a pentapeptide-repeat protein in mycobacteria, protects gyrase from FQs, but its molecular mechanism remains unknown. Here, we show that Mycobacterium smegmatis MfpA (MsMfpA) inhibits negative supercoiling by M. smegmatis gyrase (Msgyrase) in the absence of FQs, while in their presence, MsMfpA decreases FQ-induced DNA cleavage, protecting the enzyme from these drugs. MsMfpA stimulates the ATPase activity of Msgyrase by directly interacting with the ATPase domain (MsGyrB47), which was confirmed through X-ray crystallography of the MsMfpA–MsGyrB47 complex, and mutational analysis, demonstrating that MsMfpA mimics a T (transported) DNA segment. These data reveal the molecular mechanism whereby MfpA modulates the activity of gyrase and may provide a general molecular basis for the action of other pentapeptide-repeat proteins.

scholarly journals Modulated control of DNA supercoiling balance by the DNA-wrapping domain of bacterial gyrase

2020 ◽  
Vol 48 (4) ◽  
pp. 2035-2049
Author(s):  
Matthew J Hobson ◽  
Zev Bryant ◽  
James M Berger
Keyword(s):  
Atp Hydrolysis ◽  
Dna Supercoiling ◽  
Thermodynamic Efficiency ◽  
Futile Cycling ◽  
Negative Supercoiling ◽  
The Stability ◽  
Species Specific ◽  
Supercoil Relaxation ◽  
Strand Passage ◽  
Dna Wrapping

Abstract Negative supercoiling by DNA gyrase is essential for maintaining chromosomal compaction, transcriptional programming, and genetic integrity in bacteria. Questions remain as to how gyrases from different species have evolved profound differences in their kinetics, efficiency, and extent of negative supercoiling. To explore this issue, we analyzed homology-directed mutations in the C-terminal, DNA-wrapping domain of the GyrA subunit of Escherichia coli gyrase (the ‘CTD’). The addition or removal of select, conserved basic residues markedly impacts both nucleotide-dependent DNA wrapping and supercoiling by the enzyme. Weakening CTD–DNA interactions slows supercoiling, impairs DNA-dependent ATP hydrolysis, and limits the extent of DNA supercoiling, while simultaneously enhancing decatenation and supercoil relaxation. Conversely, strengthening DNA wrapping does not result in a more extensively supercoiled DNA product, but partially uncouples ATP turnover from strand passage, manifesting in futile cycling. Our findings indicate that the catalytic cycle of E. coli gyrase operates at high thermodynamic efficiency, and that the stability of DNA wrapping by the CTD provides one limit to DNA supercoil introduction, beyond which strand passage competes with ATP-dependent supercoil relaxation. These results highlight a means by which gyrase can evolve distinct homeostatic supercoiling setpoints in a species-specific manner.

Recent advances in understanding structure–function relationships in the type II topoisomerase mechanism

2005 ◽  
Vol 33 (6) ◽  
pp. 1465-1470 ◽  
Author(s):  
A.J. Schoeffler ◽  
J.M. Berger
Keyword(s):  
Structural Data ◽  
Dna Gyrase ◽  
Type Ii ◽  
Family Function ◽  
Dna Binding Domains ◽  
Binding Domains ◽  
Dna Strand ◽  
Multiple Domains ◽  
Strand Passage ◽  
Bacterial Type

DNA topos (topoisomerases) are complex, multisubunit enzymes that remodel DNA topology. Members of the type II topo family function by passing one segment of duplex DNA through a transient break in another, a process that consumes two molecules of ATP and requires the co-ordinated action of multiple domains. Recent structural data on type II topo ATPase regions, which activate and enforce the directionality of DNA strand passage, have highlighted how ATP physically controls the catalytic cycle of the enzyme. Structural and biochemical studies of specialized DNA-binding domains in two paralogous bacterial type IIA topos (DNA gyrase and topo IV) show how these enzymes selectively negatively supercoil or decatenate DNA. Taken together, these findings expand our understanding of how disparate functional elements work together to co-ordinate the type II topo mechanism.

scholarly journals CryoEM structures of open dimers of gyrase A in complex with DNA illuminate mechanism of strand passage

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Katarzyna M Soczek ◽  
Tim Grant ◽  
Peter B Rosenthal ◽  
Alfonso Mondragón
Keyword(s):  
Conformational Changes ◽  
Dna Cleavage ◽  
Atp Hydrolysis ◽  
Unique Type ◽  
Dna Molecule ◽  
Gyrase A ◽  
A Subunit ◽  
Important Intermediate ◽  
Dna Strand ◽  
Strand Passage

Gyrase is a unique type IIA topoisomerase that uses ATP hydrolysis to maintain the negatively supercoiled state of bacterial DNA. In order to perform its function, gyrase undergoes a sequence of conformational changes that consist of concerted gate openings, DNA cleavage, and DNA strand passage events. Structures where the transported DNA molecule (T-segment) is trapped by the A subunit have not been observed. Here we present the cryoEM structures of two oligomeric complexes of open gyrase A dimers and DNA. The protein subunits in these complexes were solved to 4 Å and 5.2 Å resolution. One of the complexes traps a linear DNA molecule, a putative T-segment, which interacts with the open gyrase A dimers in two states, representing steps either prior to or after passage through the DNA-gate. The structures locate the T-segment in important intermediate conformations of the catalytic cycle and provide insights into gyrase-DNA interactions and mechanism.

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