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.

Differential contributions of the latch in Thermotoga maritima reverse gyrase to the binding of single-stranded DNA before and after ATP hydrolysis

2014 ◽  
Vol 395 (1) ◽  
pp. 83-93 ◽  
Author(s):  
Yoandris del Toro Duany ◽  
Agneyo Ganguly ◽  
Dagmar Klostermeier
Keyword(s):  
Dna Binding ◽  
Atp Hydrolysis ◽  
Thermotoga Maritima ◽  
Dna Supercoiling ◽  
Helicase Domain ◽  
Reverse Gyrase ◽  
Single Stranded Dna ◽  
Phosphate Release ◽  
Before And After ◽  
Strand Passage

Abstract Reverse gyrase catalyzes the ATP-dependent introduction of positive supercoils into DNA. Supercoiling requires the functional cooperation of its N-terminal helicase domain with the C-terminal topoisomerase domain. The helicase domain contains a superfamily 2 helicase core formed by two RecA domains, H1 and H2. We show here that a helicase domain lacking the latch, an insertion in H2, fails to close the cleft in the helicase core in response to nucleotide and DNA binding at the beginning of the catalytic cycle. In the presence of the pre-hydrolysis ATP analog ADP·BeFx, however, the closed conformer can still be formed in the absence of the latch. The helicase domain lacking the latch exhibits reduced DNA affinities. The energetic difference between the two nucleotide states involved in duplex separation is diminished, rationalizing the unwinding deficiency of reverse gyrase lacking the latch. The latch most strongly contributes to binding of single-stranded DNA in the post-hydrolysis state, before phosphate release. Our results are in line with contributions of the latch in determining the direction of strand passage, and in orienting the cleaved single-stranded DNA for re-ligation. At the same time, the latch may coordinate the re-ligation reaction with strand passage and with the nucleotide cycle.

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.

scholarly journals Functional interactions between gyrase subunits are optimized in a species-specific manner

2020 ◽  
Vol 295 (8) ◽  
pp. 2299-2312
Author(s):  
Daniela Weidlich ◽  
Dagmar Klostermeier
Keyword(s):  
Atp Hydrolysis ◽  
Dna Supercoiling ◽  
Structural Features ◽  
Subunit Interaction ◽  
Dna Topoisomerase ◽  
Bacterial Dna ◽  
E Coli ◽  
Functional Interactions ◽  
Species Specific ◽  
Gyrase Inhibitors

DNA gyrase is a bacterial DNA topoisomerase that catalyzes ATP-dependent negative DNA supercoiling and DNA decatenation. The enzyme is a heterotetramer comprising two GyrA and two GyrB subunits. Its overall architecture is conserved, but species-specific elements in the two subunits are thought to optimize subunit interaction and enzyme function. Toward understanding the roles of these different elements, we compared the activities of Bacillus subtilis, Escherichia coli, and Mycobacterium tuberculosis gyrases and of heterologous enzymes reconstituted from subunits of two different species. We show that B. subtilis and E. coli gyrases are proficient DNA-stimulated ATPases and efficiently supercoil and decatenate DNA. In contrast, M. tuberculosis gyrase hydrolyzes ATP only slowly and is a poor supercoiling enzyme and decatenase. The heterologous enzymes are generally less active than their homologous counterparts. The only exception is a gyrase reconstituted from mycobacterial GyrA and B. subtilis GyrB, which exceeds the activity of M. tuberculosis gyrase and reaches the activity of the B. subtilis gyrase, indicating that the activities of enzymes containing mycobacterial GyrB are limited by ATP hydrolysis. The activity pattern of heterologous gyrases is in agreement with structural features present: B. subtilis gyrase is a minimal enzyme, and its subunits can functionally interact with subunits from other bacteria. In contrast, the specific insertions in E. coli and mycobacterial gyrase subunits appear to prevent efficient functional interactions with heterologous subunits. Understanding the molecular details of gyrase adaptations to the specific physiological requirements of the respective organism might aid in the development of species-specific gyrase inhibitors.

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.

scholarly journals Single-molecule imaging of DNA gyrase activity in livingEscherichia coli

2018 ◽  
Author(s):  
Mathew Stracy ◽  
Adam J.M. Wollman ◽  
Elzbieta Kaja ◽  
Jacek Gapinski ◽  
Ji-Eun Lee ◽  
...  
Keyword(s):  
Single Molecule ◽  
High Speed ◽  
Replication Fork ◽  
Dna Gyrase ◽  
Chromosomal Dna ◽  
Replication Fork Progression ◽  
Dna Strands ◽  
Steady State Levels ◽  
Negative Supercoiling ◽  
Supercoil Relaxation

ABSTRACTBacterial DNA gyrase introduces negative supercoils into chromosomal DNA and relaxes positive supercoils introduced by replication and transiently by transcription. Removal of these positive supercoils is essential for replication fork progression and for the overall unlinking of the two duplex DNA strands, as well as for ongoing transcription. To address how gyrase copes with these topological challenges, we used high-speed single-molecule fluorescence imaging in liveEscherichia colicells. We demonstrate that at least 300 gyrase molecules are stably bound to the chromosome at any time, with ∼12 enzymes enriched near each replication fork. Trapping of reaction intermediates with ciprofloxacin revealed complexes undergoing catalysis. Dwell times of ∼2 s were observed for the dispersed gyrase molecules, which we propose maintain steady-state levels of negative supercoiling of the chromosome. In contrast, the dwell time of replisome-proximal molecules was ∼8 s, consistent with these catalyzing processive positive supercoil relaxation in front of the progressing replisome.

scholarly journals COMPENSATION EFFECT OF MORTALITY: A CHALLENGE TO LIFE EXTENSION

2019 ◽  
Vol 3 (Supplement_1) ◽  
pp. S966-S967
Author(s):  
Natalia S Gavrilova ◽  
Leonid Gavrilov
Keyword(s):  
Life Span ◽  
Compensation Effect ◽  
Life Extension ◽  
Slope Parameter ◽  
Aging Rate ◽  
Convergence Point ◽  
Gompertz Law ◽  
The 1960S ◽  
The Stability ◽  
Species Specific

Abstract In order to develop genuine anti-aging interventions it is important to find the best estimate of the aging rate in humans, which is often measured as a slope parameter of the Gompertz law. The compensation effect of mortality (CEM), refers to mortality convergence, when higher values for the slope parameter are compensated by lower values of the intercept parameter (initial mortality) in different populations of a given species. The age of this convergence point is called the "species-specific life span". Due to CEM, factors associated with life span extension are usually accompanied by paradoxical increase in actuarial aging rate. We evaluated the stability of CEM by analyzing the United Nations abridged life tables for 241 countries and regions and estimating parameters of the Gompertz-Makeham model using method of non-linear regression in the age interval 30-80 years. We found that the species-specific lifespan is equal to 94.5 ± 0.5 years, which is the same as reported in the past for years before the 1960s: 95 ± 3 years (Gavrilov, Gavrilova, 1991). Thus, the convergence point of CEM is stable despite significant mortality decline over past 50 years and is not affected by factors decreasing mortality at younger ages. Populations deviating from CEM with apparently slow aging (with both slow actuarial aging rate and low intercept parameter) have been identified. The existence of CEM in mice (ITP data) allowed us to find interventions that are able to both extend lifespan and slow the actuarial aging rate giving promise for radical life extension.

scholarly journals An ATP/ADP-Dependent Molecular Switch Regulates the Stability of p53-DNA Complexes

1999 ◽  
Vol 19 (11) ◽  
pp. 7501-7510 ◽  
Author(s):  
Andrei L. Okorokov ◽  
Jo Milner
Keyword(s):  
Dna Damage ◽  
Dna Binding ◽  
Cellular Response ◽  
Atp Hydrolysis ◽  
P53 Protein ◽  
Molecular Switch ◽  
Dna Complexes ◽  
Switch Mechanism ◽  
The Stability ◽  
First Time

ABSTRACT Interaction with DNA is essential for the tumor suppressor functions of p53. We now show, for the first time, that the interaction of p53 with DNA can be stabilized by small molecules, such as ADP and dADP. Our results also indicate an ATP/ADP molecular switch mechanism which determines the off-on states for p53-DNA binding. This ATP/ADP molecular switch requires dimer-dimer interaction of the p53 tetramer. Dissociation of p53-DNA complexes by ATP is independent of ATP hydrolysis. Low-level ATPase activity is nonetheless associated with ATP-p53 interaction and may serve to regenerate ADP-p53, thus recycling the high-affinity DNA binding form of p53. The ATP/ADP regulatory mechanism applies to two distinct types of p53 interaction with DNA, namely, sequence-specific DNA binding (via the core domain of the p53 protein) and binding to sites of DNA damage (via the C-terminal domain). Further studies indicate that ADP not only stabilizes p53-DNA complexes but also renders the complexes susceptible to dissociation by specific p53 binding proteins. We propose a model in which the DNA binding functions of p53 are regulated by an ATP/ADP molecular switch, and we suggest that this mechanism may function during the cellular response to DNA damage.

scholarly journals Active-Site Residues of Escherichia coli DNA Gyrase Required in Coupling ATP Hydrolysis to DNA Supercoiling and Amino Acid Substitutions Leading to Novobiocin Resistance

2003 ◽  
Vol 47 (3) ◽  
pp. 1037-1046 ◽  
Author(s):  
Christian H. Gross ◽  
Jonathan D. Parsons ◽  
Trudy H. Grossman ◽  
Paul S. Charifson ◽  
Steven Bellon ◽  
...  
Keyword(s):  
Amino Acid ◽  
Atp Hydrolysis ◽  
Dna Gyrase ◽  
Dna Supercoiling ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Mutant Proteins ◽  
E Coli ◽  
Novobiocin Resistance

ABSTRACT DNA gyrase is a bacterial type II topoisomerase which couples the free energy of ATP hydrolysis to the introduction of negative supercoils into DNA. Amino acids in proximity to bound nonhydrolyzable ATP analog (AMP · PNP) or novobiocin in the gyrase B (GyrB) subunit crystal structures were examined for their roles in enzyme function and novobiocin resistance by site-directed mutagenesis. Purified Escherichia coli GyrB mutant proteins were complexed with the gyrase A subunit to form the functional A2B2 gyrase enzyme. Mutant proteins with alanine substitutions at residues E42, N46, E50, D73, R76, G77, and I78 had reduced or no detectable ATPase activity, indicating a role for these residues in ATP hydrolysis. Interestingly, GyrB proteins with P79A and K103A substitutions retained significant levels of ATPase activity yet demonstrated no DNA supercoiling activity, even with 40-fold more enzyme than the wild-type enzyme, suggesting that these amino acid side chains have a role in the coupling of the two activities. All enzymes relaxed supercoiled DNA to the same extent as the wild-type enzyme did, implying that only ATP-dependent reactions were affected. Mutant genes were examined in vivo for their abilities to complement a temperature-sensitive E. coli gyrB mutant, and the activities correlated well with the in vitro activities. We show that the known R136 novobiocin resistance mutations bestow a significant loss of inhibitor potency in the ATPase assay. Four new residues (D73, G77, I78, and T165) that, when changed to the appropriate amino acid, result in both significant levels of novobiocin resistance and maintain in vivo function were identified in E. coli.

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