The mechanism of negative DNA supercoiling: A cascade of DNA-induced conformational changes prepares gyrase for strand passage

Gyrase is a bacterial type IIA topoisomerase that catalyzes negative supercoiling of DNA. The enzyme is essential in bacteria and is a validated drug target in the treatment of bacterial infections. Inhibition of gyrase activity is achieved by competitive inhibitors that interfere with ATP- or DNA-binding, or by gyrase poisons that stabilize cleavage complexes of gyrase covalently bound to the DNA, leading to double-strand breaks and cell death. Many of the current inhibitors suffer from severe side effects, while others rapidly lose their antibiotic activity due to resistance mutations, generating an unmet medical need for novel, improved gyrase inhibitors. DNA supercoiling by gyrase is associated with a series of nucleotide- and DNA-induced conformational changes, yet the full potential of interfering with these conformational changes as a strategy to identify novel, improved gyrase inhibitors has not been explored so far. This review highlights recent insights into the mechanism of DNA supercoiling by gyrase and illustrates the implications for the identification and development of conformation-sensitive and allosteric inhibitors.

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

10.1101/397000 ◽

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.

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Crystal structures of Sulfolobus solfataricus topoisomerase III reveal that its C-terminal novel zinc finger part is a unique decatenation domain

10.1101/706986 ◽

2019 ◽

Author(s):

Hanqian Wang ◽

Junhua Zhang ◽

Xin Zheng ◽

ZhenFeng Zhang ◽

Zhiyong Zhang ◽

...

Keyword(s):

Crystal Structures ◽

Zinc Finger ◽

Conformational Changes ◽

Sulfolobus Solfataricus ◽

Dna Topoisomerases ◽

Cellular Processes ◽

Topoisomerase Iii ◽

Type Ia ◽

Strand Passage ◽

Domain V

AbstractDNA topoisomerases are essential enzymes for a variety of cellular processes involved in DNA transactions. Many of the mechanistic insights into type IA DNA topoisomerases have principally come from studies on the prokaryotes and eukaryotes. However, a structural understanding of type IA topoisomerases in the Archaeal is lacking. Here we report the crystal structures of full-length Sulfolobus solfataricus topoisomerase III (Sso topo III) both by itself and in complex with an 8-base single-stranded DNA fragment, which were determined at 2.1 Å and 2.5 Å, respectively. The structures show that, as a member of type IA topoisomerases, Sso topo III adopts a torus-like architecture consisting of a four-domain core region and a novel C-terminal zinc finger domain (domain V). Upon binding to ssDNA, Sso topo III undergoes dramatic conformational changes, similar to those of other type IA topoisomerases. Structural analyses and biochemical assays revealed that domain V is essential for the DNA decatenation activity of Sso topo III. These findings establish Sso topo III as an alternative prototype of type IA topoisomerases to further understand the loop-independent decatenation mechanism in the enzyme-bridged strand passage model.

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Direct Observation of Topoisomerase IA Gate Dynamics

10.1101/356659 ◽

2018 ◽

Author(s):

Maria Mills ◽

Yuk-Ching Tse-Dinh ◽

Keir C. Neuman

Keyword(s):

Single Molecule ◽

Conformational Changes ◽

Mechanistic Model ◽

Cellular Functions ◽

Single Stranded Dna ◽

Gate Opening ◽

Type Ia ◽

Dna Strand ◽

Dynamics Simulations ◽

Strand Passage

AbstractType IA topoisomerases cleave single-stranded DNA and relieve negative supercoils in discrete steps corresponding to the passage of the intact DNA strand through the cleaved strand. Although it is assumed type IA topoisomerases accomplish this strand passage via a protein-mediated DNA gate, opening of this gate has never been observed. We developed a single-molecule assay to directly measure gate opening of the E. coli type IA topoisomerases I and III. We found that following cleavage of single-stranded DNA, the protein gate opens by as much as 6.6 nm and can close against forces in excess of 16 pN. Key differences in the cleavage, ligation and gate dynamics of these two enzymes provide insights into their different cellular functions. The single-molecule results are broadly consistent with conformational changes obtained from molecular dynamics simulations. These results allow us to develop a mechanistic model of type IA topoisomerase-ssDNA interactions.

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BZIP Transcription Factors Modulate DNA Supercoiling Transitions

10.1101/2019.12.13.875146 ◽

2019 ◽

Cited By ~ 3

Author(s):

Johanna Hörberg ◽

Anna Reymer

Keyword(s):

Transcription Factors ◽

Conformational Changes ◽

Molecular Motors ◽

Specific Binding ◽

Dna Supercoiling ◽

Bzip Transcription Factor ◽

Specific Response ◽

Torsional Stress ◽

Nucleosome Remodeling ◽

The Impact

ABSTRACTTorsional stress on DNA, introduced by molecular motors, constitutes an important regulatory mechanism of transcriptional control. Torsional stress can modulate specific binding of transcription factors to DNA and introduce local conformational changes that facilitate the opening of promoters and nucleosome remodeling. Using all-atom microsecond scale molecular dynamics simulations together with a torsional restraint that controls the total helical twist of a DNA fragment, we addressed the impact of torsional stress on DNA complexation with a human BZIP transcription factor, MafB. We gradually over- and underwind DNA alone and in complex with MafB by 5° per dinucleotide step, monitoring the evolution of the protein-DNA contacts at different degrees of torsional strain. Our computations show that MafB changes the DNA sequence-specific response to torsional stress. The dinucleotide steps that are susceptible to absorb most of the torsional stress become more torsionally rigid, as they are involved in the protein-DNA contacts. Also, the protein undergoes substantial conformational changes to follow the stress-induced DNA deformation, but mostly maintains the specific contacts with DNA. This results in a significant asymmetric increase of free energy of DNA twisting transitions, relative to free DNA, where overtwisting is more energetically unfavorable. Our data suggest that MafB could act as a torsional stress insulator, modulating the propagation of torsional stress along the chromatin fiber, which might promote cooperative binding of other transcription factors.

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

Biological Chemistry ◽

10.1515/hsz-2013-0177 ◽

2014 ◽

Vol 395 (1) ◽

pp. 83-93 ◽

Cited By ~ 4

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.

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New Insights into Structural and Functional Roles of Indole-3-acetic acid (IAA): Changes in DNA Topology and Gene Expression in Bacteria

Biomolecules ◽

10.3390/biom9100522 ◽

2019 ◽

Vol 9 (10) ◽

pp. 522 ◽

Cited By ~ 2

Author(s):

Defez ◽

Valenti ◽

Andreozzi ◽

Romano ◽

Ciaramella ◽

...

Keyword(s):

Gene Expression ◽

Acetic Acid ◽

Conformational Changes ◽

Regulation Of Gene Expression ◽

Dna Supercoiling ◽

Dna Topology ◽

In Vitro Assays ◽

Bacterial Cells ◽

Indole 3 Acetic Acid

: Indole-3-acetic acid (IAA) is a major plant hormone that affects many cellular processes in plants, bacteria, yeast, and human cells through still unknown mechanisms. In this study, we demonstrated that the IAA-treatment of two unrelated bacteria, the Ensifer meliloti 1021 and Escherichia coli, harboring two different host range plasmids, influences the supercoiled state of the two plasmid DNAs in vivo. Results obtained from in vitro assays show that IAA interacts with DNA, leading to DNA conformational changes commonly induced by intercalating agents. We provide evidence that IAA inhibits the activity of the type IA topoisomerase, which regulates the DNA topological state in bacteria, through the relaxation of the negative supercoiled DNA. In addition, we demonstrate that the treatment of E. meliloti cells with IAA induces the expression of some genes, including the ones related to nitrogen fixation. In contrast, these genes were significantly repressed by the treatment with novobiocin, which reduces the DNA supercoiling in bacterial cells. Taking into account the overall results reported, we hypothesize that the IAA action and the DNA structure/function might be correlated and involved in the regulation of gene expression. This work points out that checking whether IAA influences the DNA topology under physiological conditions could be a useful strategy to clarify the mechanism of action of this hormone, not only in plants but also in other unrelated organisms.

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