Locking the DNA Gate of DNA Gyrase:  Investigating the Effects on DNA Cleavage and ATP Hydrolysis

Williams, Nicola L.; Maxwell, Anthony

doi:10.1021/bi991478m

Cited by 41 publications

(26 citation statements)

References 26 publications

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“…As we show here, quinolones such as OXO and CFX in fact do not favor opening of the DNA-gate, but prevent large distortions of gyrase-bound DNA that accompany cleavage. These results are in agreement with biochemical data that demonstrated quinolone binding to gyrase/ DNA complexes in the absence of cleavage (33) and to gyrase with the DNA-gate cross-linked in the closed conformation (4).…”

Section: Discussionsupporting

confidence: 92%

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The DNA-gate of Bacillus subtilis gyrase is predominantly in the closed conformation during the DNA supercoiling reaction

Gubaev

Hilbert²,

Klostermeier³

2009

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

108

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Gyrase is the only type II topoisomerase that introduces negative supercoils into DNA. Supercoiling is catalyzed via a strand-passage mechanism, in which the gate DNA (gDNA) is transiently cleaved, and a second DNA segment, the transfer DNA (tDNA), is passed through the gap before the gDNA is religated. ATPase ͉ negative supercoiling ͉ topoisomerase ͉ single molecule FRET D NA topoisomerases modulate DNA structure by interconverting different DNA topoisomers (1). Gyrases are bacterial type II topoisomerases that use the chemical energy of ATP hydrolysis to introduce negative supercoils into DNA (2). The active form of gyrase is a heterotetramer formed by two GyrA and two GyrB subunits. Supercoiling is catalyzed via a strand-passage mechanism and involves the coordinated opening and closing of 3 different protein interfaces or gates, termed N-gate, DNA-gate, and C-gate (3-5) (Fig. 1A). As a first step, one segment of double-stranded DNA, the gate DNA (gDNA), binds to the DNA-gate formed by the GyrA subunits (6) that harbor the catalytic tyrosines (7). The transesterification reactions lead to the formation of a covalent protein-DNA intermediate (8). The N-gate is formed by the GyrB subunits, which dimerize in the presence of ATP and trap the transfer DNA (tDNA) segment (9-12). The tDNA can then pass through the gap created in the gDNA. After religation of the gDNA, the tDNA leaves the enzyme through the C-gate formed by the GyrA subunits (13,14). A concerted opening and closing of these gates is a prerequisite for tight coupling of DNA cleavage and strand passage during the supercoiling reaction. The so-called double lock rule has been proposed as a simple principle to ensure unidirectional strand passage in type II topoisomerases (15, 16) (19) and that have provided strong evidence that a tDNA is required for opening of the DNA-gate (20)(21)(22). Here, we present smFRET experiments that resolve this issue by directly monitoring the conformational state of the DNA-gate in Bacillus subtilis gyrase bound to dsDNA and during the relaxation and supercoiling reactions. These FRET experiments demonstrate that the DNA-gate is closed in the presence of linear dsDNA and during DNA relaxation or ATPdependent DNA supercoiling. Our results are in agreement with a severe distortion of gDNA coupled to DNA cleavage. DNA binding, distortion, and cleavage are mechanistically distinct steps that precede opening of the DNA-gate. Gate opening is a rare event during relaxation and supercoiling that only occurs briefly to allow for strand passage. Results A Small dsDNA Substrate Functions as the gDNA for B. subtilis Gyrase.Gyrases exhibit limited sequence specificity for DNA cleavage. To facilitate studies on the DNA gate conformation by FRET, we designed a 60-bp substrate for gyrase that contains a preferred cleavage site in the center (23), flanked by a donor (A488) and acceptor fluorophore (A546 or A555) (Fig. 1B). Cleavage at the preferred site would produce two fragments with 24/28 and 36/32 bases. When the anisotropy of A546 was...

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Section: Discussionsupporting

confidence: 92%

“…The active form of gyrase is a heterotetramer formed by two GyrA and two GyrB subunits. Supercoiling is catalyzed via a strand-passage mechanism and involves the coordinated opening and closing of 3 different protein interfaces or gates, termed N-gate, DNA-gate, and C-gate (3)(4)(5) (Fig. 1A).…”

mentioning

confidence: 99%

The DNA-gate of Bacillus subtilis gyrase is predominantly in the closed conformation during the DNA supercoiling reaction

Gubaev

Hilbert²,

Klostermeier³

2009

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

108

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“…This is in agreement with studies of a DNA gyrase enzyme in which the DNA gate was locked by cysteine crosslinking. The gyrase enzyme was able to cleave DNA, but still no DNA stimulation of the ATPase activity of the enzyme occurred (28). Thus, the step(s) leading to the pronounced increase in ATP hydrolysis is limited to the events that are very tightly coupled to DNA strand passage, most probably T-segment interactions that occur during the transport event.…”

Section: Discussionmentioning

confidence: 97%

Hindering the Strand Passage Reaction of Human Topoisomerase IIα without Disturbing DNA Cleavage, ATP Hydrolysis, or the Operation of the N-terminal Clamp

Oestergaard

Giangiacomo²,

Bjergbæk³

et al. 2004

Journal of Biological Chemistry

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DNA topoisomerase II is an essential enzyme that releases a topological strain in DNA by introduction of transient breaks in one DNA helix through which another helix is passed. While changing DNA topology, ATP is required to drive the enzyme through a series of conformational changes dependent on interdomain communication. We have characterized a human topoisomerase II␣ enzyme with a two-amino acid insertion at position 351 in the transducer domain. The mutation specifically abolishes the DNA strand passage event of the enzyme, probably because of a sterical hindrance of T-segment transport. Thus, the enzyme fails to decatenate and relax DNA, even though it is fully capable of ATP hydrolysis, closure of the N-terminal clamp, and DNA cleavage. The cleavage activity is increased, suggesting that the transducer domain has a role in regulating DNA cleavage. Furthermore, the enzyme has retained a tendency to increase DNA cleavage upon nucleotide binding and also responds to DNA with elevated ATP hydrolysis. However, the DNA-mediated increase in ATP hydrolysis is lower than that obtained with the wild-type enzyme but similar to that of a cleavage-deficient topoisomerase II␣ enzyme. Our results strongly suggest that the strand passage event is required for efficient DNA stimulation of topoisomerase II-mediated ATP hydrolysis, whereas the stimulation occurs independent of the DNA cleavage reaction per se. A comparison of the strand passage deficient-enzyme described here and the cleavage-deficient enzyme may have applications in other studies where a clear distinction between strand passage and topoisomerase II-mediated DNA cleavage is desirable.Type II DNA topoisomerases are highly complex enzymes that are able to change the topological conformation of DNA by introducing a transient double strand break in one DNA helix, the G-segment, whereas another intact helix, the T-segment, is transported coordinately through the gated DNA (1). The process depends on ATP, which drives the enzyme through a series of conformational changes (2-5).The homodimeric eukaryotic topoisomerase II enzyme contains two highly conserved catalytic entities, which also share homology with the bacterial DNA topoisomerase II counterpart, DNA gyrase. The ATPase activity is found in the Nterminal region, whereas the DNA cleavage and ligation activities are held by the central region (6). The extreme C termini of the type II topoisomerases are divergent and dispensable for catalytic activity in vitro (7-9).The structural data reveal that the N-terminal region of topoisomerase II forms an ATP-operated clamp, which closes upon ATP binding (10, 11). The dimeric N-terminal clamp contains two domains in each protomer (10). The most Nterminal domain is responsible for dimer interactions during clamp closure and also holds the ATP-binding site. The second domain, called the transducer domain, forms the walls of the clamp and connects it to the enzyme core. Furthermore, communication between the ATP-binding domain and the central domain of the enzyme, respon...

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“…DNA relaxation is a prerequisite for on-biased fimS inversion, and relaxation is associated with low rates of metabolic flux in the cell (41). Moreover, novobiocin treatment mimics specifically the effect of an unfavorable [ATP]/[ADP] ratio on DNA gyrase (44). This may represent a signal that the bacterium lacks energy, and a transition from a planktonic to an attached lifestyle might be advantageous.…”

Section: Vol 188 2006mentioning

confidence: 99%

DNA Supercoiling and the Lrp Protein Determine the Directionality of fim Switch DNA Inversion in Escherichia coli K-12

et al. 2006

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Site-specific recombinases of the integrase family usually require cofactors to impart directionality in the recombination reactions that they catalyze. The FimB integrase inverts the Escherichia coli fim switch (fimS) in the on-to-off and off-to-on directions with approximately equal efficiency. Inhibiting DNA gyrase with novobiocin caused inversion to become biased in the off-to-on direction. This directionality was not due to differential DNA topological distortion of fimS in the on and off phases by the activity of its resident P fimA promoter. Instead, the leucine-responsive regulatory (Lrp) protein was found to determine switching outcomes. Knocking out the lrp gene or abolishing Lrp binding sites 1 and 2 within fimS completely reversed the response of the switch to DNA relaxation. Inactivation of either Lrp site alone resulted in mild on-to-off bias, showing that they act together to influence the response of the switch to changes in DNA supercoiling. Thus, Lrp is not merely an architectural element organizing the fim invertasome, it collaborates with DNA supercoiling to determine the directionality of the DNA inversion event.Site-specific recombinases of the integrase family are usually associated with the integration and excision of DNA sequences such as bacteriophage genomes from bacterial chromosomes or other replicons. The best-studied integrase is Int, the prototypic member of the family that catalyzes the integration and excision of bacteriophage lambda from the chromosome of Escherichia coli (33,40). Although the integration and excision reactions both require Int, an additional phage-encoded factor called the excisionase (Xis) confers directionality by being specific for the excision reaction. Despite its name, the excisionase has no enzymatic activity. Instead, it is an architectural element that helps to organize the local structure of lambda DNA in a way that favors the excision reaction. The Xis protein has been classified as a recombination directionality factor (RDF), and several other proteins have been identified that provide, or may provide, an analogous function in other integrase-dependent site-specific recombination reactions (23-25). The requirement for the RDFs arises due to the similarities of the DNA substrates and products of the integration and excision reactions. The RDF confers directionality by stimulating one reaction while inhibiting the other.An integrase-mediated site-specific recombination event controls the phase-variable expression of type 1 fimbriae in E. coli. A key difference between the fimbrial and phage recombination mechanisms is that the fimbrial system involves DNA inversion and not integration/excision. The promoter for fim operon transcription (P fimA ) is carried on a 314-bp invertible DNA element called the fim switch (fimS), and expression of the fim structural genes depends on its orientation (1, 15). With P fimA directed toward the fim operon, the genes are transcribed, and when it is inverted to the opposite orientation, the fim operon is silent (Fig. ...

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Locking the DNA Gate of DNA Gyrase: Investigating the Effects on DNA Cleavage and ATP Hydrolysis

Cited by 41 publications

References 26 publications

The DNA-gate of Bacillus subtilis gyrase is predominantly in the closed conformation during the DNA supercoiling reaction

The DNA-gate of Bacillus subtilis gyrase is predominantly in the closed conformation during the DNA supercoiling reaction

Hindering the Strand Passage Reaction of Human Topoisomerase IIα without Disturbing DNA Cleavage, ATP Hydrolysis, or the Operation of the N-terminal Clamp

DNA Supercoiling and the Lrp Protein Determine the Directionality of fim Switch DNA Inversion in Escherichia coli K-12

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