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Escherichia coli gyrase holocomplex with 217 bp phage Mu SGS DNA and albicidin stabilised by ADPNP

ABSTRACT:

SUBMITTER: Dmitry Ghilarov

PROVIDER: EMPIAR-11244 | biostudies-other |

REPOSITORIES: biostudies-other

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Molecular mechanism of topoisomerase poisoning by the peptide antibiotic albicidin.

Michalczyk Elizabeth E Hommernick Kay K Behroz Iraj I Kulike Marcel M Pakosz-Stępień Zuzanna Z Mazurek Lukasz L Seidel Maria M Kunert Maria M Santos Karine K von Moeller Holger H Loll Bernhard B Weston John B JB Mainz Andi A Heddle Jonathan G JG Süssmuth Roderich D RD Ghilarov Dmitry D

Nature catalysis 20230123 1

The peptide antibiotic albicidin is a DNA topoisomerase inhibitor with low-nanomolar bactericidal activity towards fluoroquinolone-resistant Gram-negative pathogens. However, its mode of action is poorly understood. We determined a 2.6 Å resolution cryoelectron microscopy structure of a ternary complex between <i>Escherichia coli</i> topoisomerase DNA gyrase, a 217 bp double-stranded DNA fragment and albicidin. Albicidin employs a dual binding mechanism where one end of the molecule obstructs th ...[more]

PMID: 36741192

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Escherichia coli gyrase holocomplex with 217 bp phage Mu SGS DNA and Albi-1 stabilised by ADPNP

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Single-molecule imaging of DNA gyrase activity in living Escherichia coli.

Project description:Bacterial 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 live Escherichia coli cells. 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.

| S-EPMC6326794 | biostudies-literature

SbcCD-mediated processing of covalent gyrase-DNA complex in Escherichia coli.

Project description:Quinolones trap the covalent gyrase-DNA complex in Escherichia coli, leading to cell death. Processing activities for trapped covalent complex have not been characterized. A mutant strain lacking SbcCD nuclease activity was examined for both accumulation of gyrase-DNA complex and viability after quinolone treatment. Higher complex levels were found in ΔsbcCD cells than in wild-type cells after incubation with nalidixic acid and ciprofloxacin. However, SbcCD activity protected cells against the bactericidal action of nalidixic acid but not ciprofloxacin.

| S-EPMC3811449 | biostudies-literature

YacG from Escherichia coli is a specific endogenous inhibitor of DNA gyrase.

Project description:We assign a function for a small protein, YacG encoded by Escherichia coli genome. The NMR structure of YacG shows the presence of an unusual zinc-finger motif. YacG was predicted to be a part of DNA gyrase interactome based on protein-protein interaction network. We demonstrate that YacG inhibits all the catalytic activities of DNA gyrase by preventing its DNA binding. Topoisomerase I and IV activities remain unaltered in the presence of YacG and its action appears to be restricted only to DNA gyrase. The inhibition of the enzyme activity is due to the binding of YacG to carboxyl terminal domain of GyrB. Overexpression of YacG results in growth inhibition and alteration in DNA topology due to uncontrolled inhibition of gyrase.

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Phage Mu Gam protein promotes NHEJ in concert with <i>Escherichia coli</i> ligase.

Project description:The Gam protein of transposable phage Mu is an ortholog of eukaryotic and bacterial Ku proteins, which carry out nonhomologous DNA end joining (NHEJ) with the help of dedicated ATP-dependent ligases. Many bacteria carry Gam homologs associated with either complete or defective Mu-like prophages, but the role of Gam in the life cycle of Mu or in bacteria is unknown. Here, we show that MuGam is part of a two-component bacterial NHEJ DNA repair system. Ensemble and single-molecule experiments reveal that MuGam binds to DNA ends, slows the progress of RecBCD exonuclease, promotes binding of NAD+-dependent Escherichia coli ligase A, and stimulates ligation. In vivo, Gam equally promotes both precise and imprecise joining of restriction enzyme-digested linear plasmid DNA, as well as of a double-strand break (DSB) at an engineered I-SceI site in the chromosome. Cell survival after the induced DSB is specific to the stationary phase. In long-term growth competition experiments, particularly upon treatment with a clastogen, the presence of gam in a Mu lysogen confers a distinct fitness advantage. We also show that the role of Gam in the life of phage Mu is related not to transposition but to protection of genomic Mu copies from RecBCD when viral DNA packaging begins. Taken together, our data show that MuGam provides bacteria with an NHEJ system and suggest that the resulting fitness advantage is a reason that bacteria continue to retain the gam gene in the absence of an intact prophage.

| S-EPMC6294893 | biostudies-literature

ATP binding controls distinct structural transitions of Escherichia coli DNA gyrase in complex with DNA.

Project description:DNA gyrase is a molecular motor that harnesses the free energy of ATP hydrolysis to introduce negative supercoils into DNA. A critical step in this reaction is the formation of a chiral DNA wrap. Here we observe gyrase structural dynamics using a single-molecule assay in which gyrase drives the processive, stepwise rotation of a nanosphere attached to the side of a stretched DNA molecule. Analysis of rotational pauses and measurements of DNA contraction reveal multiple ATP-modulated structural transitions. DNA wrapping is coordinated with the ATPase cycle and proceeds by way of an unanticipated structural intermediate that dominates the kinetics of supercoiling. Our findings reveal a conformational landscape of loosely coupled transitions funneling the motor toward productive energy transduction, a feature that may be common to the reaction cycles of other DNA and protein remodeling machines.

| S-EPMC5660678 | biostudies-literature

Single-nucleotide-resolution mapping of DNA gyrase cleavage sites across the Escherichia coli genome.

Project description:An important antibiotic target, DNA gyrase is an essential bacterial enzyme that introduces negative supercoils into DNA and relaxes positive supercoils accumulating in front of moving DNA and RNA polymerases. By altering the superhelical density, gyrase may regulate expression of bacterial genes. The information about how gyrase is distributed along genomic DNA and whether its distribution is affected by drugs is scarce. During catalysis, gyrase cleaves both DNA strands forming a covalently bound intermediate. By exploiting the ability of several topoisomerase poisons to stabilize this intermediate we developed a ChIP-Seq-based approach to locate, with single nucleotide resolution, DNA gyrase cleavage sites (GCSs) throughout the Escherichia coli genome. We identified an extended gyrase binding motif with phased 10-bp G/C content variation, indicating that bending ability of DNA contributes to gyrase binding. We also found that GCSs are enriched in extended regions located downstream of highly transcribed operons. Transcription inhibition leads to redistribution of gyrase suggesting that the enrichment is functionally significant. Our method can be applied for precise mapping of prokaryotic and eukaryotic type II topoisomerases cleavage sites in a variety of organisms and paves the way for future studies of various topoisomerase inhibitors.

| S-EPMC6379681 | biostudies-literature

Mechanisms for defining supercoiling set point of DNA gyrase orthologs: I. A nonconserved acidic C-terminal tail modulates Escherichia coli gyrase activity.

Project description:DNA topoisomerases manage chromosome supercoiling and organization in all cells. Gyrase, a prokaryotic type IIA topoisomerase, consumes ATP to introduce negative supercoils through a strand passage mechanism. All type IIA topoisomerases employ a similar set of catalytic domains for function; however, the activity and specificity of gyrase are augmented by a specialized DNA binding and wrapping element, termed the C-terminal domain (CTD), which is appended to its GyrA subunit. We have discovered that a nonconserved, acidic tail at the extreme C terminus of the Escherichia coli GyrA CTD has a dramatic and unexpected impact on gyrase function. Removal of the CTD tail enables GyrA to introduce writhe into DNA in the absence of GyrB, an activity exhibited by other GyrA orthologs, but not by wild-type E. coli GyrA. Strikingly, a "tail-less" gyrase holoenzyme is markedly impaired for DNA supercoiling capacity, but displays normal ATPase function. Our findings reveal that the E. coli GyrA tail regulates DNA wrapping by the CTD to increase the coupling efficiency between ATP turnover and supercoiling, demonstrating that CTD functions can be fine-tuned to control gyrase activity in a highly sophisticated manner.

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