scholarly journals Topoisomerase III Acts at the Replication Fork To Remove Precatenanes

2019 ◽  
Vol 201 (7) ◽  
Author(s):  
Chong M. Lee ◽  
Guanshi Wang ◽  
Alexandros Pertsinidis ◽  
Kenneth J. Marians
Keyword(s):  
Dna Replication ◽  
Replication Fork ◽  
Dna Helicase ◽  
Holliday Junctions ◽  
Bacterial Cells ◽  
Topoisomerase Iv ◽  
Content Type ◽  
E Coli ◽  
Topoisomerase Iii

ABSTRACTThe role of DNA topoisomerase III (Topo III) in bacterial cells has proven elusive. Whereas eukaryotic Top IIIα homologs are clearly involved with homologs of the bacterial DNA helicase RecQ in unraveling double Holliday junctions, preventing crossover exchange of genetic information at unscheduled recombination intermediates, and Top IIIβ homologs have been shown to be involved in regulation of various mRNAs involved in neuronal function, there is little evidence for similar reactions in bacteria. Instead, most data point to Topo III playing a role supplemental to that of topoisomerase IV in unlinking daughter chromosomes during DNA replication. In support of this model, we show thatEscherichia coliTopo III associates with the replication forkin vivo(likely via interactions with the single-stranded DNA-binding protein and the β clamp-loading DnaX complex of the DNA polymerase III holoenzyme), that the DnaX complex stimulates the ability of Topo III to unlink both catenated and precatenated DNA rings, and that ΔtopBcells show delayed and disorganized nucleoid segregation compared to that of wild-type cells. These data argue that Topo III normally assists topoisomerase IV in chromosome decatenation by removing excess positive topological linkages at or near the replication fork as they are converted into precatenanes.IMPORTANCETopological entanglement between daughter chromosomes has to be reduced to exactly zero every time anE. colicell divides. The enzymatic agents that accomplish this task are the topoisomerases.E. colipossesses four topoisomerases. It has been thought that topoisomerase IV is primarily responsible for unlinking the daughter chromosomes during DNA replication. We show here that topoisomerase III also plays a role in this process and is specifically localized to the replisome, the multiprotein machine that duplicates the cell’s genome, in order to do so.

scholarly journals Mechanistic insights into Lhr helicase function in DNA repair

2020 ◽  
Vol 477 (16) ◽  
pp. 2935-2947
Author(s):  
Ryan J. Buckley ◽  
Kevin Kramm ◽  
Christopher D. O. Cooper ◽  
Dina Grohmann ◽  
Edward L. Bolt
Keyword(s):  
Dna Repair ◽  
Dna Replication ◽  
Single Molecule ◽  
Dna Helicase ◽  
Holliday Junctions ◽  
Single Molecule Fret ◽  
Repair Enzymes ◽  
Dna Strands

The DNA helicase Large helicase-related (Lhr) is present throughout archaea, including in the Asgard and Nanoarchaea, and has homologues in bacteria and eukaryotes. It is thought to function in DNA repair but in a context that is not known. Our data show that archaeal Lhr preferentially targets DNA replication fork structures. In a genetic assay, expression of archaeal Lhr gave a phenotype identical to the replication-coupled DNA repair enzymes Hel308 and RecQ. Purified archaeal Lhr preferentially unwound model forked DNA substrates compared with DNA duplexes, flaps and Holliday junctions, and unwound them with directionality. Single-molecule FRET measurements showed that binding of Lhr to a DNA fork causes ATP-independent distortion and base-pair melting at, or close to, the fork branchpoint. ATP-dependent directional translocation of Lhr resulted in fork DNA unwinding through the ‘parental’ DNA strands. Interaction of Lhr with replication forks in vivo and in vitro suggests that it contributes to DNA repair at stalled or broken DNA replication.

scholarly journals Shape Changes and Interaction Mechanism of Escherichia coli Cells Treated with Sericin and Use of a Sericin-Based Hydrogel for Wound Healing

2016 ◽  
Vol 82 (15) ◽  
pp. 4663-4672 ◽  
Author(s):  
Rui Xue ◽  
Yalong Liu ◽  
Qingsong Zhang ◽  
Congcong Liang ◽  
Huazhen Qin ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Escherichia Coli ◽  
Cell Membrane ◽  
Interaction Mechanism ◽  
Bacterial Cells ◽  
Content Type ◽  
E Coli ◽  
Membrane Blebbing ◽  
Sterile Gauze

ABSTRACTTo verify the interaction mechanism between sericin andEscherichia coli, especially the morphological and structural changes in the bacterial cells, the antimicrobial activity of sericin againstE. colias a model for Gram-negative bacteria was investigated. The antibacterial activity of sericin onE. coliand the interaction mechanism were investigated in this study by analyzing the growth, integrity, and morphology of the bacterial cells following treatment with sericin. The changes in morphology and cellular compositions of bacterial cells treated with sericin were observed by an inverted fluorescence microscope, scanning electron microscopy, and transmission electron microscopy. Changes in electrical conductivity, total sugar concentration of the broth for the bacteria, and protein expression of the bacteria were determined to investigate the permeability of the cell membrane. A sericin-based hydrogel was prepared for anin vivostudy of wound dressing. The results showed that the antibacterial activity of the hydrogel increased with the increase in the concentration of sericin from 10 g/liter to 40 g/liter. The introduction of sericin induces membrane blebbing ofE. colicells caused by antibiotic action on the cell membrane. The cytoplasm shrinkage phenomenon was accompanied by blurring of the membrane wall boundaries. WhenE. colicells were treated with sericin, release of intracellular components quickly increased. The electrical conductivity assay indicated that the charged ions are reduced after exposure to sericin so that the integrity of the cell membrane is weakened and metabolism is blocked. In addition, sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated that sericin hinders the expression of bacterial protein. Sericin may damage the integrity of the bacterial cell membrane, thereby eventually inhibiting the growth and reproduction ofE. coli. Compared to sterile gauze, the sericin-based hydrogel promoted fibroblast cell proliferation and accelerated the formation of granulation tissues and neovessels.IMPORTANCEThe specific relationship and interaction mechanism between sericin andE. colicells were investigated and elucidated. The results show that after 12 h of treatment, sericin molecules induce membrane blebbing ofE. colicells, and the bacteria show decreases in liquidity and permeability of biological membrane, resulting in alterations in the conductivity of the culture medium and the integrity of the outer membrane. The subsequentin vivoresults demonstrate that the sericin-poly(N-isopropylacrylamide-N,N′-methylene-bis-acrylamide [NIPAm-MBA]) hydrogel accelerated wound healing compared to that with sterile gauze, which is a beneficial result for future applications in clinical medicine and the textile, food, and coating industries.

Prokaryotic DNA replication mechanisms

1987 ◽  
Vol 317 (1187) ◽  
pp. 395-420 ◽  
Keyword(s):  
Dna Replication ◽  
Dna Polymerase ◽  
Replication Fork ◽  
Kinetic Studies ◽  
Dna Helicase ◽  
Gene 32 ◽  
Dna Template ◽  
Lagging Strand

The three different prokaryotic replication systems that have been most extensively studied use the same basic components for moving a DNA replication fork, even though the individual proteins are different and lack extensive amino acid sequence homology. In the T4 bacteriophage system, the components of the DNA replication complex can be grouped into functional classes as follows: DNA polymerase (gene 43 protein), helix-destabilizing protein (gene 32 protein), polymerase accessory proteins (gene 44/62 and 45 proteins), and primosome proteins (gene 41 DNA helicase and gene 61 RNA primase). DNA synthesis in the in vitro system starts by covalent addition onto the 3'OH end at a random nick on a double-stranded DNA template and proceeds to generate a replication fork that moves at about the in vivo rate, and with approximately the in vivo base-pairing fidelity. DNA is synthesized at the fork in a continuous fashion on the leading strand and in a discontinuous fashion on the lagging strand (generating short Okazaki fragments with 5'-linked pppApCpXpYpZ pentaribonucleotide primers). Kinetic studies reveal that the DNA polymerase molecule on the lagging strand stays associated with the fork as it moves. Therefore the DNA template on the lagging strand must be folded so that the stop site for the synthesis of one Okazaki fragment is adjacent to the start site for the next such fragment, allowing the polymerase and other replication proteins on the lagging strand to recycle.

scholarly journals Escherichia coliZipA Organizes FtsZ Polymers into Dynamic Ring-Like Protofilament Structures

mBio ◽  
2018 ◽  
Vol 9 (3) ◽  
Author(s):  
Marcin Krupka ◽  
Marta Sobrinos-Sanguino ◽  
Mercedes Jiménez ◽  
Germán Rivas ◽  
William Margolin
Keyword(s):  
Cell Division ◽  
High Surface ◽  
Bacterial Cells ◽  
Content Type ◽  
E Coli ◽  
Membrane Attachment ◽  
Lipid Surface ◽  
Confocal Fluorescence Imaging

ABSTRACTZipA is an essential cell division protein inEscherichia coli. Together with FtsA, ZipA tethers dynamic polymers of FtsZ to the cytoplasmic membrane, and these polymers are required to guide synthesis of the cell division septum. This dynamic behavior of FtsZ has been reconstituted on planar lipid surfacesin vitro, visible as GTP-dependent chiral vortices several hundred nanometers in diameter, when anchored by FtsA or when fused to an artificial membrane binding domain. However, these dynamics largely vanish when ZipA is used to tether FtsZ polymers to lipids at high surface densities. This, along with somein vitrostudies in solution, has led to the prevailing notion that ZipA reduces FtsZ dynamics by enhancing bundling of FtsZ filaments. Here, we show that this is not the case. When lower, more physiological levels of the soluble, cytoplasmic domain of ZipA (sZipA) were attached to lipids, FtsZ assembled into highly dynamic vortices similar to those assembled with FtsA or other membrane anchors. Notably, at either high or low surface densities, ZipA did not stimulate lateral interactions between FtsZ protofilaments. We also usedE. colimutants that are either deficient or proficient in FtsZ bundling to provide evidence that ZipA does not directly promote bundling of FtsZ filamentsin vivo. Together, our results suggest that ZipA does not dampen FtsZ dynamics as previously thought, and instead may act as a passive membrane attachment for FtsZ filaments as they treadmill.IMPORTANCEBacterial cells use a membrane-attached ring of proteins to mark and guide formation of a division septum at midcell that forms a wall separating the two daughter cells and allows cells to divide. The key protein in this ring is FtsZ, a homolog of tubulin that forms dynamic polymers. Here, we use electron microscopy and confocal fluorescence imaging to show that one of the proteins required to attach FtsZ polymers to the membrane duringE. colicell division, ZipA, can promote dynamic swirls of FtsZ on a lipid surfacein vitro. Importantly, these swirls are observed only when ZipA is present at low, physiologically relevant surface densities. Although ZipA has been thought to enhance bundling of FtsZ polymers, we find little evidence for bundlingin vitro. In addition, we present several lines ofin vivoevidence indicating that ZipA does not act to directly bundle FtsZ polymers.

scholarly journals In Vitro and In Vivo Profiles of ACH-702, an Isothiazoloquinolone, against Bacterial Pathogens

2011 ◽  
Vol 55 (6) ◽  
pp. 2860-2871 ◽  
Author(s):  
Michael J. Pucci ◽  
Steven D. Podos ◽  
Jane A. Thanassi ◽  
Melissa J. Leggio ◽  
Barton J. Bradbury ◽  
...  
Keyword(s):  
Antibacterial Activity ◽  
Dna Replication ◽  
Bacterial Pathogens ◽  
Biological Data ◽  
Topoisomerase Iv ◽  
Gram Positive ◽  
Gram Negative ◽  
Content Type ◽  
Good Antibacterial Activity

ABSTRACTACH-702, a novel isothiazoloquinolone (ITQ), was assessed for antibacterial activity against a panel of Gram-positive and Gram-negative clinical isolates and found to possess broad-spectrum activity, especially against antibiotic-resistant Gram-positive strains, including methicillin-resistantStaphylococcus aureus(MRSA). For Gram-negative bacteria, ACH-702 showed exceptional potency againstHaemophilus influenzae,Moraxella catarrhalis, and aNeisseriasp. but was less active against members of theEnterobacteriaceae. Good antibacterial activity was also evident against several anaerobes as well asLegionella pneumophilaandMycoplasma pneumoniae. Excellent bactericidal activity was observed for ACH-702 against several bacterial pathogens in time-kill assays, and postantibiotic effects (PAEs) of >1 h were evident with both laboratory and clinical strains of staphylococci at 10× MIC and similar in most cases to those observed for moxifloxacin at the same MIC multiple.In vivoefficacy was demonstrated againstS. aureuswith murine sepsis and thigh infection models, with decreases in the number of CFU/thigh equal to or greater than those observed after vancomycin treatment. Macromolecular synthesis assays showed specific dose-dependent inhibition of DNA replication in staphylococci, and biochemical analyses indicated potent dual inhibition of two essential DNA replication enzymes: DNA gyrase and topoisomerase IV. Additional biological data in support of an effective dual targeting mechanism of action include the following: low MIC values (≤0.25 μg/ml) against staphylococcal strains with single mutations in bothgyrAandgrlA(parC), retention of good antibacterial activity (MICs of ≤0.5 μg/ml) against staphylococcal strains with two mutations in bothgyrAandgrlA, and low frequencies for the selection of higher-level resistance (<10−10). These promising initial data support further study of isothiazoloquinolones as potential clinical candidates.

scholarly journals Structure-specific DNA replication-fork recognition directs helicase and replication restart activities of the PriA helicase

2018 ◽  
Vol 115 (39) ◽  
pp. E9075-E9084 ◽  
Author(s):  
Tricia A. Windgassen ◽  
Maxime Leroux ◽  
Kenneth A. Satyshur ◽  
Steven J. Sandler ◽  
James L. Keck
Keyword(s):  
Dna Replication ◽  
Replication Fork ◽  
Dna Helicase ◽  
Genome Replication ◽  
Duplex Dna ◽  
Winged Helix ◽  
Replication Restart ◽  
Leading Strand ◽  
Winged Helix Domain

DNA replication restart, the essential process that reinitiates prematurely terminated genome replication reactions, relies on exquisitely specific recognition of abandoned DNA replication-fork structures. The PriA DNA helicase mediates this process in bacteria through mechanisms that remain poorly defined. We report the crystal structure of a PriA/replication-fork complex, which resolves leading-strand duplex DNA bound to the protein. Interaction with PriA unpairs one end of the DNA and sequesters the 3′-most nucleotide from the nascent leading strand into a conserved protein pocket. Cross-linking studies reveal a surface on the winged-helix domain of PriA that binds to parental duplex DNA. Deleting the winged-helix domain alters PriA’s structure-specific DNA unwinding properties and impairs its activity in vivo. Our observations lead to a model in which coordinated parental-, leading-, and lagging-strand DNA binding provide PriA with the structural specificity needed to act on abandoned DNA replication forks.

scholarly journals Regulation of Cell Division in Bacteria by Monitoring Genome Integrity and DNA Replication Status

2019 ◽  
Vol 202 (2) ◽  
Author(s):  
Peter E. Burby ◽  
Lyle A. Simmons
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Cell Division ◽  
Dna Replication ◽  
Cell Cycle Progression ◽  
Genome Instability ◽  
Sos Response ◽  
Bacterial Cells ◽  
Cycle Progression ◽  
Content Type

ABSTRACT All organisms regulate cell cycle progression by coordinating cell division with DNA replication status. In eukaryotes, DNA damage or problems with replication fork progression induce the DNA damage response (DDR), causing cyclin-dependent kinases to remain active, preventing further cell cycle progression until replication and repair are complete. In bacteria, cell division is coordinated with chromosome segregation, preventing cell division ring formation over the nucleoid in a process termed nucleoid occlusion. In addition to nucleoid occlusion, bacteria induce the SOS response after replication forks encounter DNA damage or impediments that slow or block their progression. During SOS induction, Escherichia coli expresses a cytoplasmic protein, SulA, that inhibits cell division by directly binding FtsZ. After the SOS response is turned off, SulA is degraded by Lon protease, allowing for cell division to resume. Recently, it has become clear that SulA is restricted to bacteria closely related to E. coli and that most bacteria enforce the DNA damage checkpoint by expressing a small integral membrane protein. Resumption of cell division is then mediated by membrane-bound proteases that cleave the cell division inhibitor. Further, many bacterial cells have mechanisms to inhibit cell division that are regulated independently from the canonical LexA-mediated SOS response. In this review, we discuss several pathways used by bacteria to prevent cell division from occurring when genome instability is detected or before the chromosome has been fully replicated and segregated.

scholarly journals Interactions and Localization of Escherichia coli Error-Prone DNA Polymerase IV after DNA Damage

2015 ◽  
Vol 197 (17) ◽  
pp. 2792-2809 ◽  
Author(s):  
Sarita Mallik ◽  
Ellen M. Popodi ◽  
Andrew J. Hanson ◽  
Patricia L. Foster
Keyword(s):  
Dna Damage ◽  
Dna Polymerase ◽  
Dna Helicase ◽  
Dna Lesions ◽  
Replication Forks ◽  
Content Type ◽  
Pol Iv ◽  
Dna Polymerase Iv ◽  
Stalled Replication Forks

ABSTRACTEscherichia coli's DNA polymerase IV (Pol IV/DinB), a member of the Y family of error-prone polymerases, is induced during the SOS response to DNA damage and is responsible for translesion bypass and adaptive (stress-induced) mutation. In this study, the localization of Pol IV after DNA damage was followed using fluorescent fusions. After exposure ofE. colito DNA-damaging agents, fluorescently tagged Pol IV localized to the nucleoid as foci. Stepwise photobleaching indicated ∼60% of the foci consisted of three Pol IV molecules, while ∼40% consisted of six Pol IV molecules. Fluorescently tagged Rep, a replication accessory DNA helicase, was recruited to the Pol IV foci after DNA damage, suggesting that thein vitrointeraction between Rep and Pol IV reported previously also occursin vivo. Fluorescently tagged RecA also formed foci after DNA damage, and Pol IV localized to them. To investigate if Pol IV localizes to double-strand breaks (DSBs), an I-SceI endonuclease-mediated DSB was introduced close to a fluorescently labeled LacO array on the chromosome. After DSB induction, Pol IV localized to the DSB site in ∼70% of SOS-induced cells. RecA also formed foci at the DSB sites, and Pol IV localized to the RecA foci. These results suggest that Pol IV interacts with RecAin vivoand is recruited to sites of DSBs to aid in the restoration of DNA replication.IMPORTANCEDNA polymerase IV (Pol IV/DinB) is an error-prone DNA polymerase capable of bypassing DNA lesions and aiding in the restart of stalled replication forks. In this work, we demonstratein vivolocalization of fluorescently tagged Pol IV to the nucleoid after DNA damage and to DNA double-strand breaks. We show colocalization of Pol IV with two proteins: Rep DNA helicase, which participates in replication, and RecA, which catalyzes recombinational repair of stalled replication forks. Time course experiments suggest that Pol IV recruits Rep and that RecA recruits Pol IV. These findings providein vivoevidence that Pol IV aids in maintaining genomic stability not only by bypassing DNA lesions but also by participating in the restoration of stalled replication forks.

scholarly journals In VitroandIn VivoCharacterization of the Novel Oxabicyclooctane-Linked Bacterial Topoisomerase Inhibitor AM-8722, a Selective, Potent Inhibitor of Bacterial DNA Gyrase

2016 ◽  
Vol 60 (8) ◽  
pp. 4830-4839 ◽  
Author(s):  
Christopher M. Tan ◽  
Charles J. Gill ◽  
Jin Wu ◽  
Nathalie Toussaint ◽  
Jingjun Yin ◽  
...  
Keyword(s):  
Broad Spectrum ◽  
Antibacterial Agents ◽  
Cell Activity ◽  
Dna Gyrase ◽  
Topoisomerase Iv ◽  
Bacterial Dna ◽  
Whole Cell ◽  
Content Type

ABSTRACTOxabicyclooctane-linked novel bacterial topoisomerase inhibitors (NBTIs) represent a new class of recently described antibacterial agents with broad-spectrum activity. NBTIs dually inhibit the clinically validated bacterial targets DNA gyrase and topoisomerase IV and have been shown to bind distinctly from known classes of antibacterial agents directed against these targets. Herein we report the molecular, cellular, andin vivocharacterization of AM-8722 as a representative N-alkylated-1,5-naphthyridone left-hand-side-substituted NBTI. Consistent with its mode of action, macromolecular labeling studies revealed a specific effect of AM-8722 to dose dependently inhibit bacterial DNA synthesis. AM-8722 displayed greater intrinsic enzymatic potency than levofloxacin versus both DNA gyrase and topoisomerase IV fromStaphylococcus aureusandEscherichia coliand displayed selectivity against human topoisomerase II. AM-8722 was rapidly bactericidal and exhibited whole-cell activity versus a range of Gram-negative and Gram-positive organisms, with no whole-cell potency shift due to the presence of DNA or human serum. Frequency-of-resistance studies demonstrated an acceptable rate of resistance emergencein vitroat concentrations 16- to 32-fold the MIC. AM-8722 displayed acceptable pharmacokinetic properties and was shown to be efficacious in mouse models of bacterial septicemia. Overall, AM-8722 is a selective and potent NBTI that displays broad-spectrum antimicrobial activityin vitroandin vivo.

scholarly journals Biological Cost of Single and Multiple Norfloxacin Resistance Mutations in Escherichia coli Implicated in Urinary Tract Infections

2005 ◽  
Vol 49 (6) ◽  
pp. 2343-2351 ◽  
Author(s):  
Patricia Komp Lindgren ◽  
Linda L. Marcusson ◽  
Dorthe Sandvang ◽  
Niels Frimodt-Møller ◽  
Diarmaid Hughes
Keyword(s):  
Escherichia Coli ◽  
Urinary Tract ◽  
Infection Model ◽  
Resistance Mutations ◽  
Topoisomerase Iv ◽  
E Coli ◽  
Tract Infections ◽  
Subsequent Selection

ABSTRACT Resistance to fluoroquinolones in urinary tract infection (UTIs) caused by Escherichia coli is associated with multiple mutations, typically those that alter DNA gyrase and DNA topoisomerase IV and those that regulate AcrAB-TolC-mediated efflux. We asked whether a fitness cost is associated with the accumulation of these multiple mutations. Mutants of the susceptible E. coli UTI isolate Nu14 were selected through three to five successive steps with norfloxacin. Each selection was performed with the MIC of the selected strain. After each selection the MIC was measured; and the regions of gyrA, gyrB, parC, and parE, previously associated with resistance mutations, and all of marOR and acrR were sequenced. The first selection step yielded mutations in gyrA, gyrB, and marOR. Subsequent selection steps yielded mutations in gyrA, parE, and marOR but not in gyrB, parC, or acrR. Resistance-associated mutations were identified in almost all isolates after selection steps 1 and 2 but in less than 50% of isolates after subsequent selection steps. Selected strains were competed in vitro, in urine, and in a mouse UTI infection model against the starting strain, Nu14. First-step mutations were not associated with significant fitness costs. However, the accumulation of three or more resistance-associated mutations was usually associated with a large reduction in biological fitness, both in vitro and in vivo. Interestingly, in some lineages a partial restoration of fitness was associated with the accumulation of additional mutations in late selection steps. We suggest that the relative biological costs of multiple mutations may influence the evolution of E. coli strains that develop resistance to fluoroquinolones.

Sign in / Sign up

Export Citation Format

Share Document