Mechanism of action of nalidixic acid: Purification of
            <i>Escherichia coli nalA</i>
            gene product and its relationship to DNA gyrase and a novel nicking-closing enzyme

Sugino, Akio; Peebles, Craig L.; Kreuzer, Kenneth N.; Cozzarelli, Nicholas R.

doi:10.1073/pnas.74.11.4767

Cited by 724 publications

(436 citation statements)

References 18 publications

(13 reference statements)

Supporting

Mentioning

418

Contrasting

Unclassified

Order By: Relevance

“…The interactions of the w protein with DNA have been studied in detail [12] and also some of the properties of the complexes between DNA and the topoisomerase from rat liver are known [lo, 111. E. coli DNA gyrase is able to form a covalent bond with duplex DNA in the presence of nalidixic acid and oxolinic acid [3,9,32]. As mentioned before, gene A protein of 6x174 [26,33] forms a covalent complex with 4x174 DNA.…”

Section: Discussionmentioning

confidence: 99%

Analysis of Covalent Complexes Formed between Calf Thymus DNA Topisomerase and Single-Stranded DNA

Prell

Vosberg

1980

Eur J Biochem

View full text Add to dashboard Cite

DNA topoisonitrases (or nicking-closing enzymes) introduce transient swivels into DNA. We studied the reaction of the calf thymus topoisomerase with single-stranded fd DNA. The nicking reaction of this enzyme is accompanied by strong attachment of the enzyme to DNA. We report conditions under which the intermediate complexes between DNA and topoisomerase are formed and describe some of their properties. For the analyses a filter-binding assay was used which is based on the adsorption of DNA-protein associates to Whatman glass-fiber filters [see Coombs and Pearson (1978) Proc. Natl Acad. Sci. [J.S.A. 75, 5291 -52951. The rate of appearance of DNAtopoisomerase complexes on the filter is a function of the enzyme concentration in the assay. The equivalent of about 9 x lo7 fd DNA molecules/s are found complexed, if the ratio of enzyme molecules to DNA molecules is about 50. The bond between topoisomerase and DNA is stable in 100 mM KOH or 100 mM HCl and rcsists treatment with 4 M guanidinium hydrochloride, 1 M potassium phosphate (pH 6.8) or phenol. These results indicate a covalent bond between DNA and the enzyme. Furthermore, the calf thymus enzyme attaches to the 3' terminus at the nick. The 5' terminus is dephosphorylated. Filter binding of single-stranded fd DNA as well as relaxation o f superhelical PM2 DNA is selectively inhibited by poly(dG) and poly(dG) . poly(dC), but not by other polydeoxynucleotides. This suggests a preference of the enzyme for binding and/or cleaving at d G or dG-rich clusters in the DNA.The introduction of transient swivels into DNA is catalysed by at least two different types of topoisomerases. Enzymes of the first type remove positive and/ or negative superhelical turns from closed circular DNA in an ATP-independent reaction. The cr) protein of Esclzerichia coli [l ] and the various topoisomerases of higher organisms, also called DNA-nicking/ closing enzymes [2], belong to this group. The second type of enzymes consists of the DNA gyrases [3,4]. These enzymes introduce negative turns into closed circular DNA in an ATP-requiring process. So far, DNA gyrases have been detected in prokaryotic organisms only [3,4]. In contrast, topoisomerases of the first types are widespread in nature, they were Abbreviations. DNA I, 11 and IV are circular covalently closed superhelical DNA, circular nicked DNA and circular covalently closed relaxed DNA, respectively; RFI is DNA I ; RFII is DNA 11; Gdm-HC1, guanidinium hydrochloride; MalNEt, N-ethylmaleimide; HO-HgBzOH, p-hydroxymercuribenzoate; C13AcOH, trichloroacetic acid.Enzymes. E. coli DNA polymerase I (EC 2.7.7.7); calf thymus terminal transferase (EC 2.7.7.31); calf intestine alkaline phosphatase (EC 3.1.3.1); T4 polynucleotide kinase (EC 2.7.1.78); proteinase K (EC 3.4.21.14). found in prokaryotic as well as in eukaryotic organismsThe physiological substrates of topoisomerases are most probably double-stranded DNAs. A general concept for the reaction mechanism of these enzymes with duplex DNA includes cleavage of one strand, rotation of the tw...

show abstract

Section: Discussionmentioning

confidence: 99%

Analysis of Covalent Complexes Formed between Calf Thymus DNA Topisomerase and Single-Stranded DNA

Prell

Vosberg

1980

Eur J Biochem

View full text Add to dashboard Cite

show abstract

“…As a result, quinolone concentrations sufficient to block replication do not relax chromosomal DNA supercoiling (29). When the quinolone is removed, the breaks are readily resealed (11,31) and the inhibitory effects of the compounds are reversed (13,21,27). Irreversible events that lead to rapid cell death occur at higher quinolone concentrations (1,20).…”

mentioning

confidence: 99%

Effect of Anaerobic Growth on Quinolone Lethality with Escherichia coli

Malik¹,

Hussain²,

Drlica³

2007

Antimicrob Agents Chemother

View full text Add to dashboard Cite

Quinolone activity against Escherichia coli was examined during aerobic growth, aerobic treatment with chloramphenicol, and anaerobic growth. Nalidixic acid, norfloxacin, ciprofloxacin, and PD161144 were lethal for cultures growing aerobically, and the bacteriostatic activity of each quinolone was unaffected by anaerobic growth. However, lethal activity was distinct for each quinolone with cells treated aerobically with chloramphenicol or grown anaerobically. Nalidixic acid failed to kill cells under both conditions; norfloxacin killed cells when they were grown anaerobically but not when they were treated with chloramphenicol; ciprofloxacin killed cells under both conditions but required higher concentrations than those required with cells grown aerobically; and PD161144, a C-8-methoxy fluoroquinolone, was equally lethal under all conditions. Following pretreatment with nalidixic acid, a shift to anaerobic conditions or the addition of chloramphenicol rapidly blocked further cell death. Formation of quinolone-gyrase-DNA complexes, observed as a sodium dodecyl sulfate (SDS)-dependent drop in cell lysate viscosity, occurred during aerobic and anaerobic growth and in the presence and in the absence of chloramphenicol. However, lethal chromosome fragmentation, detected as a drop in viscosity in the absence of SDS, occurred with nalidixic acid treatment only under aerobic conditions in the absence of chloramphenicol. With PD161144, chromosome fragmentation was detected when the cells were grown aerobically and anaerobically and in the presence and in the absence of chloramphenicol. Thus, all quinolones tested appear to form reversible bacteriostatic complexes containing broken DNA during aerobic growth, during anaerobic growth, and when protein synthesis is blocked; however, the ability to fragment chromosomes and to rapidly kill cells under these conditions depends on quinolone structure.When DNA topoisomerases bind to bacterial DNA, they form complexes that are trapped by the quinolone antibacterials (for a review, see reference 6). These ternary drug-enzyme-DNA complexes block DNA replication (5, 27, 29, 32), RNA synthesis (22, 33), and cell growth (2, 12). A key feature of the complexes is that the trapped DNA moiety is broken, with its ends constrained through covalent linkage to the GyrA protein (24, 25). As a result, quinolone concentrations sufficient to block replication do not relax chromosomal DNA supercoiling (29). When the quinolone is removed, the breaks are readily resealed (11, 31) and the inhibitory effects of the compounds are reversed (13,21,27). Irreversible events that lead to rapid cell death occur at higher quinolone concentrations (1, 20). We have proposed that rapid cell death arises from chromosome fragmentation that occurs when doublestrand DNA breaks are released from the protein-mediated constraint present in ternary complexes. At rapidly lethal quinolone concentrations, supercoils cannot be maintained (1) and fragmented DNA is obtained under conditions that allow resealing at bacteriost...

show abstract

“…32 -35 More recently, compounds that target proteins involved in these pathways have been developed, with the most successful being the quinolone and aminocoumarin antibiotics that target both DNA gyrase and topoisomerase IV. 36,37 These compounds were initially characterized as antimicrobial before their protein targets were identified. More recently, in this early age of target-based drug discovery, there have been concerted efforts to find inhibitors for specific DNA repair enzymes, such as helicases.…”

Section: Discussionmentioning

confidence: 99%

Identification of inhibitors for single-stranded DNA-binding proteins in eubacteria

Glanzer

Endres

Byrne

et al. 2016

J. Antimicrob. Chemother.

View full text Add to dashboard Cite

Objectives: The increasing threat of drug-resistant bacteria establishes a continuing need for the development of new strategies to fight infection. We examine the inhibition of the essential single-stranded DNA-binding proteins (SSBs) SSBA and SSBB as a potential antimicrobial therapy due to their importance in DNA replication, activating the SOS response and promoting competence-based mechanisms of resistance by incorporating new DNA.Methods: Purified recombinant SSBs from Gram-positive (Staphylococcus aureus and Bacillus anthracis) and Gram-negative (Escherichia coli and Francisella tularensis) bacteria were assessed in a high-throughput screen for inhibition of duplex DNA unwinding by small molecule inhibitors. Secondary electrophoretic mobility shift assays further validated the top hits that were then tested for MICs using in vitro assays. Results:We have identified compounds that show cross-reactivity in vitro, as well as inhibition of both F. tularensis and B. anthracis SSBA. Five compounds were moderately toxic to at least two of the four bacterial strains in vivo, including two compounds that were selectively non-toxic to human cells, 9-hydroxyphenylfluoron and purpurogallin. Three of the SSBA inhibitors also inhibited S. aureus SSBB in Gram-positive bacteria. Conclusions:Results from our study support the potential for SSB inhibitors as broad-spectrum antibacterial agents, with dual targeting capabilities against Gram-positive bacteria.

show abstract

Mechanism of action of nalidixic acid: Purification of Escherichia coli nalA gene product and its relationship to DNA gyrase and a novel nicking-closing enzyme

Cited by 724 publications

References 18 publications

Analysis of Covalent Complexes Formed between Calf Thymus DNA Topisomerase and Single-Stranded DNA

Analysis of Covalent Complexes Formed between Calf Thymus DNA Topisomerase and Single-Stranded DNA

Effect of Anaerobic Growth on Quinolone Lethality with Escherichia coli

Identification of inhibitors for single-stranded DNA-binding proteins in eubacteria

Contact Info

Product

Resources

About