Negative supercoiling of DNA by gyrase is inhibited in Salmonella enterica serovar Typhimurium during adaptation to acid stress

DNA in intracellular Salmonella enterica serovar Typhimurium relaxes during growth in the acidified (pH 4–5) macrophage vacuole and DNA relaxation correlates with the upregulation of Salmonella genes involved in adaptation to the macrophage environment. Bacterial ATP levels did not increase during adaptation to acid pH unless the bacterium was deficient in MgtC, a cytoplasmic‐membrane‐located inhibitor of proton‐driven F₁F₀ ATP synthase activity. Inhibiting ATP binding by DNA gyrase and topo IV with novobiocin enhanced the effect of low pH on DNA relaxation. Bacteria expressing novobiocin‐resistant (Nov^R) derivatives of gyrase or topo IV also exhibited DNA relaxation at acid pH, although further relaxation with novobiocin was not seen in the strain with Nov^R gyrase. Thus, inhibition of the negative supercoiling activity of gyrase was the primary cause of enhanced DNA relaxation in drug‐treated bacteria. The Salmonella cytosol reaches pH 5–6 in response to an external pH of 4–5: the ATP‐dependent DNA supercoiling activity of purified gyrase was progressively inhibited by lowering the pH in this range, as was the ATP‐dependent DNA relaxation activity of topo IV. We propose that DNA relaxation in Salmonella within macrophage is due to acid‐mediated impairment of the negative supercoiling activity of gyrase.     

Staphylococcus aureus gyrase-quinolone-DNA ternary complexes fail to arrest replication fork progression in vitro. Effects of salt on the DNA binding mode and the catalytic activity of S. aureus gyrase

Type II topoisomerases bind to DNA at the catalytic domain across the DNA gate. DNA gyrases also bind to DNA at the non-homologous C-terminal domain of the GyrA subunit, which causes the wrapping of DNA about itself. This unique mode of DNA binding allows gyrases to introduce the negative supercoils into DNA molecules. We have investigated the biochemical characteristics of Staphylococcus aureus (S. aureus) gyrase. S. aureus gyrase is known to require high concentrations of potassium glutamate (K-Glu) for its supercoiling activity. However, high concentrations of K-Glu are not required for its relaxation and decatenation activities. This is due to the requirement of high concentrations of K-Glu for S. aureus gyrase-mediated wrapping of DNA. These results suggest that S. aureus gyrase can bind to DNA at the catalytic domain independent of K-Glu concentration, but high concentrations of K-Glu are required for the binding of the C-terminal domain of GyrA to DNA and the wrapping of DNA. Thus, salt modulates the DNA binding mode and the catalytic activity of S. aureus gyrase. Quinolone drugs can stimulate the formation of covalent S. aureus gyrase-DNA complexes, but high concentrations of K-Glu inhibit the formation of S. aureus gyrase-quinolone-DNA ternary complexes. In the absence of K-Glu, ternary complexes formed with S. aureus gyrase cannot arrest replication fork progression in vitro, demonstrating that the formation of a wrapped ternary complex is required for replication fork arrest by a S. aureus gyrase-quinolone-DNA ternary complex.     

Initiation of DNA replication in Bacillus subtilis. V. Role of DNA gyrase and superhelical structure in initiation

Summary When spores of a thymine-requiring mutant of Bacillus subtilis were germinated in a medium lacking thymine, an initiation potential (an ability to initiate and complete one round of replication in the presence of thymine and in the absence of protein and RNA synthesis) was formed for both chromosomal and plasmid replication. The effect of two inhibitors of DNA gyrase, novobiocin (Nov) and nalidixic acid (Nal), on the initiation potential formed during germination for chromosomal and plasmid replication was examined.Nov and Nal inhibited formation of the initiation potential completely if the drug was added at the onset of germination. In contrast, initiation of chromosomal and plasmid replication occurred in the presence of DNA gyrase inhibitors when the drug was added after the initiation potential had been fully formed. However, chromosomal replication initiated in the presence of the inhibitors ceased after a fragment of approximately 15 MD (15×10⁶ daltons) had been replicated, and plasmid replication was limited to one round of replication in approximately half of the plasmid molecules present in the spores.Furthermore the initiation potential for both chromosomal and plasmid replication though established was destroyed gradually but steadily by prolonged incubation with Nov in the absence of thymine. In addition, relaxation of the superhelical structure of plasmid DNA during incubation with Nov was observed in vivo. This relaxation was blocked by ethidium bromide, which dissociated the S-complex. On the other hand, incubation with Nal did not reduce the initiation potential nor did it change the superhelicity of the plasmid DNA in vivo. This is consistent with the known effect of gyrase inhibitors on the enzymatic activity of DNA gyrase.These results clearly demonstrate that both the action of DNA gyrase and the superhelical structure of the DNA are essential for the initiation of chromosomal and plasmid replication. The specific chromosome organization essential for initiation and elongation and the role of DNA gyrase are discussed.IV of this series is Yoshikawa et al. 1980     

Replication restart in gyrB Escherichia coli mutants

Gyrase is an essential topoisomerase in bacteria that introduces negative supercoils in DNA and relaxes the positive supercoils that form downstream of proteins tracking on DNA, such as DNA or RNA polymerases. Two gyrase mutants that suffer partial loss of function were used here to study the need for replication restart in conditions in which gyrase activity is affected. We show that the preprimosomal protein PriA is essential for the viability of these gyrB mutants. The helicase function of PriA is not essential. The lethality of the gyrB priA double mutants is suppressed by a dnaC809 mutation, indicating a requirement for primosome assembly in gyrB strains. The lethality of gyrB priA combination of mutations is independent of the level of DNA supercoiling, as gyrB and priA were also co-lethal in the presence of a DeltatopA mutation. Inactivation of homologous recombination did not affect the viability of gyrB mutants, indicating that replication restart does not require the formation of a recombination intermediate. We propose that the replisome is disassembled from replication forks when replication progression is blocked by the accumulation of positive supercoils in gyrase mutants, and that replication restarts via PriA-dependent primosome assembly, directly on the in-activated replication forks, without the formation of a recombination intermediate.     

DNA damage induced hyperphosphorylation of replication protein A. 2. Characterization of DNA binding activity, protein interactions, and activity in DNA replication and repair

Replication protein A (RPA) is a heterotrimeric protein consisting of 70-, 34-, and 14- kDa subunits that is required for many DNA metabolic processes including DNA replication and DNA repair. Using a purified hyperphosphorylated form of RPA protein prepared in vitro, we have addressed the effects of hyperphosphorylation on steady-state and pre-steady-state DNA binding activity, the ability to support DNA repair and replication reactions, and the effect on the interaction with partner proteins. Equilibrium DNA binding activity measured by fluorescence polarization reveals no difference in ssDNA binding to pyrimidine-rich DNA sequences. However, RPA hyperphosphorylation results in a decreased affinity for purine-rich ssDNA and duplex DNA substrates. Pre-steady-state kinetic analysis is consistent with the equilibrium DNA binding and demonstrates a contribution from both the k(on) and k(off) to achieve these differences. The hyperphosphorylated form of RPA retains damage-specific DNA binding, and, importantly, the affinity of hyperphosphorylated RPA for damaged duplex DNA is 3-fold greater than the affinity of unmodified RPA for undamaged duplex DNA. The ability of hyperphosphorylated RPA to support DNA repair showed minor differences in the ability to support nucleotide excision repair (NER). Interestingly, under reaction conditions in which RPA is maintained in a hyperphosphorylated form, we also observed inhibition of in vitro DNA replication. Analyses of protein-protein interactions bear out the effects of hyperphosphorylated RPA on DNA metabolic pathways. Specifically, phosphorylation of RPA disrupts the interaction with DNA polymerase alpha but has no significant effect on the interaction with XPA. These results demonstrate that the effects of DNA damage induced hyperphosphorylation of RPA on DNA replication and DNA repair are mediated through alterations in DNA binding activity and protein-protein interactions.