Nth specifically nicks AP-containing pBSK. Typical gels showing plasmid nicking by increasing concentrations of Nth (0.05, 0.1, 0.5, 1 and 5 nM) incubated with AP containing (+AP) and control (−AP) pBSK (7.14 nM) plasmid for 15 min, as indicated. After treatment with proteinase K, samples were resolved through 1% w/v agarose gels. Controls represent +AP (C+) and –AP (C−) pBSK (7.14 nM) incubated for 15 min in the absence of Nth. Quantification of the percentage nicking is shown in bar graphs with error bars indicating standard error (SE) of the mean from triplicate experiments.

DnaD stimulates the activity of Nth. Nicking assays were carried out with increasing concentrations of Nth (0.1, 0.2, 0.5 and 1.0 nM) for 5 min in the presence or absence of DnaD (10 µM) and 7.14 nM pBSK as indicated. Control reactions were carried out with 7.14 nM pBSK in the presence of DnaD (10 µM) and in the absence of Nth. The amount of nicked pBSK, as a percentage of the total, is indicated by bar graphs (white, grey and black bars show control), Nth and Nth+DnaD reactions, respectively. Quantification of the percentage nicking is shown in bar graphs with error bars indicating SE from triplicate experiments.

YonN and HBsu untwist supercoiled DNA and stimulate the activity of Nth. (A) Topoisomerase I relaxation assays of supercoiled pBSK (18 nM) in the presence of increasing concentrations of YonN (0.1, 0.2, 0.4 and 0.8 µM) or HBsu (0.0125, 0.025, 0.05, 0.1, 0.2 and 0.4 µM). Binding reactions were carried out for 20 min at 37°C in NEB buffer 4 (50 mM potassium acetate, 20 mM Tris–acetate, pH 7.9, 10 mM magnesium acetate, 1 mM DTT) before addition of topoisomerase I (0.5 units, NEB) and BSA (100 µgml⁻¹) and further incubation for 40 min. Proteinase K was added to digest all proteins at 37°C for 30 min before resolving the topoisomers in a 1% (w/v) agarose gel. Lanes C1, C2 and C3 represent pBSK, pBSK plus proteinase K, and pBSK plus topoisomerase I plus proteinase K controls, respectively. (B) Time course Nth (0.5 nM) nicking assays with AP-containing pBSK (7.14 nM) in the presence of YonN (9.72 µM) or HBsu (10 µM), as indicated. Reactions were carried out in 20 mM Tris–HCl, pH 8.0, 1 mM EDTA, 1 mM DTT, 250 mM NaCl. pBSK and YonN or HBsu were incubated for 10 min at 37°C, then Nth was added for 1, 5, 15 min before the addition of Proteinase K for 30 min. Control reactions were carried out in exactly the same manner but incubated with buffer instead of YonN or HBsu. (C) The same time course Nth assay as in panel B but with DnaB (53 µM). In all cases three independent experiments were carried out and the data were quantified by densitometry. Quantification of the percentage nicking is shown in bar graphs with error bars indicating SE from triplicate experiments.

Deletion of nth increases sensitivity to H₂O₂. Samples of 1 µl from serial dilutions (from top to bottom: undiluted, 1:10, 1:100, 1:1000, 1:10 000, 1:100 000) of wt B. subtilis 168 and Δnth control (0 mM) and H₂O₂ exposed (22, 45, 60, 90, 120, 150 and 180 mM) cultures were spotted on LA plates. Data from two independent experiments are shown in the top and bottom panels, respectively. Detailed experimental conditions are described in ‘Experimental Procedures’ section.

Constitutive expression of dnaD is transiently stimulated by H₂O₂ exposure. (A) The dnaD promoter is constitutively active. Bar charts comparing the β-galactosidase activity in the B. subtilis P_dnaD-lacZ strain treated with 0.1 mM H₂O₂ (shaded bars) with untreated B. subtilis P_dnaD-lacZ (clear bars), as indicated. Control data with the β-galactosidase activity of the isogenic wt B. subtilis and the promoterless lacZ strains are also shown. In the control wt B. subtilis and promoterless lacZ strains the β-galactosidase activity was at background levels and the bar graphs are virtually indistinguishable from the X-axis. The β-galactosidase activity was expressed in terms of Miller's units (MU). (B) The effect of H₂O₂ peroxide on the mRNA levels of dnaB, dnaD and dnaI was examined by RT–PCR. The bar graph shows relative quantification of the mRNA levels, compared to the endogenous control r16S (ΔC_t value), from B. subtilis cells after 30 min exposure to 80 mM H₂O₂ (black bars) compared to control non-exposed cells (white bars) (ΔΔC_t value). The bars represent the normalized means ± standard deviation (SD) of the results from three independent experiments. The mRNA levels of dnaB (mean reduction = 67%, SD = 0.15, P = 0.03) and dnaI (mean reduction = 69%, SD = 0.14, P = 0.03) were reduced significantly but the mRNA levels of dnaD (mean reduction 25%, SD = 0.23, P = 0.12) were only marginally affected.