Oxidation of phosphorothioate DNA modifications leads to lethal genomic instability

Abstract

Genomic modification by sulfur in the form of phosphorothioate (PT) is widespread among prokaryotes, including human pathogens. Apart from its physiological functions, PT sulfur has redox and nucleophilic properties that suggest effects on bacterial fitness in stressful environments. Here we show that PTs are dynamic and labile DNA modifications that cause genomic instability during oxidative stress. In experiments involving isotopic labeling coupled with mass spectrometry, we observed sulfur replacement in PTs at a rate of ∼2% h^-1 in unstressed Escherichia coli and Salmonella enterica. Whereas PT levels were unaffected by exposure to hydrogen peroxide (H₂O₂) or hypochlorous acid (HOCl), PT turnover increased to 3.8-10% h^-1 after HOCl treatment and was unchanged by H₂O₂, consistent with the repair of HOCl-induced sulfur damage. PT-dependent sensitivity to HOCl extended to cytotoxicity and DNA strand breaks, which occurred at HOCl doses that were orders of magnitude lower than the corresponding doses of H₂O₂. The genotoxicity of HOCl in PT-containing bacteria suggests reduced fitness in competition with HOCl-producing organisms and during infections in humans.

Figure 1. The effect of PT modifications on survival and growth of bacteria following oxidant exposures

Wild-type (PT+, solid line) and ΔdndB–H (PT–, dashed line) strains of E. coli (A, B, E, F) and S. enterica (C, D, G, H) were exposed to H₂O₂ (A–D) or HOCl (E–H). Cytotoxicity assays (A, C, E, G) were performed with the indicated concentrations of H₂O₂ and HOCl. Growth curves (B, D, F, H) were then prepared using the LD₈₀ doses of H₂O₂ (B, D) and HOCl (F, H). Red lines in the growth curves (B, D, F, H) indicate unexposed controls, with overlapping curves for wild-type and ΔdndB–H strains; black lines represent exposed bacteria, with data plotted in solid lines (PT+) distinguishing from dotted lines (PT–) only in panels F and H. Data represent mean ± SD for 3 biological replicates. Statistically significant differences among the data sets are discussed in the text.

Figure 2. PT reaction with HOCl leads to loss of PT and strand breaks in vitro and in vivo

(a) Quantification of PT in wild-type E. coli B7A DNA exposed to HOCl in vitro. Data represent mean ± SD for 3 biological replicates. (b) PT-containing dinucleotides were quantified by LC-MS/MS 3 hours (E. coli wild-type) and 5 hours (S. enterica wild-type) after exposure to their respective LD₅₀ doses of either HOCl or H₂O₂ (as specified in Supplementary Table 3; data represent mean ± SD for 3 biological replicates).

Figure 3. Comprehensive model for PT oxidation and alkylation in DNA

Analysis of degradation products arising in reactions of HOCl and H₂O₂ with PT-containing dinucleotides (inset and Supplementary Fig. 4) revealed products consistent with the reaction model shown. The sulfur in PTs can undergo two types of reactions based on nucleophilic substitution (right side) and one-electron oxidation (left side). At low concentrations of HOCl or H₂O₂, sulfur reacts by nucleophilic substitution to generate sulfonyl chloride (Cl-S) or sulfenic acid (HO-S), which subsequently undergo hydrolysis to eliminate the modified sulfur (pathway 1; X = sulfur-containing product) or to form a DNA strand-break (pathway 2) possibly with a modified sulfur (X). At high concentrations (left side), both H₂O₂ and HOCl generate radicals (hydroxyl radical, hypochoryl radical) that perform a series of one-electron oxidations leading to phosphonate formation and subsequent DNA strand-breaks containing a phosphonate (X = H), phosphate (X = O), or other product. Inset: Concentration-dependent degradation of d(G_PSA) by HOCl causes both desulfuration to a phosphate-linked dinucleotide and a strand-break with release of dG; data represent mean ± SD for 3 replicates.

Figure 4. PT reaction with HOCl leads to loss of PT and strand breaks in vitro and in vivo

(a) Isolated DNA from wild-type (PT+) and ΔdndB–H (PT–) E. coli were exposed to 0.08–0.8 mM HOCl or 0.08–8 mM H₂O₂; iodine (I₂) exposure serves as a positive control for PT-dependent strand breaks. HOCl-induced strand-breaks are apparent as smearing in the lane for DNA containing PT (PT+), but not for DNA lacking PT (PT–); strand-breaks are not detectable for H₂O₂ exposure in any case. (b) WT and ΔdndB–H E. coli cells were exposed to 7.5- and 25-times the WT LD₅₀ concentration of H₂O₂ or HOCl for 10 min (4 and 14 mM for H₂O₂; 0.13 and 0.43 for HOCl). Again, DNA isolated from HOCl-exposed WT bacteria, but not PT– bacteria, shows strand-breaks, while no strand breaks are apparent after H₂O₂ treatment in either strain. Gel images are cropped for clarity (see Supplementary Fig. 9 for unprocessed images). Data for LD₅₀ and LD₈₀ doses are presented in Supplementary Table 3.

Figure 5. Quantification of PT turnover by isotope-labeling and mass spectrometry

(a) Principle of PT turnover assay. Bacteria were grown in minimal medium containing [¹⁵N]/[³⁴S]-labeled nutrients, with resulting bacterial DNA containing [¹⁵N]-labeled nucleobases with [³⁴S]-labeled PTs (Original PT). To initiate an experiment, cells are transferred to minimal medium with [¹⁴N]/[³²S]-labeled constituents and subjected to stresses. Newly synthesized PT-linked dinucleotides (New PT) are labeled with [¹⁴N] and [³²S]. The [³⁴S] in the original PTs was found to undergo replacement with [³²S] (Turnover PT) in both untreated and oxidant-treated cells. We also observed low levels of PT dinucleotides labeled with [³⁴S] and [¹⁴N] (Cryptic PT) resulting from reuse of [³⁴S] from nutrient pools or other PT modification sites. In all cases, isolated DNA was hydrolyzed to canonical mononucleotides and PT-containing dinucleotides, and quantities of various isotope-labeled dinucleotides were determined by LC-MS/MS analysis. (b) Validation of PT turnover method. Cultures of wild-type E. coli B7A were grown with [¹⁵N]/[³⁴S]-containing medium, and then cells were transferred to [¹⁴N]/[³²S]-containing medium. At various times, genomic DNA was isolated and subjected to LC-MS/MS analysis of PT-containing dinucleotides. The plot shows cell growth (OD₆₀₀; blue line), original PT (red line) and new PT (black line). (c) The level of [¹⁵N]/[³³S]-labeled, PT-containing dinucleotides (“turnover” PT) were determined in wild-type, untreated E. coli B7A as a function of time. The level of this turnover PT as a function of total PT was used to calculate the %PT turnover. (d) Estimates of the rate of PT turnover per hour were calculated by performing linear regression analysis of plots of %PT turnover as a function of time in unstressed E. coli B7A and S. enterica Cerro 87 (black) and for bacteria exposed to HOCl (red) and H₂O₂ (blue), with regression analysis illustrated in Supplementary Figure 7 for wild-type, untreated Cerro 87. Data represent mean ± SD for 3 biological replicates and values for HOCl-treated samples are significantly different from untreated and H2)2-treated by Student’s t-test at P < 0.05.