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Oxidation of phosphorothioate DNA modifications leads to lethal genomic instability

Genomic modification with sulfur as phosphorothioate (PT) is widespread among prokaryotes, including human pathogens. Apart from its physiological functions, the redox and nucleophilic properties of PT sulfur 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. Using coupled isotopic labeling-mass spectrometry, we observed sulfur replacement in PTs at a rate of ~2%/h in unstressed Escherichia coli and Salmonella enterica. While PT levels were unaffected by exposure to hydrogen peroxide (H2O2) or hypochlorous acid (HOCl), PT turnover increased to 3.8–10%/h for HOCl and was unchanged for H2O2, consistent with repair of HOCl-induced sulfur damage. PT-dependent HOCl sensitivity extended to cytotoxicity and DNA strand-breaks, which occurred at orders-of-magnitude lower doses of HOCl than H2O2. The genotoxicity of HOCl in PT-containing bacteria suggests reduced fitness in competition with HOCl-producing organisms and during human infections.


INTRODUCTION
While bacterial DNA modifications that function in restriction-modification (R-M) and epigenetics typically comprise simple nucleobase methylations, 1-4 a growing variety of modification chemistries have introduced new layers of complexity in our understanding of bacterial genome protection and regulation. [5][6][7][8] Here we show that phosphorothioate (PT) modifications of DNA, in which a non-bridging phosphate oxygen is replaced with sulfur, are dynamic modifications that are highly labile during oxidative stress. Although widespread among bacteria and archaea, 9 including human pathogens, 10,11 little is known about the fitness advantage conferred by these DNA modifications other than their role in R-M systems. PTs are synthesized by a complex of proteins (Supplementary Results, Supplementary Fig. 1), DndABCDE, 9 with DndA often replaced by other cysteine desulfurases, such as IscS in Escherichia coli. 12 A highly unusual feature of PTs is that, even in the presence of the cognate restriction proteins DndFGHI in those bacteria in which PTs function in R-M, 13,14 only a small fraction (10-20%) of modification consensus sequences contain a PT. 6 While this points to PT involvement in an atypical R-M system, many bacteria lack obvious restriction activity (DndFGHI), 6,9 which suggests an alternative function for PTs, such as epigenetics.
Another unusual feature of PTs as physiological DNA modifications involves the redox and nucleophilic properties of the sulfur, which are largely unexplored in terms of biological implications. The nucleophilicity of the sulfur has been exploited to sequence RNA and DNA by alkylation to form an unstable PT triester. 15 In terms of redox behavior, the PT sulfur should be readily oxidized by analogy to biological thiols, 16 though there are few studies of PT oxidation under biochemically or biologically relevant conditions. 6,[17][18][19] Here we report the application of genetic and analytical tools to discover that PTs are labile DNA modifications that turn over at a significant rate in unstressed cells and that are selectively sensitive to oxidization by hypochlorous acid (HOCl), which causes genomic instability under biologically relevant conditions.

PT increases sensitivity of bacteria to HOCl but not H 2 O 2
The two bacterial species chosen for study of PT effects on bacterial oxidative stress response, E. coli B7A and Salmonella enterica serovar Cerro 87, naturally possess dnd genes A-I as a R-M system and have been well characterized [6][7][8]13,14 in terms of both quantities of PT (875 ± 68 and 620 ± 41 PT per 10 6 nt, respectively) and PT modifications consensus sequences (G ps AAC/G ps TTC). The choice of mutant strains lacking PTs was highly critical in light of a previous study in which S. enterica strains lacking individual dnd genes A, C, D and E were found to be more sensitive to H 2 O 2 than wild-type S. enterica, which suggested that PTs and/or Dnd proteins were protective against oxidative stress. 18 The problem with these studies is that, while deletion of individual dnd genes indeed prevents PT synthesis, the restriction activity of DndF-I proteins remains intact so that loss of PT results in significant genotoxicity and greatly reduced fitness even in the absence of oxidant stress. 20 Here we used the ΔdndB-H deletion mutants of E. coli and S. enterica, which lack both PT modifications (dndB-E) and PT restriction activity (dndF-H). The choice of exposure conditions was also critical in light of the 1000-fold variation in sensitivity of wild-type E. coli to HOCl (LD 50 6 μM to 6 mM) depending upon the use of phosphate-buffered saline or lysogeny broth (LB medium) for the exposure; [21][22][23][24] LB is known to quench HOCl in exposure studies. 22,24 To assess growth during exposures, we used M9 minimal medium 25 since it sustained equivalent growth of wild-type and ΔdndB-H strains of both bacteria ( Fig.   1) and caused minimal interference with oxidants during exposures.
Using these conditions, H 2 O 2 did not cause PT-dependent cytotoxicity following exposure of either wild-type or ΔdndB-H strains of E. coli and S. enterica (Fig. 1a,c) in either loggrowth or stationary phase ( Supplementary Fig. 2). This is consistent with the conclusion that the apparent protection afforded by PT against H 2 O 2 toxicity as reported previously 18 was actually caused by heightened H 2 O 2 sensitivity of mutant strains lacking individual dnd genes (i.e., lacking PT) as a result of unrestrained restriction enzyme activity from DndF-H. In contrast, PT-containing wild-type bacteria were more sensitive to HOCl than the ΔdndB-H strains lacking PT (Fig. 1e,g). The 1.2-fold increase in LD 50 dose of HOCl for the ΔdndB-H E. coli was not statistically significant (21 ± 2.8 μM versus 17 ± 0.9 μM; mean ± SD for N=3). However, the PT-containing wild-type S. enterica was 4.8-fold more sensitive to HOCl than the ΔdndB-H mutant (LD 50 6.0 ± 0.3 μM versus 29 ± 0.2 μM). That HOCl sensitivity depends on the level of PT modifications was demonstrated using the S. enterica ΔdndB strain in which loss of the DndB transcription factor causes a 2-fold increase in PT levels compared to wild-type cells (1236 ± 53 versus 620 ± 41, respectively; Supplementary  Table 1). 7,8 The LD 50 for HOCl decreased from 6 ± 0.3 μM in the wild-type strain to 3.7 ± 1.8 μM in the ΔdndB strain (p<0.09, Student's t-test; Supplementary Fig. 3). HOCl sensitivity differences due to genetic manipulations were ruled out by the similar HOCl sensitivities of wild-type cells and S. enterica ΔdndF-H cells lacking only the restriction activity ( Supplementary Fig. 3); both strains possess identical levels of PT (Supplementary  Table 1). While the presence of PT did not affect the apparent growth rate of the bacteria during LD 50 exposures to either H 2 O 2 or HOCl, PT-dependent growth effects become apparent at LD 80 doses of HOCl (Fig. 1b, d, f, h). These results demonstrate that PT DNA modifications compromise bacterial fitness in the face of HOCl exposure, which raises questions about the mechanism underlying PT-dependent HOCl toxicity.

HOCl causes PT-dependent DNA damage in vitro and in vivo
As the first step in defining the mechanisms linking PT modifications with HOCl cytotoxicity, we exposed intact DNA isolated from wild-type E. coli to HOCl and quantified d(G PS A) and d(G PS T) dinucleotides by LC-MS. As shown in Fig. 2a, all PTs were consumed at less than 1.6 μM HOCl, which is well below the LD 50 concentration of 6-17 μM for B7A ( Fig. 1) even at a 9-fold higher DNA concentration than in the in vitro experiment (50 μg/mL vs. ~6-7 μg/mL). This points to an HOCl reaction that degrades PTcontaining nucleotides. However, treatment of wild-type B7A and Cerro 87 cells with LD 50 doses of either HOCl or H 2 O 2 did not cause a reduction in the steady-state level of PT (Fig.  2b). This suggests that HOCl does not access DNA in vivo, that PT is unreactive with HOCl in vivo, or that damaged PT in cells is repaired or replaced.
To begin to resolve these mechanistic possibilities, we characterized the chemical mechanisms driving PT-dependent sensitivity to HOCl exposure. The reactions are summarized in Fig. 3, which represents the most comprehensive model for PT oxidation chemistry to date. The first step was to use chromatography-coupled mass spectrometry (LC-MS) to define the products arising in reactions of d(G PS A) and d(G PS T) dinucleotides with H 2 O 2 and HOCl. Reaction of d(G PS A) with either H 2 O 2 or HOCl resulted in a dosedependent disappearance of the PT-containing dinucleotide and formation of desulfurated dinucleotide d(G PO A), a phosphonate-bridged dinucleotide d(G PH A), and free 2deoxyguanosine (dG) and 2′-deoxyadenosine (dA) ( Fig. 3; Supplementary Fig. 4a). The formation of desulfurated and phosphonate products in the dinucleotide reactions is consistent with previously reported observations with H 2 O 2 . 18 Higher concentrations of H 2 O 2 than HOCl were required for d(G PS A) consumption and d(G PO A) and d(G PH A) formation ( Supplementary Fig. 4c), with consumption of the latter products at higher HOCl concentrations. This is consistent with the higher rate constant for HOCl reaction with thiols, such as cysteine, compared to H 2 O 2 (2.9 vs. 3 × 10 −7 M −1 s −1 ; pH 7.4, 37°C). 26,27 Our observation of complete PT consumption by four equivalents of HOCl (corresponds to 1 μM HOCl in Fig. 3) is consistent with the similar reactivity of HOCl with thiols such as cysteine. 28 The heightened reactivity of HOCl compared to H 2 O 2 is likely explained by the fact that, while both H 2 O 2 and HOCl can be reduced to form hydroxyl radical and other reactive species (left pathway in Fig. 3), HOCl is also capable of halogenating thiols by direct reaction with the nucleophilic sulfur (right pathway in Fig. 3). 29 This direct halogenation reaction is faster than HOCl reduction to release free radical species. 30,31 To determine if the phosphonate in d(G PH A) is a precursor of the desulfurated phosphate in d(G PO A), HPLC-purified d(G PH A) was reacted with H 2 O 2 and HOCl, with LC-MS analysis ruling out d(G PO A) formation and establishing d(G PO A) and d(G PH A) as independent reaction products. While these results suggest that the halogenation pathway (right side Fig.  3) will predominate at biologically relevant HOCl concentrations, DNA strand-breaks represent a common feature of both the phosphonate and halogenation pathways. Though it is highly unlikely that HOCl-induced nucleobase halogenation products (e.g., 5chlorocytosine, 8-chloroguanine, 8-chloroadenine) influence the oxidation of PTs by HOCl, we note that no products of nucleobase halogenation were detected in MS CID scans or in UV absorbance chromatograms (in-line HPLC; 260 nm) of HOCl oxidation of PTcontaining dinucleotides ( Supplementary Fig. 4). Small amounts of nucleobase halogenation products are likely to have arisen in the reactions, but are apparently below the detection limit of the analyses performed for the PT oxidation products.
Two sets of studies were performed to assess the formation of DNA strand-breaks caused by PT oxidation by HOCl and H 2 O 2 in vitro and in vivo. First, DNA isolated from wild-type and ΔdndB-H E. coli strains was exposed to the oxidants, including I 2 as positive control for PT-dependent strand breaks, 6 and resolved on a 0.7% agarose gel as a low-sensitivity method to detect DNA strand breaks as smearing. As shown in Fig. 4a, treatment of PTcontaining DNA with I 2 and micromolar concentrations of HOCl caused extensive genomic DNA degradation and smearing, which was not apparent in DNA lacking PTs. Strand-breakinduced smearing is not apparent for H 2 O 2 -exposed DNA at concentrations up to 7.8 mM (Fig. 4a). The basis for the higher reactivity of H 2 O 2 with PT in dinucleotides compared to DNA is uncertain but may relate to contamination with redox-active metals in the synthetic dinucleotides or issues of DNA secondary structure. However, similar behavior was observed in bacteria exposed for 10 min to HOCl and H 2 O 2 concentrations 7.5-and 25-fold higher than LD 50 doses (E. coli B7A wild-type and ΔdndB-H shown in Fig. 4b). The DNA damage caused by HOCl was complete within 10 min of exposure, as judged by the lack of additional DNA strand-breaks in longer incubations ( Supplementary Fig. 5). The basis for the lack of DNA damage by H 2 O 2 may relate to the presence of catalase or other H 2 O 2 degrading activities. We conclude that HOCl, but not H 2 O 2 , produces significant amounts of PT-dependent DNA damage at biologically relevant concentrations. The evidence for HOClinduced PT desulfuration and DNA strand-breaks coupled with unchanging levels of PT following HOCl exposure suggest a mechanism of PT damage and repair or replacement in response to the genomic instability introduced by PT.

PTs are labile and turnover during normal growth
The evidence for a constant steady-state level of PTs in oxidatively stressed cells in the face of oxidative PT desulfuration and strand break formation suggests a repair-like turnover of PTs. To test this model, we developed an isotope labeling and mass spectrometry method to quantify PT dynamics in vivo, as illustrated in Figure Table 2), we were able to quantify (PT per 10 6 nt) all PT isotopomeric dinucleotides within a single DNA sample. 32 The isotope labeling method was validated in log-growing, unstressed E. coli. As shown in

HOCl causes PT turnover at biologically relevant doses
If PTs are more reactive with HOCl than H 2 O 2 , as suggested by the cytotoxicity and DNA damage results, then we would expect HOCl exposure to cause a higher rate of PT turnover reflecting increased damage and subsequent repair by Dnd proteins. We tested this hypothesis by applying the PT turnover assay following exposure of wild-type E. coli and S. enterica to biologically relevant (LD 50 ) doses of HOCl and H 2 O 2 . Oxidant-induced changes in the percentage of replaced PTs as a function of time are shown in Supplementary Fig. 8 and the PT turnover rates derived from linear regression of these data are plotted in Fig. 5d. Exposure of E. coli and S. enterica to H 2 O 2 did not significantly (p=0.97) affect PT turnover, with untreated and H 2 O 2 -treated S. enterica showing turnover rates of 2.1% (± 0.3%) hr −1 and 2.1% (± 0.4%) hr −1 , respectively. Similarly, H 2 O 2 exposure did not change PT turnover in E. coli B7A: 2.5% (± 0.2%) hr −1 and 1.4% (± 0.04%) hr −1 for untreated and H 2 O 2 -treated cells, respectively. These results are consistent with lack of PT-dependent H 2 O 2 toxicity in wild-type and ΔdndB-H strains of both E. coli and S. enterica (Fig. 1). However, HOCl treatment significantly increased the PT turnover to 3.8% (± 0.3%; p<0.005) hr −1 in E. coli and 10% (± 0.8%; p<0.001) hr −1 for S. enterica. This increase can be explained by desulfuration of PT to form phosphate 17 or by repair of HOCl-induced strand breaks, followed by PT reinsertion with [ 32 S] by the Dnd enzymes. The results are also consistent with the greater HOCl sensitivity of the wild-type, PT-containing bacteria compared to ΔdndB-H strains in the survival (Fig. 1) and DNA damage ( Fig. 2 and Fig. 4) assays.

DISCUSSION
Here we have shown that PTs are labile DNA modifications that compromise bacterial fitness in environments that risk oxidative stress and exposure to hypohalous acids. Given the widespread horizontal transfer of dnd genes among bacteria and archaea, including human pathogens, 10,11 PTs clearly confer benefits to bacteria in terms of restrictionmodification activity that protects against pathogenic DNA invasion or by virtue of controlling gene expression in the sizable portion of organisms that lack restriction enzymes. 9 In spite of these benefits, however, we have demonstrated a direct chemical link between PTs and lethal genomic instability caused by exposure to HOCl, but not H 2 O 2 , at levels encountered in many biologically relevant environments. One of the most well defined environments for HOCl exposure involves granulocytes of the human innate immune system, in which activated neutrophils produce up to 10 [36][37][38] For bacteria without the PT modifications, the extensive production of extracellular HOX would be a clear advantage.
Even without exogenous exposure to oxidants, PT modifications in the bacterial strains studied here turn over relatively frequently, with ~2% of PTs replaced every hour, the time of one cell division in minimal media. There is no known PT removal enzyme, so it is likely that PT turnover results from endogenous oxidants, metals, or alkylating agents modifying the PT sulfur, with this damage leading to desulfuration, phosphonate formation, or DNA strand-break chemistry that we observed in vitro (Fig. 3). Another possible scenario to explain PT turnover is that PTs move from one location to another, though maintaining a constant steady-state level. While this scenario is plausible for bacteria lacking PTdependent restriction activity, which represent a significant portion of PT-containing bacteria, 6 this would seem unlikely when sequence-specific restriction activity is present as in the E. coli and S. enterica strains studied here. Unlike chemically stable methyl modifications of DNA, the PT modifications thus present the first case of a physiological DNA modification that is subject to damage and repair.
In parallel with this PT damage and repair activity, PT oxidation produces significant levels of DNA damage, which almost certainly contributes to the fitness cost of bearing PT modifications. Exposure to HOCl caused high levels of DNA strand-breaks that were detectable with low-sensitivity agarose gel analysis, while H 2 O 2 exposure did not produce detectable damage (Fig. 4). Though our characterization of PT oxidation chemistry is the most comprehensive to date (Fig. 3), the presence of undefined chemical structures at some of the 5′-and 3′-ends of the strand-breaks could alter DNA repair efficiency. Notably, the formation of phosphonates in the intact DNA backbone is completely unexplored in terms of effects on protein binding, the existence of repair mechanisms, and their genetic toxicology. The few reports that exist for methylphosphonates (H replaced with CH 3 , Fig. 3) point to significant changes in DNA structure, with bending and deviations from B-like structure near the phosphonate, which could contribute to toxic disruption of DNA replication and transcription. 39,40 Nonetheless, while we do not know the chemistry of the PT-dependent strand-break ends, it is possible that these modified ends could be removed by known 5′and 3′-trimming DNA repair activities, such as endonuclease IV or exonuclease III for 3′termini clean-up and DNA pol I for both 3′-and 5′-termini clean-up. 41 The PT oxidation products may thus present novel challenges to the DNA repair machinery in bacteria, with damage occurring even under unstressed conditions as a result of PT reaction with endogenous oxidants and alkylating agents. Finally, HOCl is known to produce other types of PT-independent DNA damage, such as halogenated and oxidized nucleobases (e.g., 5chlorocytosine, 8-chloroguanine, 8-oxoguanine, 8-chloroadenine). For several reasons, it is highly unlikely that these nucleobase damage products play a role in the differences in HOCl toxicity or DNA strand-break formation in wild-type bacteria compared to bacteria lacking PT modification genes. First, repair of halogenated nucleobases as a cause of the DNA strand-breaks observed in HOCl-treated cells is unlikely given the low level of nucleobase damage expected in the bacteria. Previous studies demonstrated that treating mammalian cells with HOCl at 500 μM led to levels of halogenated nucleobases ranging over 1-10 lesions per 10 5 nt, with damage in RNA only 50% higher. 42 This is too low to account for the high levels of DNA strand-breaks observed at lower HOCl concentrations on the insensitive agarose gels in Figure 4. More importantly, the HOCl-induced DNA strandbreaks were entirely PT-dependent, as was the increase in cytotoxicity in the PT-containing bacterial strains.
We have demonstrated that PTs are highly dynamic DNA modifications that, while conferring benefits to host bacteria, compromise bacterial fitness due to lethal genomic instability under commonly encountered chemical stresses. How the lability of PTs affects the ecological niche of bacteria remains a mystery awaiting quantitative analysis. Along with questions about the evolutionary adaptation of PT-containing bacteria to hostile environments, 43 our observations have implications for the virulence of bacterial pathogens during human infections and for bacterial fitness in other environments.

Bacterial growth assays
Single colonies of the respective bacterial strain were grown in 5 mL isotopically unlabeled M9 medium overnight at 37 °C. Cells (2.7 mL) were pelleted in 50 mL falcon tubes at 3000×g for 8 min at ambient temperature (unless indicated otherwise) and resuspended with fresh M9 medium to a starting optical density at 600 nm (OD 600 ) of 0.2, followed by growth at 37°C, 230 rpm for 60 min.  45 . The stocks were diluted with sterile water immediately before the experiment and distributed in flat-bottom 96 well-plates. Cell stocks were added to each well containing the oxidant, mixed rapidly, and growth was monitored in 10 min increments at 600 nm in an EL X 808iu plate-reader (BIO-TEK Instrument Inc.) set for 37 °C. The OD of each biological replicate was averaged (N=3) and plotted with the standard deviation.

Survival assay and LD 50 calculation
Cells were prepared as described in the growth curve assay. After 60 min oxidant exposure, cells were removed and diluted 1:10 5 with fresh M9 and 70 μL plated using 4 mm glassbeads (VWR 26396-456) on LB-agar plates. Plates were incubated over-night at 37 °C and colonies counted using a FluorChem 8900 (Alpha Innotech). Colony counts were plotted over dose range and the LD 50 dose was calculated from the logarithmic regression equation for each biological replicate (N=3), with data plotted as mean ± standard deviation. The formation of previously established reaction products dG and d(G PO A) were monitored by LC-MS/MS.

Preparation of [ 13 C]-labeled PT dinucleotides for absolute quantification of PT
Wild-type E. coli B7A was grown overnight in 5 mL [ 13 C 6 ]-glucose M9 medium and 1 mL of this culture was used to inoculate 100 mL [ 13 C 6 ]-glucose M9 medium for growth to OD 600 ~1.4 at 37 °C and 230 rpm shaking. Bacteria were then centrifuged (3500×g) at 4 °C for 10 min. DNA was isolated with the Qiagen genomic tip 500G kit, according to manufacturer's instructions. Isolated DNA (~1 mg) was digested with 10u Nuclease P1 (Sigma-Aldrich) in 30 mM NH 4 OAc pH 5.3 and 20 μM ZnCl 2 at 55 °C for 5 h. Calf intestine alkaline phosphatase (34 units; Sigma-Aldrich) and 1/10 volume of 1 M Tris pH 8 were added, with incubation for an additional 4 h at 37 °C to release 2′-deoxynucleosides and PT dinucleotides. Enzymes were removed by centrifugation of the digest through a 10 kDa MWCO column at 12,000×g for 10 min. A 100 μL volume of the digest was injected onto a Hydro-RP HPLC column (4 μm particle size, 80 Å pore size, 250 mm length, 4.6 mm inner diameter; Phenomenex, Torrance, CA) with an Agilent 1200 HPLC with binary pump, degasser, autoinjector, DAD detector, and fraction collector. Ammonium acetate (5 mM, pH 5.3) was used as solvent A at 1 mL/min flow rate and 35 °C column temperature. Elution started with 3% acetonitrile (ACN; solvent B) for 5 min, increasing to 10% ACN after 30 min, followed by a 2 min column rinsing with 20% ACN and then back to 3% ACN at 34 min. Eluting 2′-deoxyribonucleosides and PT dinucleotides were monitored at 260 nm and collected using a fraction collector. Under these conditions, d(G PS A) eluted at 25.8 min and d(G PS T) at 28.0 min. Fractions containing each dinucleotide were combined and lyophilized, and the concentration of PT was adjusted to roughly 100 fmol/μL; 10 mM theophylline was added as an external standard. The resulting internal standard mix is considered a 10× SILIS (stable isotope-labeled internal standard) that results in a final concentration of 10 fmol/μL in the sample.

LC-MS/MS analysis of PT-containing dinucleotides
Synthetic PT dinucleotides or nuclease P1 hydrolyzed DNA (1-10 μg depending on the growth state noted in each experiment) was analyzed by LC-MS/MS on an Agilent 1290 series HPLC system equipped with a Synergy Fusion RP HPLC column (2.5 μm particle size, 100 Å pore size, 100 mm length, 2 mm inner diameter) with a guard column (Phenomenex) and a diode array detector (DAD). The HPLC was coupled to an Agilent 6430 triple quadrupole mass spectrometer (MS). The column was eluted at 0.35 mL/min at 35 °C with a linear gradient of 3-8% acetonitrile in 97% solvent A (5 mM ammonium acetate buffer, pH 5.3) over 11 min. The column is rinsed with 40% acetonitrile in A for 1 min and initial conditions were regenerated by rinsing with 97% solvent A for 2 min. Canonical ribonucleosides eluting from the column were quantified by 260 nm UV absorbance using the DAD. PT-containing dinucleotides were identified and quantified by tandem quadrupole mass spectrometry, with electrospray ionization (ESI) operated with the following parameters: N 2 temperature 350 °C, N 2 flow 10 L/min, nebulizer pressure 40 psi, 3500 V capillary voltage and 2 V cell accelerator voltage. For product identification the MS was operated in positive ion by scanning a 200-1000 Da range or product ion scan mode, respectively. For quantification and PT metabolism assays, the MS was operated mode by monitoring multiple fragmentation reactions (MRM mode) under previously optimized MS conditions. The transitions and retention times used for identification of dinucleotides and nucleosides can be found in Supplementary Table 2.

HOCl exposure of isolated DNA
Genomic DNA (1 μg) from wild-type and ΔdndB-H E. coli were incubated with 0-60 pmol of HOCl in 20 μL total for 60 min at 37°C. The reaction was stopped by addition of 1/10 volume (2 μL) of 5 M NH 4 OAC and 10 volumes (22 μL) of isopropanol. The precipitated DNA was pelleted by centrifugation at 16,000×g for 15 min at 4 °C, washed with isopropanol, and dried under vacuum. Following resuspension in water, DNA was enzymatically hydrolyzed for PT quantification by LC-MS/MS according to the method below. For analysis by gel electrophoresis, DNA (10 μg) was incubated with 360 pmol HOCl as described above, precipitated, washed, resuspended in water with gel loading buffer, and resolved on a 0.7% agarose gel. As a positive control, the same amount of DNA was incubated with a final concentration of 3 mM iodine (Fluka, NY) and 50 mM Na 2 HPO 4 buffer for 5 min at 65 °C to cause strand-breaks at PT sites, as described elsewhere. 6

Analysis of DNA strand-breaks in HOCl-and H 2 O 2 -exposed bacteria
S. enterica wild-type, ΔdndB and ΔdndB-H, or E. coli B7A wild-type and ΔdndB-H were prepared as described for the survival assay. After 60 min of growth, cells were placed on ice and exposed to various doses of HOCl or H 2 O 2 . HOCl dependent strand-breaks were first observed at 7.5-fold×LD 50 for each wild-type strain (see Supplementary Table 3), and these doses were then used for each corresponding ΔB-H strain. Effects caused by the genetic background of the strains and by the resulting difference in LD 50 doses were ruled out by exposure of all bacterial strains to higher doses (25-fold×LD 50 see Supplementary Table 3). Cells (in 1 mL LB) were pelleted at 8000×g for 2 min at 4 °C. Using reagents from the EZNA Bacterial DNA Isolation Kit (Omega Biotek), DNA was isolated by adding 2 μL lysozyme and 20 μL BTL digestion buffer for 10 min at 37 °C, followed by 20 μL proteinase K for 40 min at 55 °C and then 2 μL RNaseA for 2 min at 24 °C. The digestion mix was filtered through a 0.2 μm spin filter at 10,000×g for 10 min to remove insoluble cellular debris. A 40 μL aliquot was mixed with 5 μL of loading dye (6× dye from New England Biolabs) and loaded on a 0.7% agarose gel. A 1kb extended size marker from NEB was used. DNA was stained with ethidium bromide and visualized with FluorChem 8900 (Alpha Innotech).

PT metabolism assay
Cells from a single-colony were grown in 6 mL of [

Statistical analyses
Significance testing was performed using an unpaired, two-tailed Student's t-test based on data from at least three replicate analyses with technical duplicates.

Supplementary Material
Refer to Web version on PubMed Central for supplementary material.  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 H 2 O 2 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 H 2 O 2 , 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 H 2 O 2 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 PS A) 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.