RESULTS The cleavage by PAA–TAE of natural and synthetic PT DNA Distinctive features are associated with PT modiﬁcation: DNA degradation during normal or pulse-ﬁeld gel elec- trophoresis (the Dnd phenotype) which is not inhibited by formaldehyde and proteinase K treatment; the Dnd phenotype can be repressed if the electrophoretic buffer is supplemented with a small amount of reducing agents, especially those containing sulfur, like thiourea, or if Tris is replaced by HEPES in the electrophoresis buffer. The DNA degradation is due to the DNA cleavage speciﬁcally at the site of PT modiﬁcation (1,11,12). The enteropathogenic S. enterica serovar Cerro 87 and the soil-dwelling S. lividans, both with heavily PT modiﬁed DNA, were chosen to investigate the DNA cleavage reaction. Dinucleotide PT d(GPSA) was synthesized to analyze the reaction products. Cleaved genomic DNA was monitored using agarose gel electrophoresis, while the reaction products of d(GPSA) were analyzed using HPLC/MS. We treated puriﬁed genomic DNA and syn- thetic PT dinucleotide using PAA–TAE buffer (PAA, peracetic; TAE (Tris, Acetic acid, EDTA); PAA–TAE can be generated either subjecting TAE to 2 h of electro- phoresis or mixing PAA with TAE) (1,11,12). Exposure of a linearized 5.4-kb plasmid pHZ209 from S. lividans to either PAA–TAE or PAA–TAE mixture causes extensive DNA cleavage (see Supplementary Figure 1SA for the site-speciﬁc cleavage of a 9.7 kb linearized plasmid carrying the Salmonella dpt gene cluster), while plasmid treated with TAE buffer alone remains uncut (Figure 1A). Interestingly, the banding patterns caused by PAA–TAE buffer and PAA–TAE mixture are very similar (Figure 1A). Figure 1B and Supplementary Figure S1B show the cleavage of the synthesized PT d(GPSA) using PAA–TAE. Dinucleotide d(GPSA) (peak 1) was completely degraded by PAA–TAE, producing six new UV254 absorbing peaks (2–7, Figure 1B). The cleavage of R or S conﬁguration of d(GPSA) generated the same six reaction products (Figure 1B). The reaction products PAA–TAE is a mixture of chemicals, including H2O2, PAA, acetic acid, Tris and EDTA. To clarify which chemical is involved in the cleavage reaction, reactivity of d(GPSA) with H2O2 was analyzed using HPLC/MS. Figure 2A shows that the same six UV254 absorbing peaks were generated (peak 2–7) with H2O2 treatment, although the HPLC peak pattern is different. One main reaction product of H2O2 oxidation was d(GA) (peak 3, 60% of reaction products under the reaction condition), with the PT restored to a normal DNA phosphodiester bond. In order to determine what inﬂuences the HPLC peak pattern and how is the PT bond cleaved in the reaction, we incubated d(GPSA) using increasing amount of H2O2 or PAA and analyzed the reaction products using HPLC/ MS. We found that an increment in concentrations of H2O2 (from 3.4 to 880 mM, Supplementary Figure 2S1) Nucleic Acids Research, 2012, Vol. 40, No. 18 9117 or PAA (from 0.14 to 30 mM, Figure 2B), causes peak 3 (corresponding to d(GA)), to gradually become dominant followed by an increase of peak 2. Structural mass spectrum analysis revealed that the sub- stance in peak 2 contained a hydrogen-phosphonate bond dinucleotide d(GHA) (Figure 2C). The structure of d(GHA) was further conﬁrmed by the synthesized of a standard compound. Release of synthesized d(GHA) from the resin using ammonium hy- droxide resulted in its hydrolysis, generating peak 3–7, identical to the PAA degradation products of d(GPSA) (Figure 2D). The H–phosphonate compound d(GHA) is stable under acidic conditions and is the likely intermediate in the cleavage of d(GPSA). High resolution Mass analysis of the reaction products contained in peak 4–7 revealed that d(GHA) was hydrolyzed at either the 30- or 50-side of H–phosphonate bond, but not at both sides, generating the four hydrolyzed products (Figure 2C and D and Supplementary Figure 2S2). d(GPSA) as a reductant The above data established that the PT bond could be oxidized by H2O2 and peracetic acid, and that there are two outcomes of the oxidative reaction: sulfur exchange with oxygen and restoration of a phosphodiester bond, or removal of sulfur generating H–phosphonate bond that is susceptible to hydrolysis. These results suggest that the reaction is a reduction–oxidation reaction. The possibility that the PT linkage can function as a reductant and consumption of H2O2 was further investigated. The reduction of H2O2 by d(GPSA) was monitored using HPLC. Figure 3 shows that, in sharp contrast to the d(GA) control, which has no effect on H2O2, increasing the concentration of d(GPSA) directly correlated with a decrease in H2O2 levels. H2O2 resistance of S. enterica As shown above, oxidization of PT DNA backbone by H2O2 or peracetic acid will result in either the restoration to a normal phosphodiester linkage, or the cleavage of the backbone. In the former case, when the modiﬁcation- hosting bacteria are in the presence of peroxides, like H2O2, PT bonds can function as a reductant: speciﬁcally under the condition of H2O2. The restoration to a normal DNA backbone is innocuous. In the later case, although PT modiﬁed bonds can still function as a reductant, the cleavage of the DNA backbone might be lethal, since in vivo repair of double-strand DNA breaks could be dif- ﬁcult—even a single non-repaired break might result in the death of the bacterium. We asked what is the actual response of the modiﬁcation-hosting bacterial cell to a H2O2 containing environment. Salmonella enterica serovar Cerro 87 contains the four clustered dptB-E genes which are orthologous to the dndB–E genes shared by many PT hosting bacteria. Hypothetically, DptB could be a regulator whose absence increases the amount of DNA phosphorothioa- tion, and DptC, D and E are required for replacing a non-bridging O with S in the DNA backbone (3,5).