9118 Nucleic Acids Research, 2012, Vol. 40, No. 18 Figure 1. Cleavage reaction of PT DNA. (A) Agarose gel showing the effect of treating a linearized plasmid pHZ209 isolated from S. lividans with PAA–TAE buffer or by mixing PAA with TAE buffer (normal 40 mM TAE buffer pH 7.5 containing 5 mM per-acetic acid). First lane, PAA and TAE mixture. Second lane, pre-run TAE buffer. Third lane, untreated TAE buffer. (B) HPLC traces showing R or S conﬁguration form of synthesized PT dinucleotide d(GPSA) ([M+H]+ m/z 597, 1) oxidized by PAA–TAE. Six new reaction products were identiﬁed by MS, with m/z 565 (2), 581 (3), 332 (4), 252 (5), 316 (6) and 268 (7), respectively. H2O2 peak can also be observed if excess of H2O2 was added to the reaction mixture. Growth curves showed that the S. enterica serovar Cerro 87 wild-type and dptB mutant that contains PT DNA are quite resistant to H2O2, and started growth quickly at higher H2O2 concentrations (Figure 4A and B). Inactivating dptC-E in S. enterica individually by non-polar in-frame deletions showed that loss of phosphorothioation correlated with an increasing lag phase after the addition of H2O2, and cessation of growth at 4.4 mM H2O2. (Note that at the highest H2O2 concentration (4.4 mM), the OD decreased slightly, possibly due to bleaching of the medium.) These results suggested that PT modiﬁcation increased the H2O2 resist- ance of S. enterica. Resistance to DNA double-strand break caused by H2O2 The in vivo susceptibility of PT genomic DNA to double-strand break caused by peroxide was further investigated. Salmonella enterica WT and dptB-E gene mutant cells were treated using increasing amount of H2O2. Chromosomal DNAs were then extracted and DNA double-strand breaks were monitored by gel electrophor- esis. The gels were run in TAE buffer supplemented with thiourea to protect the modiﬁed DNA from degradation during electrophoresis. Figure 5 shows more degradation can be observed in unmodiﬁed compared to PT modiﬁed DNA, indicating that the phosphorothioation protected DNA from oxidative double-strand breakage in vivo. The function of sulfur on DNA as a reducing reagent We suspect that sulfur on the modiﬁed DNA could function as a biological reductant in vivo. A prediction of this model is that, when bacterial cells are facing H2O2 containing environments, the sulfur on the modiﬁed DNA should be consumed to neutralize the oxidant. In order to test this hypothesis, S. enterica and its dptB mutant cells were again incubated with H2O2 and the sulfur content on chromosomal DNA was monitored using Dnd phenotype analysis. Figure 6 shows that phosphorothioation on chromosomal DNA, both in the wild-type strain and its dptB mutant, decreased with the addition of increasing amounts of H2O2, suggesting that sulfur on the modiﬁed DNA was indeed used to neutralize H2O2. DISCUSSION By performing in vitro experiments we discovered, that the peroxides, including H2O2 and peracetic acid, react with and are consumed by PT modiﬁed DNA. The reduction– oxidation reaction, originally identiﬁed as Dnd pheno- type, is manifested in every DNA degradation gel from diverse bacteria (8,14–31). In our hands, this reaction observed in TAE buffer, Tris buffer, glycine buffer (11,12,32) and water. None of the reaction conditions is greatly deviate from physiological conditions. It is predict- able that when PT DNA meets H2O2 and possibly other peroxides, the redox reaction observed here will happen, suggesting that PT DNA could function as a peroxide reducing reagent in vivo. Growth curve experiments showed that S. enterica con- taining naturally phosphorothioated DNA were more re- sistant to H2O2 than their mutant derivatives that lack the modiﬁcation, suggesting that PT modiﬁcation has anti-oxidative physiological function in the hosting bacteria. It is possible that anti-oxidative function could come directly from its reducing chemical properties. Consistent with this view is, sulfur on the modiﬁed DNA is consumed upon incubation of the bacterial cells with H2O2 (Figure 6). We used high concentration of H2O2 (3.4–880 mM) and peracetic acid (0.14–30 mM) to test their reactivity with PT DNA (Figure 2B and Supplementary Figure 2S1). However, the double bonds of DNA, unsaturated membrane lipids and the cysteine SH groups of proteins are prone to oxidation and growth is inhibited when