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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.

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Figure 1: The effect of PT modifications on the survival and growth of bacteria after exposure to oxidants.
Figure 2: PT reaction with HOCl leads to a loss of PTs and strand breaks in vitro and in vivo.
Figure 3: A comprehensive model for PT oxidation and alkylation in DNA.
Figure 4: PT reaction with HOCl leads to a loss of PT and strand breaks in vitro and in vivo.
Figure 5: Quantification of PT turnover by isotope labeling and mass spectrometry.

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Acknowledgements

The authors gratefully acknowledge funding from the Deutsche Forschungsgemeinschaft (S.K.), the US National Science Foundation (CHE-1019990; P.C.D.), the US National Institute of Environmental Health Science (ES002109; P.C.D.), the US National Institute of Allergy and Infectious Diseases (AI112711; P.C.D.), the National Natural Science Foundation of China (31630002; D.Y.), and the Singapore-MIT Alliance for Research and Technology sponsored by the National Research Foundation of Singapore (P.C.D.).

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Correspondence to Peter C Dedon.

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Kellner, S., DeMott, M., Cheng, C. et al. Oxidation of phosphorothioate DNA modifications leads to lethal genomic instability. Nat Chem Biol 13, 888–894 (2017). https://doi.org/10.1038/nchembio.2407

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