The defense mechanism of phosphorothioated DNA under peroxynitrite-mediated oxidative stress.

DNA phosphorothioation (PT) exists in many pathogenic bacteria; however, the mechanism of PT-DNA resistance to the immune response is unclear. In this work, we meticulously investigated the peroxynitrite (PN) tolerance using PT-bioengineered E. coli strains. The in-vivo experiment confirms that the S+ strain survives better than the S- strain under moderately oxidative stress. The LCMS, IC, and GCMS experiments demonstrated that phosphorothioate partially converted to phosphate, and by-product included sulfate and elemental sulfur. By using O,O-diethyl thiophosphate ester (DETP), the reaction rates k1 was determined to be 4.3 ± 0.5 M-1s-1 in the first-order to both phosphorothioate and peroxynitrite at 35 oC and pH=8.0. The IC50 values of phosphorothioate di-nucleotides are dramatically increased by 400 to 700 folds compared to DETP. The SH/OH Yin-Yang mechanism rationalizes the in-situ DNA self-defense against PN-mediated oxidative stress at the extra bioenergetic cost of DNA modification.

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