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 the byproduct included sulfate
and elemental sulfur. When O,O-diethyl thiophosphate
ester (DETP) was used, the reaction rate k1 was determined to be 4.3 ± 0.5 M–1 s–1 in the first-order for both phosphorothioate and
peroxynitrite at 35 °C and pH of 8.0. The IC50 values
of phosphorothioate dinucleotides are dramatically increased by 400–700-fold
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.