Defense Mechanism of Phosphorothioated DNA under Peroxynitrite-Mediated Oxidative Stress

Abstract

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 k 1 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 IC 50 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.

Origin of iodine preferential attack at sulfur in phosphorothioate and subsequent P-O or P-S bond dissociation

“…S8–S10 , figure S2 in 7 , and figure S3 in ref. 8 ). Two PT dinucleotides (G PS G and G PS A, the most abundant PT-modified sites in dnd gene–bioengineering S. lividans 1326 and Escherichia coli B7A) were used as the PT model compounds for iodine-induced reactions, and the iodine-induced product profiles were measured using high-pressure liquid chromatography-mass spectrometry (HPLC-MS) ( 8 ).…”

“…The DNA phosphorothioation is physically recognized by the molecular interaction of the sulfur binding domain (SBD) in the Streptomyces coelicolor type IV restriction enzyme ScoMcrA ( 4 , 5 ). Moreover, phosphorothioate chemistry confers PT-DNA antioxidant activity to repair oxidative damage caused by hydrogen peroxide and peracetic acid ( 6 , 7 ) and even peroxynitrious acid ( 8 ), in which PT-DNA is converted back to normal DNA and forms sulfur oxide products. As shown in Fig.…”

“…1 , path 4) is illusive, in part because the P-S/P-O reversion usually dominates, and the P-O scission rarely occurs during most antioxidant reactions ( Fig. 1 , path 2) ( 6 – 8 ). For instance, in the presence of hydrogen peroxide and peroxynitrite, the P-S/P-O reversion converts the phosphorothioate diester to a normal phosphate DNA linkage (loss of the PT handle) and provides multiple electrons to quench additional reactive oxygen species (ROS).…”

Low Energy Photoionization of Phosphorothioate DNA-Oligomers and Ensuing Hole Transfer

“…40 In analogy, it is expected that the potential (A-PS) CT states would contribute to the emission of PS-modified adenine oligomers and change the profile of the fluorescence spectra if the (A-PS) CT states are involved substantially. To prove the existence of CT states, fluorescence spectroscopy and global analysis were measured for d(A ps ) 20 in comparison to that of d(A) 20 .…”

Molecular recognition between bacterial phosphorothioate DNA and sulfur-binding domain (SBD): competition between the water cage and chalcogen-hydrophobic packet

“…PT-DNA) is produced by a series of Dnd proteins encoded in gene cluster dndABCDE 4 and possesses a variety of biological functions. [5][6][7][8][9] In certain bacterial strains another gene cluster dndFGH distinguishes and attacks non-PT-modified foreign DNA, constituting restriction-modification (R-M) systems. 10 Thus, the PT-dependent restriction endonucleases (REases) were believed to restrict PT-modified mobile genetic elements in horizontal gene transfers and prevent foreign DNA invasion.…”