Phosphorothioate DNA As An Antioxidant In Bacteria

Phosphorothioation modification on the bacterial genome DNAleads to the often-observed Dnd phenotype: DNA degradation duringelectrophoresis. DNA phosphorothioation is controlled by five clusteredgenes dndA-E. Four of the genes, dndA and dndC-E, are essential for thephosphorothioate modification. Biological function（s） of the modificationremained elusive until the recent discoveries of a Streptomyces coelicolorgene that specifically restricts phosphorothioate DNA from S. lividans,and a restriction system in S. enterica serovar Cerro87against PT-freeheterologous DNA. What is chemical property of phosphorothioationleading to the DNA ds breakage （Dnd phenotype）? Whether thissensitivity to dsDNA cleavage makes the host bacterial cell sensitive orinsensitive to oxidative environment?The both synthesized R and S configuration of phosphorothioatedinucleotide dG^SA were completely degraded by PAA-TAE, producing six new UV₂₅₄absorbing peaks by HPLC. The reaction reactivity of dG^SAwith H₂O₂or PAA was analyzed using HPLC-MS. Phosphorothioatedinucleotide dG^SA was directly reacted with PAA in water to produce sixcompounds. The reaction products of dG^SA and PAA were analyzed byHPLC-MS, and the results showed that there are different HPLC patternsin different pH Tris-HCl buffer. The H-phosphonate compound dG^HA isstable under acidic conditions and is likely the intermediate in thecleavage of dG^SA. The structure of compound in peak2was analyzed byhigh resolution mass spectrometry. Mass spectrum analysis revealed thatthe compoud in peak2maybe contain a hydrogen-phosphonatebond（dG^HA）. The structures of dG^HA and compounds4-7were furtherconfirmed by the synthesized standard compounds.The reaction products of dG^SA and PAA in1%HAc was lyophilized.The lyophilized powder was dissolved in different pH buffer andanalyzed by HPLC. The results showed that hydrogen-phosphonate bondof dinucleotide dG^HA can be hydrolyzed in pH>5.0solution. Thephosphorothioate diester was treated by PAA or H₂O₂and then waslyophilized to pungent yellow lyophilized powder. The yellow powderwas identified as elementary sulfur using GC-MS. H-phosphonatecompound dG^HA can be oxidized into normal dinucleotide dG^OA by PAAor H₂O₂in pH7.0buffer.Phosphorothioate DNA also can be directly reacted with H₂O₂in different solution. The products of reaction between phosphorothioateDNA and H₂O₂have two possible fates: backbone cleavage or transferredinto normal backbone.Base on above results, we proposed the mechanism ofphosphorothioate DNA cleavage during electrophoresis in TAE buffer. Invitro oxidation of phosphorothioate （PT） DNA by H₂O₂or peracetic acidhas two possible outcomes: DNA backbone cleavage or sulfur removalresulting in restoration of normal DNA backbone.The possibility that phosphorothioate linkage can function as areductant and consume H₂O₂was analyzed. The consumption of H₂O₂by dG^SA was monitored by HPLC. The results showed that, remarkablycontrast to the dG^OA control, with the treatment of increasing dG^SA, theamount of H₂O₂was increasingly consumed. Detected by HPLC, thelower limit of the reaction for PAA is0.14mM, while monitoring usingmass, as low as10μM PAA can still be detected to react with thedinucleotide. Growth curves showed that the S. enterica serovar Cerro87wild-type and dpt B mutant that contains PT DNA are quite resistant toH₂O₂, and started growth quickly at higher H₂O₂concentrations.Inactivating dpt C-E in S. enterica individually by non-polar in-framedeletions showed that loss of phosphorothioation correlated with anincreasing lag phase after the addition of H₂O₂, and cessation of growth at4.4mM H₂O₂. These results suggest that the phosphorothioate modification increased the H₂O₂resistance of S. enterica.S. enteric wild-type and the dpt B-E gene mutants were treated withincreasing amount of H₂O₂. Chromosomal DNAs were then isolated andmonitored using gel electrophoresis. The results showed that more DNAdouble strands break can be observed by DNA withoutphosphorothioation than DNA modified, indicating that phosphorothioatemodification could protect DNA from oxidative damage in vivo. When S.enterica and its dpt B mutant cells were again incubated with H₂O₂, thesulfur modified abundance on chromosomal DNA was decreased withincreasing amounts of H₂O₂treatment, suggesting that sulfur on themodified DNA was used to consume H₂O₂as an antioxidant.Introducing DNA phosphorothioate modification into E. coli DH10Bcan enhance its anti-oxidation capability. This will explain why the dptgene cluster is located on genomic island and why it is kept in diversemicroorganisms while the modification is not essential.