Phosphorothioation Of DNA Backbone By Bacterial Dnd Genes

The DNA degradation (Dnd) phenotype was originally observed during electrophoresis of genomic DNA from Streptomyces lividans and was thought to involve a post-replicative DNA modification that reacted with a peracid derivative of Tris formed at the electrophoretic anode. This phenotype was more recently discovered to involve incorporation of sulfur into DNA of strains of S. lividans 66, S. avermitilis NRRL8165, and P. fluorescens PfO-1 by ³⁵S feeding experiment and to be governed by a five gene cluster, dndA-E, in S. lividans. However, the precise chemical nature of DNA sulfur modification remained obscure although extensive effort had been made.Here we set out to define the chemical structure of the DNA sulfur modification by first isolating ³⁵S labeled nucleosides from Dnd phenotype bacterial strains E. coli B7A and DH10B(pJTU1238) cultured with ³⁵S-cysteine. DNA isolated from these strains was hydrolyzed and dephosphorylated and the resulting nucleosides resolved by HPLC followed by scintillation counting to localize ³⁵S-containing fractions.Having established chromatographic parameters for ³⁵S-containing species, we next characterized the chemical properties of the molecules by LC-MS using the same HPLC conditions which revealed reproducible m/z values of 597, 446, 348, 152 and 136. These data suggested the presence of a G- and A-containing dideoxynucleotide structure for the m/z 597 molecular ion, with loss of guanine yielding the ion at m/z of 446. The 16 mass unit increase over a canonical dG-dA dinucleotide, the expected masses for G and A released from the putative dinucleotide, and the nuclease resistance of the putative dinucleotide species suggested the presence of a sulfur atom in the sugar-phosphate backbone, most likely the phorphorothioate-containing species. The involvement of sulfur atom causes two stereoisomer of phosphorothioate bond, R_P and SP. To corroborate the phosphorothioate structure and to define its stereochemistry, we employed synthetic phosphorothioate-containing dideoxynucleotides 5'-d(G_PSA)-3' and 5'-d(A_PSG)-3', each with the chiral R_P or SP configuration of the phosphorous-sulfur bond. Of these model compounds, 5'-d(G_PSA)-3' R_P had an HPLC retention time, high-resolution mass spectral characteristics and LC/MS(4) pattern identical to material observed in B7A and DH10B(pJTU1238).When DH10B(pJTU1238) and B7A genomic DNA were hydrolyzed with only snake venom phosphodiesterase (specific to R_P configuration) and alkaline phosphatase, we could not detect either the R_P or SP isomers of 5'-d(G_PSA)-3' by LC-MS. These results suggest that phosphorothioation of DNA by the Dnd modification system is at least stereo-selective for the R_P configuration of the phosphorothioate. We thus conclude that the S-containing species from the B7A and DH10B(pJTU1238) DNA is a 5'-d(G_PSA)-3' dideoxynucleoside with an R_P phosphorothioate bond.While the present results do not allow us to conclude that the dnd-dependent phosphorothioation of G-A sequences in the E. coli strains occurs sequence specifically, an identical set of studies performed with Streptomyces lividans 1326, which displays the Dnd phenotype, revealed a 5'-d(G_PSG)-3' R_P species. No phosphorothioate form could be detected in a mutant (ZX1) deficient in the dnd gene cluster.The widespread presence of the phenotype in more than 100 microbe isolates and of sets of dnd gene homologs in diverse microorganisms suggests that the phosphorothioation may represent a type of restriction modification system. In addition to the structural and replicative compatibility of phosphorothioate-containing DNA molecules, the known resistance of phosphorothioate linkages in nucleic acids to a variety of nuclease activities, and the post-replicative and site-specific nature of the modification suggest that phosphorothioates inserted by the dnd modification system might play a protection mechanism for specific DNA against nucleases. To our knowledge, unlike other DNA or RNA modification systems, DNA phosphorothioation by dnd modification system is the first physiological modification on the DNA backbone.