The Physiology, Biochemistry And Genomic Distribution Of DNA Phosphorthioate Modifications

PT modifications, a non-bridging phosphate oxygen in DNA replaced with sulphur, are widespread aomong prokaryotes with a diversity of unique PT sequence contexts and three discrete genomic frequencies in different bacteria. However, the present technique to detected PT is based on a tandem quadrupole mass spectrometry approach, by which PT dinulceotides is identified in the nuclease digested genomic DNA. Thus, the consensus sequence and genome distribution of PT in baterial genome are still unknown. PT modifications are required the participation of five-gene cluster(dnd A-E family), but the mechanism of enzymatic PT modification remain largely unclear. In terms of physiological functions of PT modifications, there is evidence that in some bacteria, PT modifications are part of a novel R-M system, but the mechanism of this widespread, PT-dependent restriction system remains unclear. To better understand PT biology, we peformed three studies in this project: genomic mapping of PT across bacterial genome; enzymatic mechanism of PT modification; mechanism of PT-dependent restriction system.To begin with, we firstly placed the modifications in the context of the genomic landscape by developing two highly novel, orthogonal technologies to quantitatively map PT locations in bacterial genomes: single-molecule, real-time(SMRT) sequencing and iodine-cleavage based deep DNA sequencing(ICDS). By SMRT method, PT was detected in GpsAAC/GpsTTC sequences at a frequency of 4855 sites out of 40,701 total(~12% modification) in the 5.2 Mb genome of E. coli B7 A. In addition, singlemolecule analysis of SMRT suggested partial or incomplete modification of these modified sites with PT, i.e., PT modification did not occur consistently at a given site in a genome in a population of bacteria cells-the phenomenon of partial modification. Meanwhile, ICDS was based on the PT-specific cleavage character of iodine, and the cleavage sites were then specifically labled and sequenced. In this method, we detected about 7,000 PT sites in E. coli B7 A genome, with 90% overlap between SMRT and ICDS. More PT sites dectected by ICDS was due to its high-thoughput analysis of DNA molecules, which enable ICDS to identify these sites with highly low modification frequencies.Next, we performed a series of studies to characterize the restriction mechanism of the unusual PT-dependent R-M system in Salmonella, and found that, unlike traditional restriction-modification(R-M) systems, the presence of restriction system in the PT-deficient mutant was not lethal, but instead resulted in several pathological phenotypes. Subsequent transcriptional profiling analysis revealed that more than 600 gene expression were affected by restriction system in this mutant, including the activation of SOS and DNA repair related genes, which was consistent with that the unrestrained restriction enzymes caused extensive DNA cleavage in the absence of PT modifications. ATP hydrolysis activity in two of the restriction proteins suggested an ATP-dependent mechanism of the restriction system. These results suggested that PT-dependent restriction system functioned in an ATP-driven and unusual DNA cleavage-based mechanism and provided new insights into R-M systems.In addition, partial modifcaiton of PT raised a question about the mechanism of PT-modifying enzymes. In this study, the substrate specificity of PT-modifying enzymes was investigated in a cell-free reaction system from Salmonella enterica serovar Cerro 87. The results revealed that doublestranded DNA(ds-DNA) oligonucleotides underwent de novo PT modification by the cell-free system, with the same modification pattern as in vivo, i. e., GpsAAC/GpsTTC motifs. Interestingly, PT modification also occurred in the GAAC/GTTC motif that could not be modified in vivo, with no significant effect by its flanking regions. In addition, hemi-PT DNA also served as substrate of the PT-modifying enzymes, but not single-stranded DNA(ss-DNA). Subsequent heterologous over-expression and purification of Dpt enzymes revealed that Dpt C, D, and E formed a large protein complex, with all of subunits in tetrameric conformations. We also found that purified Dpt CDE complex possessed non-sequence-specificity binding activity to DNA oligonucleotides.In summary, DNA phosphorothiote modification attracted wide interest since it was the first discovery of sulphur in DNA backbone. This study provided better understanding of the PT biology: Fistly, the genomic distribution of PT modification provided new insights into this novel epigenetic modification and helped us to explore its physiological functions; Secondly, the demonstration of PT-dependent R-M system revealed a novel bacterial defense system with distinct feature from traditional R-M systems, which expanded our knowledge about the natual defense systems. Thirdly, the elucidation of PT-modifying enzymes provided new insights into the biochemycal mechanism of PT modifications, and also provided a potential application for producing diverse site- and stereo-specific PT DNAs.