In July 27st issue of Scientific Reports, Prof. Delin You’s Group and Peter Dedon’s lab from MIT collaborated in publishing the article entitled “In vitro analysis of phosphorothioate modification of DNA reveals substrate recognition by a multiprotein complex”. In this article, their labs provided the first demonstration of in vitro DNA PT modification by PT-modifying enzymes that function as a large protein complex.
A wide variety of prokaryotes possess DNA modifications consisting of sequence-specific phosphorothioates (PT) inserted by members of five-gene cluster. The highly partial PT modification of short consensus motifs in bacterial genomes suggests unusual target selection by PT-modifying enzymes. In this study, Prof. Delin You’s Group characterized the substrate recognition of the PT-modifying enzymes termed DptC, D and E in a cell-free system from Salmonella enterica serovar Cerro 87. The results revealed that double-stranded oligodeoxynucleotides underwent de novo PT modification in vitro, with the same modification pattern as in vivo, i. e., GpsAAC/GpsTTC motif. Hemi-PT DNA also served as substrate of the PT-modifying enzymes, but not single-stranded DNA. Unexpectedly, PT modification also occurred in the GAAC/GTTC motif that could not be modified in vivo, with no significant effect by its flanking regions. The PT-modifying enzymes were then found to function as a large protein complex, with all of three subunits in tetrameric conformations. These studies open the door to understanding the biochemical process and the biological function of this novel epigenetic modification.
PhD students Bo Cao is the first author of this article. This research was supported by grants from the National Science Foundation of China, the Ministry of Science and Technology, Shanghai Pujiang Program from the Shanghai Municipal Council of Science and Technology.