In vitro analysis of phosphorothioate modification of DNA reveals substrate recognition by a multiprotein complex

• Main Authors: Cao, Bo (Contributor), Zheng, Xiaoqing (Author), Cheng, Qiuxiang (Author), Yao, Fen (Author), Zheng, Tao (Author), Ramesh Babu, I. (Contributor), Zhou, Huchen (Author), You, Delin (Author), Dedon, Peter C. (Contributor)
• Other Authors: Massachusetts Institute of Technology. Department of Biological Engineering (Contributor)
• Format: Article
• Language: English
• Published: Nature Publishing Group, 2015-09-10T13:22:14Z.
• Subjects: Article
• Online Access: Get fulltext

 A wide variety of prokaryotes possess DNA modifications consisting of sequence-specific phosphorothioates (PT) inserted by members of a five-gene cluster. Recent genome mapping studies revealed two unusual features of PT modifications: short consensus sequences and partial modification of a specific genomic site in a population of bacteria. To better understand the mechanism of target selection of PT modifications that underlies these features, we characterized the substrate recognition of the PT-modifying enzymes termed DptC, D and E in a cell extract system from Salmonella. 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. Unexpectedly, in these in vitro analyses we observed no significant effect on PT modification by sequences flanking GAAC/GTTC motif, while PT also occurred in the GAAC/GTTC motif that could not be modified in vivo. Hemi-PT DNA also served as substrate of the PT-modifying enzymes, but not single-stranded DNA. The PT-modifying enzymes were then found to function as a large protein complex, with all of three subunits in tetrameric conformations. This study provided the first demonstration of in vitro DNA PT modification by PT-modifying enzymes that function as a large protein complex.