In vitro analysis of phosphorothioate modification of DNA reveals substrate recognition by a multiprotein complex
Author(s)Cao, Bo; Zheng, Xiaoqing; Cheng, Qiuxiang; Yao, Fen; Zheng, Tao; Ramesh Babu, I.; Zhou, Huchen; You, Delin; Dedon, Peter C.; ... Show more Show less
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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.
DepartmentMassachusetts Institute of Technology. Department of Biological Engineering
Nature Publishing Group
Cao, Bo, Xiaoqing Zheng, Qiuxiang Cheng, Fen Yao, Tao Zheng, I. Ramesh Babu, Huchen Zhou, Peter Dedon, and Delin You. “In Vitro Analysis of Phosphorothioate Modification of DNA Reveals Substrate Recognition by a Multiprotein Complex.” Scientific Reports 5 (July 27, 2015): 12513.
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