1. In vitro analysis of phosphorothioate modification of DNA reveals substrate recognition by a multiprotein complex.
- Author
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Cao, Bo, Zheng, Xiaoqing, Cheng, Qiuxiang, Yao, Fen, Zheng, Tao, Ramesh Babu, I., Zhou, Huchen, Dedon, Peter, and You, Delin
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THIOPHOSPHATES ,DNA ,PROTEINS ,PROKARYOTES ,ENZYMES - Abstract
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., G
ps AAC/Gps TTC 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. [ABSTRACT FROM AUTHOR]- Published
- 2015
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