title: In vitro analysis of phosphorothioate modification of DNA reveals substrate recognition by a multiprotein complex.
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., 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.
authors.affiliation: 1] State Key Laboratory of Microbial Metabolism, and School of Life Sciences &Biotechnology, Shanghai Jiao Tong University, Shanghai, China 200030 [2] Department of Biological Engineering, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139.
authors.forename: Bo
authors.initials: B
authors.lastname: Cao
authors.affiliation: State Key Laboratory of Microbial Metabolism, and School of Life Sciences &Biotechnology, Shanghai Jiao Tong University, Shanghai, China 200030.
authors.forename: Xiaoqing
authors.initials: X
authors.lastname: Zheng
authors.affiliation: State Key Laboratory of Microbial Metabolism, and School of Life Sciences &Biotechnology, Shanghai Jiao Tong University, Shanghai, China 200030.
authors.forename: Qiuxiang
authors.initials: Q
authors.lastname: Cheng
authors.affiliation: State Key Laboratory of Microbial Metabolism, and School of Life Sciences &Biotechnology, Shanghai Jiao Tong University, Shanghai, China 200030.
authors.forename: Fen
authors.initials: F
authors.lastname: Yao
authors.affiliation: State Key Laboratory of Microbial Metabolism, and School of Life Sciences &Biotechnology, Shanghai Jiao Tong University, Shanghai, China 200030.
authors.forename: Tao
authors.initials: T
authors.lastname: Zheng
authors.affiliation: Department of Biological Engineering, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139.
authors.forename: I
authors.initials: I
authors.lastname: Ramesh Babu
authors.affiliation: State Key Laboratory of Microbial Metabolism, and School of Life Sciences &Biotechnology, Shanghai Jiao Tong University, Shanghai, China 200030.
authors.forename: Huchen
authors.initials: H
authors.lastname: Zhou
authors.affiliation: 1] Department of Biological Engineering, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139 [2] Singapore-MIT Alliance for Research and Technology, 1 CREATE Way, Singapore 138602.
authors.forename: Peter
authors.initials: P
authors.lastname: Dedon
authors.affiliation: 1] State Key Laboratory of Microbial Metabolism, and School of Life Sciences &Biotechnology, Shanghai Jiao Tong University, Shanghai, China 200030 [2] Joint International Research Laboratory of Metabolic and Developmental Sciences, Shanghai Jiao Tong University, Shanghai, China 200240.
authors.forename: Delin
authors.initials: D
authors.lastname: You
mesh_terms: D001483:Base Sequence; D004269:DNA, Bacterial / Q000737:chemistry*; D008969:Molecular Sequence Data; D046912:Multiprotein Complexes / Q000737:chemistry*; D010710:Phosphates / Q000737:chemistry*; D054735:Phosphorothioate Oligonucleotides / Q000737:chemistry*; D019779:Salmonella enterica / Q000235:genetics*
chemical_list: D004269:DNA, Bacterial; D046912:Multiprotein Complexes; D010710:Phosphates; D054735:Phosphorothioate Oligonucleotides; C035638:thiophosphoric acid