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

Sci Rep. 2015 Jul 27:5:12513. doi: 10.1038/srep12513.

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Base Sequence
  • DNA, Bacterial / chemistry*
  • Molecular Sequence Data
  • Multiprotein Complexes / chemistry*
  • Phosphates / chemistry*
  • Phosphorothioate Oligonucleotides / chemistry*
  • Salmonella enterica / genetics*

Substances

  • DNA, Bacterial
  • Multiprotein Complexes
  • Phosphates
  • Phosphorothioate Oligonucleotides
  • thiophosphoric acid