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Nat Commun. 2014 Jun 5;5:3951. doi: 10.1038/ncomms4951.

Genomic mapping of phosphorothioates reveals partial modification of short consensus sequences.

Author information

1
1] State Key Laboratory of Microbial Metabolism and School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200233, China [2] Department of Biological Engineering, Center for Environmental Health Science, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA [3].
2
1] Key Laboratory of Combinatorial Biosynthesis and Drug Discovery, Ministry of Education and School of Pharmaceutical Sciences, Wuhan University, Wuhan 430071, China [2].
3
1] Department of Biological Engineering, Center for Environmental Health Science, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA [2].
4
State Key Laboratory of Microbial Metabolism and School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200233, China.
5
Pacific Biosciences, Menlo Park, California 94025, USA.
6
Key Laboratory of Combinatorial Biosynthesis and Drug Discovery, Ministry of Education and School of Pharmaceutical Sciences, Wuhan University, Wuhan 430071, China.
7
Department of Biological Engineering, Center for Environmental Health Science, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.

Abstract

Bacterial phosphorothioate (PT) DNA modifications are incorporated by Dnd proteins A-E and often function with DndF-H as a restriction-modification (R-M) system, as in Escherichia coli B7A. However, bacteria such as Vibrio cyclitrophicus FF75 lack dndF-H, which points to other PT functions. Here we report two novel, orthogonal technologies to map PTs across the genomes of B7A and FF75 with >90% agreement: single molecule, real-time sequencing and deep sequencing of iodine-induced cleavage at PT (ICDS). In B7A, we detect PT on both strands of GpsAAC/GpsTTC motifs, but with only 12% of 40,701 possible sites modified. In contrast, PT in FF75 occurs as a single-strand modification at CpsCA, again with only 14% of 160,541 sites modified. Single-molecule analysis indicates that modification could be partial at any particular genomic site even with active restriction by DndF-H, with direct interaction of modification proteins with GAAC/GTTC sites demonstrated with oligonucleotides. These results point to highly unusual target selection by PT-modification proteins and rule out known R-M mechanisms.

PMID:
24899568
PMCID:
PMC4322921
DOI:
10.1038/ncomms4951
[Indexed for MEDLINE]
Free PMC Article
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