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A Novel Saturation Mutagenesis Approach: Single Step Characterization of Regulatory Protein Binding Sites in RNA Using Phosphorothioates
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Genomic mapping of phosphorothioates reveals partial modification of short consensus sequences

Bo Cao1, Chao Chen2, Michael S DeMott3

  • 11] 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].

Nature Communications
|June 6, 2014
PubMed

Insights

Bacterial phosphorothioate DNA modifications, essential for various functions, were mapped using novel sequencing technologies. These methods revealed unusual target selection by modification proteins, challenging existing restriction-modification system models.

Area of Science:

  • Microbiology
  • Genomics
  • Molecular Biology

Background:

  • Bacterial phosphorothioate (PT) DNA modifications are synthesized by Dnd proteins A-E.
  • These modifications often function within restriction-modification (R-M) systems, involving DndF-H proteins.
  • The absence of dndF-H in some bacteria, like Vibrio cyclitrophicus FF75, suggests alternative roles for PT modifications.

Purpose of the Study:

  • To develop and apply novel, orthogonal technologies for genome-wide mapping of bacterial PT modifications.
  • To investigate the genomic distribution and characteristics of PT modifications in Escherichia coli B7A and Vibrio cyclitrophicus FF75.
  • To elucidate the target selection mechanisms of PT modification proteins and assess their relationship with R-M systems.

Main Methods:

  • Development of two novel technologies: single molecule, real-time sequencing and deep sequencing of iodine-induced cleavage at PT (ICDS).
  • Application of these methods to map PT modifications across the genomes of E. coli B7A and V. cyclitrophicus FF75.
  • Oligonucleotide-based assays to demonstrate direct interaction between modification proteins and specific DNA motifs.

Main Results:

  • High agreement (>90%) between the two novel mapping technologies.
  • In E. coli B7A, PT modifications were found on both strands of GpsAAC/GpsTTC motifs, with only 12% of potential sites modified.
  • In V. cyclitrophicus FF75, PT modifications occurred as single-strand modifications at CpsCA motifs, with 14% of potential sites modified.
  • Single-molecule analysis revealed that PT modification can be partial even at actively restricted sites.
  • Demonstration of direct interaction between PT modification proteins and GAAC/GTTC motifs.

Conclusions:

  • The developed sequencing and ICDS methods provide accurate genome-wide mapping of PT modifications.
  • PT modification in bacteria exhibits unusual target selection patterns, deviating from typical R-M system behaviors.
  • The findings suggest that PT modifications have functions beyond canonical R-M systems and highlight the complexity of bacterial DNA modification.

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