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  • Genome-wide protein-DNA interaction site mapping in bacteria using a double-stranded DNA-specific cytosine deaminase.

Genome-wide protein-DNA interaction site mapping in bacteria using a double-stranded DNA-specific cytosine deaminase.

Nature microbiology (2022-06-02)
Larry A Gallagher, Elena Velazquez, S Brook Peterson, James C Charity, Matthew C Radey, Michael J Gebhardt, FoSheng Hsu, Lauren M Shull, Kevin J Cutler, Keven Macareno, Marcos H de Moraes, Kelsi M Penewit, Jennifer Kim, Pia A Andrade, Thomas LaFramboise, Stephen J Salipante, Michelle L Reniere, Victor de Lorenzo, Paul A Wiggins, Simon L Dove, Joseph D Mougous
ABSTRACT

DNA-protein interactions are central to fundamental cellular processes, yet widely implemented technologies for measuring these interactions on a genome scale in bacteria are laborious and capture only a snapshot of binding events. We devised a facile method for mapping DNA-protein interaction sites in vivo using the double-stranded DNA-specific cytosine deaminase toxin DddA. In 3D-seq (DddA-sequencing), strains containing DddA fused to a DNA-binding protein of interest accumulate characteristic mutations in DNA sequence adjacent to sites occupied by the DNA-bound fusion protein. High-depth sequencing enables detection of sites of increased mutation frequency in these strains, yielding genome-wide maps of DNA-protein interaction sites. We validated 3D-seq for four transcription regulators in two bacterial species, Pseudomonas aeruginosa and Escherichia coli. We show that 3D-seq offers ease of implementation, the ability to record binding event signatures over time and the capacity for single-cell resolution.

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Millipore
Anti-VSV-Glycoprotein−Agarose antibody, Mouse monoclonal, clone P5D4, purified from hybridoma cell culture, PBS suspension