Structural interpretation of DNA–protein hydroxyl-radical footprinting experiments with high resolution using HYDROID

Alexey K. Shaytan, Hua Xiao, Grigoriy A. Armeev, Daria Gaykalova, Galina A. Komarova, Carl Wu, Vasily M. Studitsky, David Landsman, Anna R. Panchenko

Research output: Contribution to journalArticle

Abstract

Hydroxyl-radical footprinting (HRF) is a powerful method for probing structures of nucleic acid–protein complexes with single-nucleotide resolution in solution. To tap the full quantitative potential of HRF, we describe a protocol, hydroxyl-radical footprinting interpretation for DNA (HYDROID), to quantify HRF data and integrate them with atomistic structural models. The stages of the HYDROID protocol are extraction of the lane profiles from gel images, quantification of the DNA cleavage frequency at each nucleotide and theoretical estimation of the DNA cleavage frequency from atomistic structural models, followed by comparison of experimental and theoretical results. Example scripts for each step of HRF data analysis and interpretation are provided for several nucleosome systems; they can be easily adapted to analyze user data. As input, HYDROID requires polyacrylamide gel electrophoresis (PAGE) images of HRF products and optionally can use a molecular model of the DNA–protein complex. The HYDROID protocol can be used to quantify HRF over DNA regions of up to 100 nucleotides per gel image. In addition, it can be applied to the analysis of RNA–protein complexes and free RNA or DNA molecules in solution. Compared with other methods reported to date, HYDROID is unique in its ability to simultaneously integrate HRF data with the analysis of atomistic structural models. HYDROID is freely available. The complete protocol takes ~3 h. Users should be familiar with the command-line interface, the Python scripting language and Protein Data Bank (PDB) file formats. A graphical user interface (GUI) with basic functionality (HYDROID_GUI) is also available.

Original languageEnglish (US)
JournalNature Protocols
DOIs
StateAccepted/In press - Jan 1 2018

Fingerprint

DNA Footprinting
Hydroxyl Radical
DNA
Experiments
Structural Models
Network protocols
DNA Cleavage
Nucleotides
Graphical user interfaces
Gels
Boidae
Molecular Models
Aptitude
Nucleosomes
Information Storage and Retrieval

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)

Cite this

Structural interpretation of DNA–protein hydroxyl-radical footprinting experiments with high resolution using HYDROID. / Shaytan, Alexey K.; Xiao, Hua; Armeev, Grigoriy A.; Gaykalova, Daria; Komarova, Galina A.; Wu, Carl; Studitsky, Vasily M.; Landsman, David; Panchenko, Anna R.

In: Nature Protocols, 01.01.2018.

Research output: Contribution to journalArticle

Shaytan, Alexey K. ; Xiao, Hua ; Armeev, Grigoriy A. ; Gaykalova, Daria ; Komarova, Galina A. ; Wu, Carl ; Studitsky, Vasily M. ; Landsman, David ; Panchenko, Anna R. / Structural interpretation of DNA–protein hydroxyl-radical footprinting experiments with high resolution using HYDROID. In: Nature Protocols. 2018.
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