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Nat Protoc. 2018 Nov;13(11):2535-2556. doi: 10.1038/s41596-018-0048-z.

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

Author information

1
National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, MD, USA. shaytan_ak@mail.bio.msu.ru.
2
Department of Biology, Lomonosov Moscow State University, Moscow, Russia. shaytan_ak@mail.bio.msu.ru.
3
Laboratory of Biochemistry and Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA.
4
Department of Biology, Lomonosov Moscow State University, Moscow, Russia.
5
Department of Otolaryngology-Head and Neck Surgery, Johns Hopkins School of Medicine, Baltimore, MD, USA.
6
Department of Physics, Lomonosov Moscow State University, Moscow, Russia.
7
Department of Biology, Johns Hopkins University, Baltimore, MD, USA.
8
Department of Molecular Biology & Genetics, Johns Hopkins University School of Medicine, Baltimore, MD, USA.
9
Fox Chase Cancer Center, Philadelphia, PA, USA.
10
National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, MD, USA.
11
National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, MD, USA. panch@ncbi.nlm.nih.gov.

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.

PMID:
30341436
PMCID:
PMC6322412
DOI:
10.1038/s41596-018-0048-z
[Indexed for MEDLINE]
Free PMC Article

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