UMMS Affiliation

Department of Biochemistry and Molecular Pharmacology

Publication Date

2020-02-20

Document Type

Article

Disciplines

Amino Acids, Peptides, and Proteins | Biochemical Phenomena, Metabolism, and Nutrition | Biochemistry | Biophysics | Cell Biology | Cells | Genetic Phenomena | Genomics | Molecular Biology

Abstract

We here describe a technique termed STRIDE (SensiTive Recognition of Individual DNA Ends), which enables highly sensitive, specific, direct in situ detection of single- or double-strand DNA breaks (sSTRIDE or dSTRIDE), in nuclei of single cells, using fluorescence microscopy. The sensitivity of STRIDE was tested using a specially developed CRISPR/Cas9 DNA damage induction system, capable of inducing small clusters or individual single- or double-strand breaks. STRIDE exhibits significantly higher sensitivity and specificity of detection of DNA breaks than the commonly used terminal deoxynucleotidyl transferase dUTP nick-end labeling assay or methods based on monitoring of recruitment of repair proteins or histone modifications at the damage site (e.g. gammaH2AX). Even individual genome site-specific DNA double-strand cuts induced by CRISPR/Cas9, as well as individual single-strand DNA scissions induced by the nickase version of Cas9, can be detected by STRIDE and precisely localized within the cell nucleus. We further show that STRIDE can detect low-level spontaneous DNA damage, including age-related DNA lesions, DNA breaks induced by several agents (bleomycin, doxorubicin, topotecan, hydrogen peroxide, UV, photosensitized reactions) and fragmentation of DNA in human spermatozoa. The STRIDE methods are potentially useful in studies of mechanisms of DNA damage induction and repair in cell lines and primary cultures, including cells with impaired repair mechanisms.

Keywords

Repair, Targeted gene modification

Rights and Permissions

Copyright The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/),whichpermitsnon-commercialre-use,distribution,andreproductioninanymedium,providedtheoriginalwork is properly cited. For commercial re-use, please contact journals.permissions@oup.com

DOI of Published Version

10.1093/nar/gkz1118

Source

Kordon MM, Zarębski M, Solarczyk K, Ma H, Pederson T, Dobrucki JW. STRIDE-a fluorescence method for direct, specific in situ detection of individual single- or double-strand DNA breaks in fixed cells. Nucleic Acids Res. 2020 Feb 20;48(3):e14. doi: 10.1093/nar/gkz1118. PMID: 31832687; PMCID: PMC7026605. Link to article on publisher's site

Journal/Book/Conference Title

Nucleic acids research

Related Resources

Link to Article in PubMed

PubMed ID

31832687

Creative Commons License

Creative Commons Attribution-Noncommercial 4.0 License
This work is licensed under a Creative Commons Attribution-Noncommercial 4.0 License

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