STRIDE-a fluorescence method for direct, specific in situ detection of individual single- or double-strand DNA breaks in fixed cells
Authors
Kordon, Magdalena M.Zarebski, Miroslaw
Solarczyk, Kamil
Ma, Hanhui
Pederson, Thoru
Dobrucki, Jurek W.
UMass Chan Affiliations
Department of Biochemistry and Molecular PharmacologyDocument Type
Journal ArticlePublication Date
2020-02-20Keywords
RepairTargeted gene modification
Amino Acids, Peptides, and Proteins
Biochemical Phenomena, Metabolism, and Nutrition
Biochemistry
Biophysics
Cell Biology
Cells
Genetic Phenomena
Genomics
Molecular Biology
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Show full item recordAbstract
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.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
DOI
10.1093/nar/gkz1118Permanent Link to this Item
http://hdl.handle.net/20.500.14038/41387PubMed ID
31832687Related Resources
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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.comDistribution License
http://creativecommons.org/licenses/by-nc/4.0/ae974a485f413a2113503eed53cd6c53
10.1093/nar/gkz1118
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Except where otherwise noted, this item's license is described as 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