STRIDE—a fluorescence method for direct, specific in situ detection of individual single- or double-strand DNA breaks in fixed cells

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. γH2AX). 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.

Do TUNEL and Other Apoptosis Assays Detect Cell Death in Preclinical Studies?

“…In the same year, Liu et al [9] demonstrated that apoptosis in tumors of breast cancer patients is associated with poor prognosis. More recently, the TUNEL assay has been used to detect DNA breakage in studies unrelated to cell death [10,11]. These include chromothripsis, which is characterized by extensive genomic rearrangements and provides cells with a growth advantage over those without such rearrangements [10].…”

Sequence Preference and Initiator Promiscuity for De Novo DNA Synthesis by Terminal Deoxynucleotidyl Transferase

“…This activity is of major importance in the diversification of immunoglobulins and T cell receptors in the process of V(D)J recombination of the adaptive immune system via random addition of nucleotides to nicked DNA strands. 4 , 5 TdT’s unique ability to mediate template-independent polymerization has made it a valuable tool in a variety of molecular biology applications including finding strand breaks, 6 modifying DNA oligomers with various NTPs, 7 and identifying DNA damage and epigenetic modifications. 8 Furthermore, the enzyme has proven useful for the generation of polynucleotides of high molecular weight 9 and amphiphilic structures upon extension with BODIPY-dUTP, 10 for detection of DNA and RNA on surfaces, 11 , 12 and immobilization of DNA on solid supports.…”

Breakage of CRISPR/Cas9-Induced Chromosome Bridges in Mitotic Cells

“…In addition to methods based on monitoring of histone modification at the damage sites, we used a method for the direct detection of DNA DSBs (double-strand breaks) in chromosome bridges during mitosis. The method we used is abbreviated STRIDE (section “Sensitive Recognition of Individual DNA Ends), and it enables the direct in situ detection of 3′OH DNA ends even when they are individual DNA double-strand cuts ( Kordon et al, 2019 ). It consists of the conjugation of deoxynucleotide analogues to free DNA ends and their detection by hybridization with fluorescent nucleotides after rolling circle amplification.…”

“…Samples were fixed in PFA 4%, and coverslips were sent embedded in 1x PBS to intoDNA (Krakow, Poland). The dSTRIDE methodology was applied according to the protocol previously described by Kordon et al (2019) .…”