Visualizing DNA single- and double-strand breaks in the Flash comet assay by DNA polymerase-assisted end-labelling

Abstract

In the comet assay, tails are formed after single-cell gel electrophoresis if the cells have been exposed to genotoxic agents. These tails include a mixture of both DNA single-strand breaks (SSBs) and double-strand breaks (DSBs). However, these two types of strand breaks cannot be distinguished using comet assay protocols with conventional DNA stains. Since DSBs are more problematic for the cells, it would be useful if the SSBs and DSBs could be differentially identified in the same comet. In order to be able to distinguish between SSBs and DSBs, we designed a protocol for polymerase-assisted DNA damage analysis (PADDA) to be used in combination with the Flash comet protocol, or on fixed cells. By using DNA polymerase I to label SSBs and terminal deoxynucleotidyl transferase to label DSBs with fluorophore-labelled nucleotides. Herein, TK6-cells or HaCat cells were exposed to either hydrogen peroxide (H2O2), ionising radiation (X-rays) or DNA cutting enzymes, and then subjected to a comet protocol followed by PADDA. PADDA offers a wider detection range, unveiling previously undetected DNA strand breaks.

Graphical Abstract

Figure 1.

(A) A general overview of how the Flash comet protocol is combined with the PADDA-technique. (B) When comets are stained in a conventional comet assay, fluorescent stains such as Hoechst 33342 or SYBR Gold are used for a global staining of DNA, not allowing any discrimination between DNA single-strand breaks (SSBs) and double-strand breaks (DSBs). (C, D) SSBs and DSBs are selectively labelled in two steps using the PADDA protocol. In the first step (C), SSBs are recognized by DNA polymerase I (Pol I) which starts a template dependent polymerization of new fluorescently labelled nucleotides (fluorophores). This elongation will be terminated by a DSB. As a consequence of this, Pol I will blunt DSBs and thus prime the DSBs for the second step in the PADDA (D). The latter step is mediated by the template independent elongation of free 3′OH-groups by terminal deoxynucleotidyl transferase (TdT) which incorporates another fluorophore than the one used in step one, during the elongation in the second step.

Figure 2.

Conventional staining of comets in combination with a selective labelling of DNA-double strands (DSBs, in green): TK6-cells were subjected to single-cell gel electrophoresis using the Flash comet protocol. After the electrophoresis, DSBs were labelled using the PADDA technique where only the fluorophore AF647 was used to label the DSBs. The four different fluorescent stains that were used for the global staining were: SYBR Gold, Hoechst 33342, propidium iodide and acridine orange (all red). In these experiments, the cells had been exposed to 50 μM H₂O₂ for 15 min.

Figure 3.

(A) shows the labelling of DNA single-strand breaks (SSBs, in green) and double-strand breaks (DSBs, in red) in comets from TK6-cells that had been exposed to vehicle alone or hydrogen peroxide (H₂O₂) for 15 min. After exposure, the cells were subjected to single-cell gel electrophoresis using the Flash comet protocol. After the electrophoresis, SSBs and DSBs were labelled using the PADDA technique where the fluorophore AF555 was used to label SSBs and AF647 to label DSBs. Hoechst 33342 (blue) was used for the global staining of DNA. The intensities of the labelled SSBs (B) and DSBs (C) were quantified using a modified comet assay pipeline for CellProfiler. Mean values for the integrated intensities of SSBs and DSBs are marked with a red line (B, C). A minimum of 100 cells were acquired per condition per experiment. Statistical significance was evaluated using the Mann–Whitney test. ***P < 0.001

Figure 4.

(A) shows the labelling of DNA single-strand breaks (SSBs, in green) and double-strand breaks (DSBs, in red) in comets from TK6-cells that had been exposed to vehicle or ionizing radiation from X-rays (0, 10 or 100 Gy). After exposure, the cells were subjected to single-cell gel electrophoresis using the Flash comet protocol. After the electrophoresis, SSBs and DSBs were labelled using the PADDA technique where the fluorophore AF488 was used to label SSBs and AF555 to label DSBs. Hoechst 33342 (Blue) was used for the global staining of DNA. The intensities of the labelled SSBs (B) and DSBs (C) were quantified using a modified comet assay pipeline for CellProfiler. Mean values for the intensities of SSBs and DSBs are marked with a red line (B, C). A minimum of 100 cells were acquired per condition per experiment. Statistical significance was evaluated using the Kruskal-Wallis test followed by Dunn's multiple comparisons test. ***P < 0.001

Figure 5.

(A) shows the labelling of DNA single-strand breaks (SSBs, in green) and double-strand breaks (DSBs, in red) in comets from TK6-cells that had been exposed to vehicle or increasing concentrations of the nickase Nt.BsmAI. After lysis and enzyme reaction, the cells were subjected to single-cell gel electrophoresis using the Flash comet protocol. After the electrophoresis, SSBs and DSBs were labelled using the PADDA technique where the fluorophore AF488 was used to label SSBs and AF555 to label DSBs. Hoechst 33342 (blue) was used for the global staining of DNA. The intensities of the labelled SSBs (B) and DSBs (C) were quantified using a modified comet assay pipeline for CellProfiler. Mean values for the intensities of SSBs and DSBs are marked with a red line (B, C). A minimum of 100 cells were acquired per condition per experiment. Statistical significance was evaluated using the Kruskal–Wallis test followed by Dunn's multiple comparisons test. ***P < 0.001

Figure 6.

(A) shows the labelling of DNA single-strand breaks (SSBs, in green) and double-strand breaks (DSBs, in red) in comets from TK6-cells that had been exposed to vehicle or increasing concentrations of the restriction enzyme RsaI. After lysis and enzyme reaction, the cells were subjected to single-cell gel electrophoresis using the Flash comet protocol. After the electrophoresis, SSBs and DSBs were labelled using the PADDA technique where the fluorophore AF488 was used to label SSBs and AF555 to label DSBs. Hoechst 33342 (blue) was used for the global staining of DNA. The intensities of the labelled SSBs (B) and DSBs (C) were quantified using a modified comet assay pipeline for CellProfiler. Mean values for the intensities of SSBs and DSBs are marked with a red line (B, C). A minimum of 100 cells were acquired per condition per experiment. Statistical significance was evaluated using the Kruskal–Wallis test followed by Dunn's multiple comparisons test. ***P < 0.001

Figure 7.

(A, D) shows the labelling of DNA single-strand breaks on slide (SSBs, in green) and double-strand breaks (DSBs, in red) in fixed HaCat cells that had been exposed to vehicle, Nt.BsmAI or RsaI. After fixation the cells were exposed to DNA damage treatment and SSBs and DSBs were labelled using the PADDA technique where the fluorophore AF488 was used to label SSBs and AF555 to label DSBs. Hoechst 33342 (blue) was used for the global staining of DNA. The intensities of the labelled SSBs (B or E) and DSBs (C or F) were quantified using a modified comet assay pipeline for CellProfiler. Mean values for the intensities of SSBs and DSBs are marked with a red line (B, C, E, F). A minimum of three pictures were acquired per condition per experiment. Statistical significance was evaluated using the Mann–Whitney test. ***P < 0.001