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. 2020 Sep 21;9:e60498. doi: 10.7554/eLife.60498

Figure 6. In situ detection of the cellular endogenous U-DNA content.

(A). Scheme represents that genomic uracil residues can be visualized in situ using our further developed U-DNA sensor construct via immunocytochemistry (through FLAG-tag) or directly via SNAP-tag chemistry. (B) HCT116 cells expressing UGI and treated with 5FdUR show efficient staining with the uracil sensor compared to non-treated cells, detected by confocal microscopy. Uracil residues are labelled by our FLAG-ΔUNG-SNAP sensor protein visualized by the SNAP647 substrate. DAPI was used for DNA counterstaining. Our optimized staining method is capable of comparable, specific uracil detection in HCT116 cells even with paraformaldehyde (PFA) fixation compared to the Carnoy fixation applied previously (Róna et al., 2016). Scale bar represents 40 µm. Note that the nuclei of the treated cells (5FdUR_UGI) are enlarged as compared to the non-treated ones (NT_UGI) presumably due to cell cycle arrest (Huehls et al., 2016; Yan et al., 2016).

Figure 6.

Figure 6—figure supplement 1. Design and validation of the new FLAG-ΔUNG-SNAP uracil-sensor.

Figure 6—figure supplement 1.

(A) Scheme of the used constructs in this study, with their applications highlighted. The catalytically inactive (D154N and H277N, mutated sites indicated with black lines) and truncated ΔUNG (the N-terminal 84 residues, responsible for the binding to RPA and PCNA, were also removed) was created from human UNG2. The ΔUNG uracil-recognizing core was fused to different epitope tags: His-tag for affinity purification, 1x/3xFLAG and Au1 for antibody-based detection, DsRed-monomer or SNAP-tag for direct fluorescent detection. Our newly developed FLAG-ΔUNG-SNAP sensor construct is indicated with blue frame, separately from the constructs characterized previously (Róna et al., 2016). (B) Dot blot assay was used to compare uracil binding capability of FLAG-ΔUNG-DsRed and FLAG-ΔUNG-SNAP sensor constructs. CJ236 [dut−, ung−] (positive control) and XL1-Blue [dut+, ung+] (negative control) E. coli genomic DNA samples (8 ng) were measured in two-third dilution series. (C) Comparison of the FLAG-ΔUNG-DsRed and the FLAG-ΔUNG-SNAP constructs in detecting uracil-rich plasmid DNA aggregates in HCT116 in an immunocytochemistry assay. The sensors were visualized through the FLAG epitope tag. DAPI was used to counterstain DNA. Scale bar represents 10 μm.