Fluorescence detection of cellular nucleotide excision repair of damaged DNA

Abstract

To maintain genetic integrity, ultraviolet light-induced photoproducts in DNA must be removed by the nucleotide excision repair (NER) pathway, which is initiated by damage recognition and dual incisions of the lesion-containing strand. We intended to detect the dual-incision step of cellular NER, by using a fluorescent probe. A 140-base pair linear duplex containing the (6-4) photoproduct and a fluorophore-quencher pair was prepared first. However, this type of DNA was found to be degraded rapidly by nucleases in cells. Next, a plasmid was used as a scaffold. In this case, the fluorophore and the quencher were attached to the same strand, and we expected that the dual-incision product containing them would be degraded in cells. At 3 h after transfection of HeLa cells with the plasmid-type probes, fluorescence emission was detected at the nuclei by fluorescence microscopy only when the probe contained the (6-4) photoproduct, and the results were confirmed by flow cytometry. Finally, XPA fibroblasts and the same cells expressing the XPA gene were transfected with the photoproduct-containing probe. Although the transfer of the probe into the cells was slow, fluorescence was detected depending on the NER ability of the cells.

Figure 1. Fluorescent probes to detect the NER dual incisions.

(a) A linear duplex containing the (6–4) photoproduct and the fluorophore–quencher pair. Detection of the dual incisions in cells was not successful due to nonspecific degradation. (b) A plasmid-type probe containing the fluorophore and the quencher in the same strand. Degradation of the dual-incision product by cellular nucleases was expected to obtain the positive signal.

Figure 2. Plasmid-type fluorescent probe to detect the NER dual incisions.

(a) Structure of the plasmid. The green and black circles represent fluorescein and Dabcyl, respectively. The italicized letters represent the BstNI recognition sequence, and the underlined sequence was added to the original pBSII KS (-) UV plasmid to incorporate the modified nucleosides. (b) Chemical structures of the (6–4) photoproduct and the modified nucleosides bearing fluorescein and Dabcyl. (c) UVDE treatment of pBSII KS (-) FQTT (TT) and pBSII KS (-) FQ64 ((6–4)). (d) Repair synthesis on pBSII KS (-) FQTT (TT) and pBSII KS (-) FQ64 ((6–4)), followed by BstNI digestion.

Figure 3. Detection of the NER dual incisions in HeLa cells.

(a) Fluorescence images of the cells cultured at 37°C for 3 h, after transfection with pBSII KS (-) FQTT (upper panels) or pBSII KS (-) FQ64 (lower panels). (b) Flow cytometry analysis of the HeLa cells transfected with pBSII KS (-) FQTT (blue) or pBSII KS (-) FQ64 (red), together with the transfection reporter bearing Cy5.

Figure 4. Detection of the cellular NER ability.

(a) The XPA cells (upper panels) and the same cells in which the XPA gene was expressed (lower panels) were cultured at 37°C for 6 h after transfection with pBSII KS (-) FQ64. (b) The fluorescein and Cy5 emissions from the pBSII KS (-) FQ64 probe and the transfection reporter, respectively, in the XP12ROSV (NER –) and XP12ROSV/XPA (NER +) cells, shown in panel a, were quantified. The data were statistically analyzed by Student's t test (n = 10; *, p < 0.01), and error bars represent standard deviation. Background correction was not performed.