Fluorescent probes for the analysis of DNA strand scission in base excision repair

Abstract

We have developed fluorescent probes for the detection of strand scission in the excision repair of oxidatively damaged bases. They were hairpin-shaped oligonucleotides, each containing an isomer of thymine glycol or 5,6-dihydrothymine as a damaged base in the center, with a fluorophore and a quencher at the 5'- and 3'-ends, respectively. Fluorescence was detected when the phosphodiester linkage at the damage site was cleaved by the enzyme, because the short fragment bearing the fluorophore could not remain in a duplex form hybridized to the rest of the molecule at the incubation temperature. The substrate specificities of Escherichia coli endonuclease III and its human homolog, NTH1, determined by using these probes agreed with those determined previously by gel electrophoresis using (32)P-labeled substrates. Kinetic parameters have also been determined by this method. Since different fluorophores were attached to the oligonucleotides containing each lesion, reactions with two types of substrates were analyzed separately in a single tube, by changing the excitation and detection wavelengths. These probes were degraded during an incubation with a cell extract. Therefore, phosphorothioate linkages were incorporated to protect the probes from nonspecific nucleases, and the base excision repair activity was successfully detected in HeLa cells.

Figure 1.

Scheme of the BER pathway. The short-patch sub-pathway is shown. The filled and open circles represent damaged and undamaged bases, respectively.

Figure 2.

Structures of the oligonucleotide probes (A) and the damaged bases (B) used in this study.

Figure 3.

(A, B) Cleavage of Fl-RTg (circles), Cy3-STg (squares), Cy5-DHT (triangles), and Fl-T (diamonds) by E. coli Endo III (A) and human NTH1 (B), as determined by fluorescence measurement. (C) Dual-probe analysis (bold lines) of the human NTH1 activity for Fl-RTg (circles) and Cy5-DHT (triangles). The thin lines are the same as those shown in (B).

Figure 4.

Electrophoretic analysis of DNA glycosylase/AP lyase reactions in a cell extract. (A) Fl-Tg-PO (lanes 1–4) and Fl-Tg-PS (lanes 5–8) were incubated with a HeLa cell extract at 30°C for 0 (lanes 1 and 5), 30 (lanes 2 and 6), 60 (lanes 3 and 7) and 90 min (lanes 4 and 8). Lanes 9 and 10 are Fl-GCGCGA and Fl-GCGCG markers, respectively. (B) Fl-T-PS-Dab (lanes 1–4), Fl-Tg-PS-Dab2 (lanes 5–8) and Fl-Tg-PS-Dab1 (lanes 9–12) were incubated with a HeLa cell extract at 30°C for 0 (lanes 1, 5 and 9), 30 (lanes 2, 6 and 10), 60 (lanes 3, 7 and 11) and 90 min (lanes 4, 8 and 12). Lanes 13 and 14 are Fl-GCGCGA and Fl-GCGCG markers, respectively. The probes are listed in Table 3.

Figure 5.

Detection of DNA glycosylase/AP lyase reactions in cells. HeLa cells were incubated at 37°C for 5 h after transfection of Fl-T-PS-Dab (A) and Fl-Tg-PS-Dab2 (B).