e101 Nucleic Acids Research, 2010, Vol. 38, No. 7 to some extent (8). On the other hand, (5S)-Tg was a very poor substrate for human NTH1, as compared to the 5R isomer (8). The activity of the E. coli enzyme for DHT was extremely low, whereas DHT was a good substrate for mouse NTH1 (23). These results agreed with the substrate speciﬁcity of each enzyme determined by ﬂuorescence measurements in this study. To conﬁrm whether the pref- erence of E. coli Endo III for (5S)-Tg, which was observed previously (8,9), could be shown by using the ﬂuorescent probes, we performed kinetic analysis of the enzyme reaction. Fl-RTg and Fl-STg were incubated with E. coli Endo III, and the ﬂuorescence intensity was measured at 1-min intervals to obtain the velocity at each substrate concentration. The parameters determined from Lineweaver–Burk plots (Supplementary Figure S6) are listed in Table 2. This analysis revealed that the kcat/Km value for the 5S probe was larger by one order of magni- tude than that for the 5R isomer, mainly due to the dif- ference in the Km values, and this tendency was similar to the results obtained by using the 32P-labeled substrates (9). These identical substrate speciﬁcities and comparable kinetic parameters demonstrated that our method using the ﬂuorophore/quencher oligonucleotide probes is reliable, even for a quantitative analysis. Dual-probe analysis of NTH1 reactions Since each ﬂuorophore is excited and detected at speciﬁc wavelengths, we expected that the enzyme reactions with two probes could be analyzed in a single tube, unless the emission band of one ﬂuorophore overlaps with the exci- tation band of the other. To demonstrate that such an analysis could be performed successfully, Fl-RTg and Cy5-DHT, which were good and intermediate substrates for human NTH1, respectively (Figure 3B), were mixed at the same concentration (50 nM), and the NTH1 reactions were analyzed at the excitation/emission wavelengths for each ﬂuorophore. In this case, the excitation wavelengths for ﬂuorescein and Cy5 were suﬃciently separated, so that Table 2. Kinetic parameters of the E. coli Endo III reactions determined with the ﬂuorescent probes Substrate Fl-RTg Fl-STg Km (nM) 8.12 0.402 kcat (min–1) 0.496 0.286 kcat/Km (nM–1 min–1) 0.0611 0.713 PAGE 6 OF 8 each ﬂuorophore could be excited independently. As shown in Figure 3C, the chain cleavage of Fl-RTg was only slightly aﬀected in the presence of Cy5-DHT, but the reaction with Cy5-DHT, which was recognized less eﬃciently by NTH1 than Fl-RTg, was inhibited to some extent, according to our expectation. Since this type of study cannot be performed with 32P-labeled substrates, this new method provides various novel application possibilities. Detection of cellular BER using phosphorothioate-containing probes The probes used in the above-mentioned experiments may be applied to the analysis of strand scission by the DNA glycosylases/AP lyases in cells because product separation is not required for the detection of the enzyme activity. Therefore, the probes were investigated by using a HeLa cell extract. First, an Fl-RTg-type oligonucleotide without the quencher (Fl-Tg-PO in Table 3) was prepared to facil- itate the detection of the bands after gel electrophoresis, and this oligonucleotide was incubated with a HeLa cell extract. Analysis by gel electrophoresis revealed that this type of oligonucleotide was degraded, probably by exonucleases (Figure 4A, lanes 1–4). This result indicated that ﬂuorescence would be detected non-speciﬁcally even when a damaged base is not present in the probe. Therefore, phosphorothioate linkages, which have been used to protect antisense oligonucleotides from nucleases (25,26), were incorporated. Considering the enzyme–phos- phate interactions found in the crystal structure of a complex between Bacillus stearothermophilus Endo III and its substrate DNA (27), ﬁve phosphodiester linkages around the damaged base in each strand were kept intact. An incubation of this phosphorothioate-modiﬁed oligonucleotide (Fl-Tg-PS in Table 3) with a cell extract generated several products, including the Fl-GCGCGA 6-mer and the Fl-GCGCG 5-mer, in which the italicized letters represent nucleoside 30-phosphorothioate (Figure 4A, lanes 5–8). The 6-mer is the product generated by the chain cleavage by a bifunctional enzyme, most likely NTH1 in this case, followed by the removal of the a,b-unsaturated aldehyde by AP endonuclease, and the 5-mer is produced by hydrolysis of the phosphodiester linkage in the Fl-GCGCGA 6-mer by an exonuclease. Since non-speciﬁc degradation could be prevented by the phosphorothioate modiﬁcation, the quencher was attached to the 30-end of Fl-Tg-PS, and this probe (Fl-Tg-PS-Dab1 in Table 3) was treated in the same Table 3. Oligonucleotide probes used for the cell extract analysis Designation Fl-Tg-PO Fl-Tg-PS Fl-Tg-PS-Dab1 Fl-Tg-PS-Dab2 Fl-T-PS-Dab Sequencea Fluorescein-d(GCGCGA-(5R)Tg-ACGCCGCCCCCGGCGTATCGCGC) Fluorescein-d(GCGCGA-(5R)Tg-ACGCCGCCCCCGGCGTATCGCGC) Fluorescein-d(GCGCGA-(5R)Tg-ACGCCGCCCCCGGCGTATCGCGC)-Dabcyl Fluorescein-d(GCGCGA-(5R)Tg-ACGCCGCCCCCGGCGTATCGCGC)-Dabcyl Fluorescein-d(GCGCGA-T-ACGCCGCCCCCGGCGTATCGCGC)-Dabcyl aThe underlined letters and the italicized letters represent the double-stranded region and the 30-phosphorothioate, respectively.