PAGE 5 OF 8 Nucleic Acids Research, 2010, Vol. 38, No. 7 e101 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. containing undamaged thymine at position X was used as a negative control, as listed in Table 1. These oligonucleotides were synthesized on a DNA synthesizer, as described in the ‘Materials and Methods’ section. It was conﬁrmed by gel electrophoresis that the probes could function as substrates for Endo III, as shown in Supplementary Figure S3. Fluorescence analysis of Endo III and NTH1 reactions Prior to the ﬂuorescence analysis of the enzyme reactions, one of the probes, Fl-RTg, was dissolved in a pH 8.0 buﬀer, and its ﬂuorescence was measured at various tem- peratures. The intensity changed only slightly when the temperature was lower than 50 C, but it increased steeply when the solution was heated to 50–65 C. Since this temperature dependence was identical to the transi- tion from the hairpin structure to the single strand deter- mined by ultraviolet (UV) absorption (data not shown), the increase in the ﬂuorescence intensity was obviously caused by the dissociation of the stem region. A similar increase in the ﬂuorescence intensity was observed when the same probe was incubated with E. coli Endo III in a pH 8.0 buﬀer at 37 C. Figure 3A (circles) shows the results after a 30-min incubation with various concentrations of the enzyme. In this case, the excitation wavelength was 494 nm, and the ﬂuorescence was measured at 520 nm, as shown in Table 1. Since the chain cleavage by E. coli Endo III under similar conditions was conﬁrmed by gel electrophoresis, as shown in Supplementary Figure S3, it could be reasoned that the observed increase in the ﬂuo- rescence intensity was caused by dissociation of the ﬂuorescein-bearing fragment due to the thermal instability of the Endo III-cleaved probe, and, hence, the ﬂuores- cence increase could be correlated directly to the DNA glycosylase/AP lyase activity of this enzyme. The vertical axes of the graphs in Figure 3 show the product yield, in which 100% cleavage was derived from the ﬂuorescence intensity of Y-ACGCGA, measured under the same con- ditions. A very similar result was obtained when human NTH1 was used (Figure 3B, circles), but such an increase was not observed for Fl-T, which did not contain a damaged base, in the cases of both E. coli Endo III and human NTH1 (Figure 3, A and B, diamonds). In order to analyze the eﬀect of the ﬂuorophore type, ﬂuorescein/ Dabcyl and Cy3/BHQ2 were attached to the same hairpin oligonucleotide containing (5S)-Tg (Fl-STg and Cy3-STg, respectively), and the ﬂuorescence was measured at the wavelengths shown in Table 1. When these probes were incubated with E. coli Endo III, the results were very similar to those obtained for Fl-RTg, regardless of the ﬂuorophore type (Supplementary Figure S5, A and C). This result agreed with the observa- tion that Fl-RTg and Cy3-STg were cleaved by E. coli Endo III with similar eﬃciencies, as detected in the analysis by gel electrophoresis (Supplementary Figure S3). In the case of human NTH1, only a small increase in the ﬂuorescence intensity was observed (Supplementary Figure S5, B and D), and this property was similar between Fl-STg and Cy3-STg, although the deviation was large at the highest enzyme concentration in the Cy3 case. These results conﬁrmed that the ﬂuorophore type did not aﬀect the analysis by this method. Substrate speciﬁcity determined by ﬂuorescence measurement Although the ﬂuorescence intensity was increased when Cy3-STg was incubated with E. coli Endo III (Figure 3A, squares), the increase was small in the case of human NTH1 (Figure 3B, squares). A diﬀerence between the E. coli and human enzymes was also found for the other probe, Cy5-DHT. An incubation with E. coli Endo III resulted in a very small increase in the ﬂuores- cence intensity (Figure 3A, triangles), whereas the increase by human NTH1 was intermediate (Figure 3B, triangles) between Fl-RTg (circles) and Cy3-STg (squares). In previous studies (8,23), the substrate speciﬁcities of Endo III and NTH1 were analyzed by gel electrophoresis using 32P-labeled substrates. E. coli Endo III recognized both (5R)-Tg and (5S)-Tg well, although (5S)-Tg was preferred