e101 Nucleic Acids Research, 2010, Vol. 38, No. 7 PAGE 4 OF 8 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). Analysis of the BER reaction in a HeLa cell extract The probes listed in Table 3 (30 and 12.5 pmol for gel electrophoresis and ﬂuorescence measurement, respec- tively) were incubated with a whole-cell extract (20 mg) (19) at 30 C in a buﬀer (5 ml) containing 50 mM HEPES-KOH (pH 7.8), 70 mM KCl, 9.8 mM MgCl2, 1.3 mM DTT, 2 mM ATP, 22 mM phosphocreatine, 0.2 mM glycine, 0.4 mM EDTA, 0.36 mg/ml BSA, 0.05 mg/ml creatine phosphokinase and 7.8% glycerol. After the reaction (times shown in Figure 4 and Supplementary Figure S7), these mixtures were heated to 95 C for 5 min. Gel electrophoresis was performed as described above, after the addition of 90% formamide (5 ml), heating to 95 C for 3 min, and centrifugation at 15 000 r.p.m. for 3 min. The ﬂuorescence intensity was measured at 37 C with excitation and emission bandwidths of 5 nm, after dilution of each sample with 1 M Tris–HCl (pH 8.0, 195 ml). Detection of the BER reaction in living HeLa cells HeLa S3 cells were grown in Dulbecco’s modiﬁed Eagle’s medium supplemented with 10% fetal bovine serum, 100 U/ml penicillin and 100 mg/ml streptomycin at 37 C in a humidiﬁed 5% CO2 incubator. The cells were seeded into a four-well slide lumox (Greiner Bio-One) and grown to 30–50% conﬂuence. The Fl-Tg-PS-Dab2 probe and the Fl-T-PS-Dab control were transfected into the cells using Lipofectamine 2000 (Invitrogen), according to the manufacturer’s instructions. At 5 h after transfection, the cells were analyzed on an Olympus IX71 ﬂuorescence microscopy system. RESULTS AND DISCUSSION Design of oligonucleotide probes To detect the Endo III-type enzyme reactions by ﬂuores- cence using the ﬂuorophore–quencher system, we designed the oligonucleotide probes shown in Figure 2A. The duplex must be suﬃciently stable at the incubation tem- perature of 37 C, but the ﬂuorophore-bearing oligo- nucleotide on the 50 side of the damage site, which is the product of the enzyme reaction, must be able to dissociate from the rest of the molecule at this temperature. Since the melting temperature of 11-bp duplexes containing Tg in the center was reportedly about 25 C (20), we chose the 13-bp duplex that was used for our previous study on E. coli Endo III (9), and linked the two strands with a tetrathymidylate loop, to avoid dissociation without chain cleavage by the enzyme. Three types of damaged bases, (5R)-Tg, (5S)-Tg and (5S)-DHT (Figure 2B), were incorporated at position X in the hairpin oligonucleotide shown in Figure 2A. Tg is known to be a good substrate for Endo III (8,21), and it exists as either the 5R cis–trans pair (5R,6S and 5R,6R) or the 5S cis–trans pair (5S,6R and 5S,6S), as a result of epimerization at the C6 position in solution (22). DHT is also reportedly recognized by Endo III (21,23). Three types of commercially-available ﬂuorophores, ﬂuorescein, Cy3 and Cy5 (Supplementary Figure S1A), which had diﬀerent excitation/emission wavelengths, were attached to the 50-end (shown as Y in Figure 2A), diﬀering with the lesion types (Table 1), and the appropriate quenchers, Dabcyl and BHQ2 (Supplementary Figure S1B), at the 30-end (shown as Z) were chosen according to the molecular beacon report (24). In addition to these three probes (Fl-RTg, Cy3-STg and Cy5-DHT), Fl-STg, in which ﬂuorescein was attached to the (5S)-Tg-containing oligonucleotide, was prepared to analyze the eﬀect of the ﬂuorophore type, and Fl-T