PAGE 7 OF 8 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). manner. In the analysis by gel electrophoresis, the bands showing the chain cleavage by a DNA glycosylase/AP lyase were detected, but an unquenched band was also detected, with a mobility similar to that of the full-length probe (Figure 4B, lanes 9–12). This unexpected product was considered to be generated by hydrolysis of the phosphodiester linkage between the 30-end nucleoside and the Dabcyl linker. As shown in Supplementary Figure S1B, this linker had a hydroxyl group and a bridging oxygen, and was diﬀerent from that of ﬂuor- escein. This oxygen-containing part might be recognized as a nucleoside. Therefore, the phosphodiester linkage at this site was changed into a phosphorothioate. This improved probe (Fl-Tg-PS-Dab2 in Table 3) yielded only the expected products (Figure 4B, lanes 5–8). Using Fl-Tg-PS-Dab2, the strand scission in the BER reactions in the HeLa cell extract was monitored by ﬂuorescence. The ﬂuorescence intensity increased depend- ing on the incubation time, whereas no increase was observed for the negative control (Fl-T-PS-Dab), as shown in Supplementary Figure S7. In the experiments using puriﬁed enzymes, analysis by gel electrophoresis revealed that the products had the a,b-unsaturated aldehyde (Supplementary Figure S3), and thus, the increase in the ﬂuorescence intensity observed in Nucleic Acids Research, 2010, Vol. 38, No. 7 e101 Figure 3 showed only the chain cleavage of the probes. On the other hand, the reactions in the cell extract included the 30-end processing of the 50 fragment, as shown in Figure 4, although it did not aﬀect the ﬂuores- cence. Finally, the Fl-Tg-PS-Dab2 probe, as well as the Fl-T-PS-Dab control, was transfected into HeLa cells. After 5 h, ﬂuorescence emission was distinctly detected at the nuclei of the Fl-Tg-PS-Dab2-transfected cells (Figure 5B), whereas ﬂuorescence was hardly observed for the control cells (Figure 5A). CONCLUSION Oligonucleotide-based probes for the detection of strand scission in the BER reactions by ﬂuorescence were developed in this study. The substrate speciﬁcities of E. coli Endo III and human NTH1 were successfully deter- mined, and the presence of the DNA glycosylase/AP lyase activity in living HeLa cells was detected by using a phosphorothioate-modiﬁed probe. The initial step of the BER pathway in cells, especially the cellular recognition of damaged bases, can be studied in detail using this new method. SUPPLEMENTARY DATA Supplementary Data are available at NAR Online. FUNDING The Ministry of Education, Culture, Sports, Science, and Technology, Japan (Grant-in-Aid for Scientiﬁc Research 21310142 to S.I.). Funding for open access charge: Ministry of Education, Culture, Sports, Science, and Technology, Japan. Conﬂict of interest statement. None declared. REFERENCES 1. Garinis,G.A., van der Horst,G.T.J., Vijg,J. and Hoeijmakers,J.H.J (2008) DNA damage and ageing: new-age ideas for an age-old problem. Nature Cell Biol., 10, 1241–1247. 2. Zharkov,D.O. (2008) Base excision DNA repair. Cell. Mol. Life Sci., 65, 1544–1565. 3. Wilson,D.M. and Bohr,V.A. (2007) The mechanics of base excision repair, and its relationship to aging and disease. DNA Repair, 6, 544–559. 4. Rechkunova,N.I., Maltseva,E.A. and Lavrik,O.I. (2008) Nucleotide excision repair in higher eukaryotes: mechanism of primary damage recognition. Mol. Biol., 42, 20–26. 5. Leibeling,D., Laspe,P. and Emmert,S. (2006) Nucleotide excision repair and cancer. J. Mol. Hist., 37, 225–238. 6. Hegde,M.L., Hazra,T.K. and Mitra,S. (2008) Early steps in the DNA base excision/single-strand interruption repair pathway in mammalian cells. Cell Res., 18, 27–47. 7. Mazumder,A., Gerlt,J.A., Absalon,M.J., Stubbe,J., Cunningham,R.P., Withka,J. and Bolton,P.H. (1991) Stereochemical studies of the b-elimination reactions at aldehydic abasic sites in DNA: endonuclease III from Escherichia coli, sodium hydroxide, and Lys-Trp-Lys. Biochemistry, 30, 1119–1126. 8. Katafuchi,A., Nakano,T., Masaoka,A., Terato,H., Iwai,S., Hanaoka,F. and Ide,H. (2004) Diﬀerential speciﬁcity of human and Escherichia coli endonuclease III and VIII homologues for oxidative base lesions. J. Biol. Chem., 279, 14464–14471.