Published online 27 January 2010 Nucleic Acids Research, 2010, Vol. 38, No. 7 e101 doi:10.1093/nar/gkq022 Fluorescent probes for the analysis of DNA strand scission in base excision repair Naoyuki Matsumoto1, Tatsuya Toga1, Ryosuke Hayashi1, Kaoru Sugasawa 2 , Katsuo Katayanagi3, Hiroshi Ide3, Isao Kuraoka1 and Shigenori Iwai1,* 1Division of Chemistry, Graduate School of Engineering Science, Osaka University, 1-3 Machikaneyama, Toyonaka, Osaka 560-8531, 2Biosignal Research Center, Organization of Advanced Science and Technology, Kobe University, 1-1 Rokkodai, Nada-ku, Kobe, Hyogo 657-8501 and 3Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, Higashi-Hiroshima 739-8526, Japan Received October 6, 2009; Revised January 7, 2010; Accepted January 11, 2010 ABSTRACT We have developed fluorescent probes for the detec- tion 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 50- and 30-ends, respectively. Fluorescence was detected when the phospho- diester 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 incuba- tion 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 32P-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. INTRODUCTION DNA is subjected to chemical reactions with both endogenous and exogenous factors in cells, and the resultant damaged nucleobases cause genetic mutations that lead to carcinogenesis (1). These lesions in DNA are repaired by several pathways to maintain the genetic integrity. Damaged base moieties with a relatively small change in the chemical structure, which are produced by hydrolysis, oxidation and alkylation, are removed by the base excision repair (BER) pathway (2,3), whereas those with a larger structural change, formed by exposure to ultraviolet light and reactions with carcinogens, are the substrates for nucleotide excision repair (4,5). In the former pathway, DNA glycosylases ﬁrst recognize the damaged base and cleave its glycosidic bond. Some of these enzymes have an additional apurinic/apyrimidinic (AP) lyase activity that causes a strand break by b- or b,d-elimination at the AP site formed by the ﬁrst reaction (6). In the case of the endonuclease (Endo) III-type enzymes, a trans a,b-unsaturated aldehyde (7) is report- edly formed by the b-elimination, as shown in Figure 1. The Endo VIII-type enzymes catalyze the b,d-elimination to produce a 30 phosphate at the cleavage site. Mono- functional DNA glycosylases without the associated AP lyase activity require another enzyme, AP endonuclease, to make an incision at the AP site. AP endonuclease and polynucleotide kinase remove the a,b-unsaturated aldehyde and the phosphate, respectively, which are produced at the 30-end by the reactions of the bifunctional enzymes. Subsequently, the gap is ﬁlled with the correct nucleotide by DNA polymerase. In the case of the monofunctional enzymes, the remaining 20-deoxyribose 50-phosphate (dRP) and the ﬂap structure formed as a repair intermediate are removed by the dRP lyase activity of DNA polymerase b and ﬂap endonuclease, respectively. Finally, a phosphodiester linkage is formed by DNA ligase at the nick site, as shown in Figure 1. The strand scission by the DNA glycosylases/AP lyases has been analyzed by using 32P-labeled oligonucleotides. Usually, the 50-end of the damaged base-containing strand is labeled using [g-32P]ATP and polynucleotide kinase, and then this oligonucleotide is hybridized to the unlabeled *To whom correspondence should be addressed. Tel: +81 6 6850 6250; Fax: +81 6 6850 6240; Email: iwai@chem.es.osaka-u.ac.jp The Author(s) 2010. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/2.5), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.