title: Fluorescence detection of cellular nucleotide excision repair of damaged DNA.
abstract: To maintain genetic integrity, ultraviolet light-induced photoproducts in DNA must be removed by the nucleotide excision repair (NER) pathway, which is initiated by damage recognition and dual incisions of the lesion-containing strand. We intended to detect the dual-incision step of cellular NER, by using a fluorescent probe. A 140-base pair linear duplex containing the (6-4) photoproduct and a fluorophore-quencher pair was prepared first. However, this type of DNA was found to be degraded rapidly by nucleases in cells. Next, a plasmid was used as a scaffold. In this case, the fluorophore and the quencher were attached to the same strand, and we expected that the dual-incision product containing them would be degraded in cells. At 3 h after transfection of HeLa cells with the plasmid-type probes, fluorescence emission was detected at the nuclei by fluorescence microscopy only when the probe contained the (6-4) photoproduct, and the results were confirmed by flow cytometry. Finally, XPA fibroblasts and the same cells expressing the XPA gene were transfected with the photoproduct-containing probe. Although the transfer of the probe into the cells was slow, fluorescence was detected depending on the NER ability of the cells.
authors.affiliation: Division of Chemistry, Graduate School of Engineering Science, Osaka University, 1-3 Machikaneyama, Toyonaka, Osaka 560-8531, Japan.
authors.forename: Tatsuya
authors.initials: T
authors.lastname: Toga
authors.affiliation: Division of Chemistry, Graduate School of Engineering Science, Osaka University, 1-3 Machikaneyama, Toyonaka, Osaka 560-8531, Japan.
authors.forename: Isao
authors.initials: I
authors.lastname: Kuraoka
authors.affiliation: Division of Chemistry, Graduate School of Engineering Science, Osaka University, 1-3 Machikaneyama, Toyonaka, Osaka 560-8531, Japan.
authors.forename: Shun
authors.initials: S
authors.lastname: Watanabe
authors.affiliation: Division of Dermatology, Graduate School of Medicine, Kobe University, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe, Hyogo 650-0017, Japan.
authors.forename: Eiji
authors.initials: E
authors.lastname: Nakano
authors.affiliation: Division of Dermatology, Graduate School of Medicine, Kobe University, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe, Hyogo 650-0017, Japan.
authors.forename: Seiji
authors.initials: S
authors.lastname: Takeuchi
authors.affiliation: Division of Dermatology, Graduate School of Medicine, Kobe University, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe, Hyogo 650-0017, Japan.
authors.forename: Chikako
authors.initials: C
authors.lastname: Nishigori
authors.affiliation: Biosignal Research Center, Kobe University, 1-1 Rokkodai, Nada-ku, Kobe, Hyogo 657-8501, Japan.
authors.forename: Kaoru
authors.initials: K
authors.lastname: Sugasawa
authors.affiliation: Division of Chemistry, Graduate School of Engineering Science, Osaka University, 1-3 Machikaneyama, Toyonaka, Osaka 560-8531, Japan.
authors.forename: Shigenori
authors.initials: S
authors.lastname: Iwai
mesh_terms: D001483:Base Sequence; D015374:Biosensing Techniques; D053837:DNA Cleavage; D004249:DNA Damage; D015342:DNA Probes / Q000737:chemistry; D004260:DNA Repair; D005456:Fluorescent Dyes / Q000737:chemistry; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D010957:Plasmids / Q000737:chemistry / Q000235:genetics
chemical_list: D015342:DNA Probes; D005456:Fluorescent Dyes