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Nucleic Acids Research, 2001, Vol. 29, No. 24 4948-4954
© 2001 Oxford University Press

Structural study of DNA duplexes containing the (6–4) photoproduct by fluorescence resonance energy transfer

Toshimi Mizukoshi1,*, Takashi S. Kodama2,3, Yoshie Fujiwara1, Tadahide Furuno4, Mamoru Nakanishi4 and Shigenori Iwai1,5

1Department of Bioorganic Chemistry, Biomolecular Engineering Research Institute, 6-2-3 Furuedai, Suita, Osaka 565-0874, Japan, 2Department of Medical Genetics, Biomedical Research Center, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan, 3CREST JST (Japan Science and Technology Corporation), 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan, 4Faculty of Pharmaceutical Sciences, Nagoya City University, 3-1 Tanabedori, Mizuho-ku, Nagoya 467-8603, Japan and 5Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan

Fluorescence resonance energy transfer (FRET) experiments have been performed to elucidate the structural features of oligonucleotide duplexes containing the pyrimidine(6–4)pyrimidone photoproduct, which is one of the major DNA lesions formed at dipyrimidine sites by UV light. Synthetic 32mer duplexes with and without the (6–4) photoproduct were prepared and fluorescein and tetramethylrhodamine were attached, as a donor and an acceptor, respectively, to the aminohexyl linker at the C5 position of thymine in each strand. Steady-state and time-resolved analyses revealed that both the FRET efficiency and the fluorescence lifetime of the duplex containing the (6–4) photoproduct were almost identical to those of the undamaged duplex, while marked differences were observed for a cisplatin-modified duplex, as a model of kinked DNA. Lifetime measurements of a series of duplexes containing the (6–4) photoproduct, in which the fluorescein position was changed systematically, revealed a small unwinding at the damage site, but did not suggest a kinked structure. These results indicate that formation of the (6–4) photoproduct induces only a small change in the DNA structure, in contrast to the large kink at the (6–4) photoproduct site reported in an NMR study.

* To whom correspondence should be addressed. Tel: +81 6 6872 8218; Fax: +81 6 6872 8219; Email: toshimi{at}beri.co.jp


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[Abstract] [Full Text] [PDF]


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