Structural study of DNA duplexes containing the (6-4) photoproduct by fluorescence resonance energy transfer.
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.
Mizukoshi
T
T
Department of Bioorganic Chemistry, Biomolecular Engineering Research Institute, 6-2-3 Furuedai, Suita, Osaka 565-0874, Japan. toshimi@beri.co.jp
Kodama
T S
TS
Fujiwara
Y
Y
Furuno
T
T
Nakanishi
M
M
Iwai
S
S
D002945:Cisplatin / Q000737:chemistry; D004247:DNA / Q000737:chemistry*; D005453:Fluorescence; D009690:Nucleic Acid Conformation; D009841:Oligonucleotides / Q000737:chemistry; D013050:Spectrometry, Fluorescence / Q000379:methods*
D009841:Oligonucleotides; D004247:DNA; D002945:Cisplatin