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

Abstract

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.

Figure 1

Sequences and structures of the duplexes used for the FRET experiments.

Figure 2

Structure models of the DNA duplexes. (A) TT-L; (B) Pt-L. Fluorescein and tetramethylrhodamine attached to the linker are shown at the top and bottom of each duplex, respectively.

Figure 3

FRET experiments using the cisplatin-modified duplex. (A and B) Steady-state fluorescence emission spectra of GG-L (A) and Pt-L (B). Thin lines are the spectra of the duplexes without tetramethylrhodamine. (C) Fluorescein emission decay curves of GG-L (circles) and Pt-L (squares). Open and closed markers represent the duplexes without and with tetramethylrhodamine, respectively. Single exponential curve fits are overlaid.

Figure 4

FRET experiments using the (6–4) photoproduct-containing duplexes. (A–E) Steady-state fluorescence emission spectra of TT-L (A), (6–4)-L1 (B), (6–4)-L2 (C), TT-S (D) and (6–4)-S (E). (F) Fluorescein emission decay curves of TT-L (circles) and (6–4)-L1 (squares) with single exponential curve fits. Thin lines in (A–E) and open and closed markers in (F) are explained in the legend to Figure 3.

Figure 5

Determination of the unwinding caused by (6–4) photoproduct formation. (A) Duplexes used for the systematic experiments. The positions of the fluorescein attachment are indicated by arrows with the distances from tetramethylrhodamine. TT represents the site of the (6–4) photoproduct. (B) The distances between the fluorescent dyes in the undamaged (open squares) and (6–4) photoproduct-containing (solid squares) duplexes. Each value is within ±1.2 Å. (C) DNA models used for the data fitting in (B). Unkinked models of the (6–4) photoproduct-containing duplex (left) and the B-DNA duplex (right) are illustrated. Fitting parameters are described in the text.