Detection of DNA bending in a DNA-PAP1 protein complex by fluorescence resonance energy transfer.

The structure of DNA in a DNA-protein complex was studied by means of fluorescence resonance energy transfer (FRET) method. Oligonucleotide phosphorothioates were labeled with fluorescein and eosin to obtain a donor- and acceptor-labeled DNA. The formation of a complex of the DNA with PAP1(70), which is a DNA binding site fragment derived from transcription regulatory protein, PAP1, of fission yeast, was confirmed by gel retardation analysis and fluorescence measurements. FRET of the donor- and acceptor-labeled DNA with and without PAP1(70) indicated that the DNA in the complex was bent about 26 degrees toward the protein-binding surface.