Amplified primer extension assay for psoralen photoproducts provides a sensitive assay for a (CG)6TA(CG)2(TG)8 Z-DNA torsionally tuned probe: preferential psoralen photobinding to one strand of a B-Z junction.
An amplified primer extension assay has been developed for quantitatively mapping the sites of psoralen photoaddition to DNA. This assay was applied to a torsionally tuned Z-DNA-probe that was specifically designed for the primer extension assay. The torsionally tuned Z-DNA forming sequence, (CG)6TA(CG)2(TG)8, forms Z-DNA in vitro at negative superhelical density: sigma = -0.05. The internal 5'-TA dinucleotide was reactive to psoralen when it existed as B-DNA. Upon the formation of Z-DNA, the internal 5'-TA no longer photobound psoralen. The torsionally tuned sequence was synthesized as an EcoRI fragment such that, when Z-DNA formed, the central 5'-AATT of the EcoRI sites was part of the B-Z junctions. The 5'-AATT sequence was not reactive with psoralen when it existed as B-DNA. When the 5'-AATT sequence existed as a B-Z junction, one strand of each junction became hyperreactive to psoralen. The TT directly 5' to the B-DNA-Z-DNA junction photobound psoralen in a strand-specific fashion. Quantitation of the relative rate of psoralen photobinding to the internal 5'-TA and the 5'-AATT at the B-Z junctions provides relationships that are characteristic of the level of supercoiling in DNA.
Hoepfner
R W
RW
Department of Molecular Genetics, Biochemistry and Microbiology, College of Medicine, University of Cincinnati, Ohio 45267-0524.
Sinden
R R
RR
D001482:Base Composition; D001483:Base Sequence; D001665:Binding Sites; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D015342:DNA Probes; D004278:DNA, Superhelical / Q000737:chemistry / Q000378:metabolism; D005363:Ficusin / Q000378:metabolism*; D008027:Light; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009693:Nucleic Acid Hybridization; D010777:Photochemistry
D015342:DNA Probes; D004278:DNA, Superhelical; D004247:DNA; D005363:Ficusin