Hyperreactivity of B-Z junctions to 4,5',8-trimethylpsoralen photobinding assayed by an exonuclease III/photoreversal mapping procedure.
We have developed an exonuclease III/photoreversal procedure to map, with base-pair resolution, the bases that have photoreacted with 4,5',8-trimethylpsoralen (Me3-psoralen) forming either monoadducts or interstrand crosslinks in DNA. This assay allows quantification of relative rates of Me3-psoralen photobinding to bases in DNA at levels less than one crosslink per 8000 base-pairs. We demonstrate the applicability of the Me3-psoralen mapping procedure on the Z-forming sequence GAATT(CG)6-TA(CG)6AATTC. The results confirm our previous findings that Me3-psoralen forms crosslinks in the 5'TA within the (CG)6TA(CG)6 sequence when it exists in the B conformation but not when it exists in the Z conformation. In addition, with increasing superhelical density we observe at least a hundred-fold increased Me3-psoralen presumably represent B-Z junctions. The two presumed junctions respond differently with increasing negative superhelical tension, however, suggesting that the structures of these negative superhelical tension, however, suggesting that the structures of these junctions differ. This increased Me3-psoralen photoreactivity provides a positive signal for the presence of Z-DNA. The sequence and assay described here provide a "torsionally tuned probe" for determining the effective superhelical density of DNA in vivo.
Kochel
T J
TJ
Department of Biochemistry and Molecular Biology, College of Medicine, University of Cincinnati, OH 45267-0522.
Sinden
R R
RR
D001483:Base Sequence; D001665:Binding Sites; D003432:Cross-Linking Reagents; D004247:DNA / Q000235:genetics* / Q000378:metabolism; D005090:Exodeoxyribonucleases / Q000235:genetics*; D011564:Furocoumarins / Q000378:metabolism*; D008027:Light; D008969:Molecular Sequence Data; D010957:Plasmids; D015183:Restriction Mapping; D014307:Trioxsalen / Q000378:metabolism*
D003432:Cross-Linking Reagents; D011564:Furocoumarins; D004247:DNA; D005090:Exodeoxyribonucleases; C020133:exodeoxyribonuclease III; D014307:Trioxsalen