Analysis of trimethylpsoralen photoreactivity to Z-DNA provides a general in vivo assay for Z-DNA: analysis of the hypersensitivity of (GT)n B-Z junctions.
We have described an exonuclease III/photoreversal procedure to map, with base pair resolution, the bases which have photoreacted with 4,5',8-trimethylpsoralen (Me3-psoralen) forming either monoadducts or interstrand cross-links in DNA (20). This assay allows quantitation of relative rates of Me3-psoralen photobinding to bases in DNA at levels as low as one cross-link per 8,000 base pairs. This assay should be useful for a wide variety of applications of Me3-psoralen photobinding to DNA. Here, we demonstrate the applicability of the Me3-psoralen exo III assay for analysis of the conformation of the Z forming sequences (GT)12ATGT and GAATTC(TG)6TA(TG)6. We have shown previously 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 (34). More recently we have confirmed this result with the exo III assay and have shown at least a hundred fold increase in Me3-psoralen photoreactivity at the 5'AT sequence within the EcoR I sites (GAATTC) which presumably represent B-Z junctions flanking (CG)6TA(CG)6 (20). Here we demonstrate both the characteristic decrease in psoralen photobinding to 5'TAs within (GT)12ATGT and (TG)6TA(TG)6 and the hyperreactivity of B-Z junctions. These characteristic properties of Me3-psoralen photobinding provide an assay for Z-DNA that is applicable in vivo. The general applicability of this approach for assaying Z-DNA in vivo is discussed.
Kochel
T J
TJ
University of Cincinnati, College of Medicine.
Sinden
R R
RR
D001709:Biotechnology; D003432:Cross-Linking Reagents; D004247:DNA / Q000032:analysis*; D004278:DNA, Superhelical / Q000032:analysis; D009690:Nucleic Acid Conformation; D010777:Photochemistry; D010957:Plasmids; D015183:Restriction Mapping; D014307:Trioxsalen
D003432:Cross-Linking Reagents; D004278:DNA, Superhelical; D004247:DNA; D014307:Trioxsalen