We have developed a new genomic sequencing method for detecting, with resolution at the nucleotide level, the interstrand DNA cross-links induced by 4,5',8-trimethylpsoralen along single-copy genes in mammalian cells. The cross-links (diadducts) initially formed are converted into monoadducts by alkali reversal prior to the use of terminal transferase-dependent PCR (TD-PCR). After alkali reversal, but not before, the DNA strands can be separated and used as templates for gene-specific primer extension, which is the first step in the TD-PCR procedure. The converted psoralen adducts block primer extension, and the prematurely terminated single-stranded products are then amplified by TD-PCR and visualized on a sequencing gel. Adducts formed by angelicin, a psoralen derivative that forms only monoadducts, were also investigated by use of TD-PCR. Comparison of the adduct distribution patterns of in vivo-treated DNA with those of in vitro-treated DNA revealed that the binding of transcription factors inhibited both psoralen cross-linking and angelicin monoadduct formation in the c-JUN and c-FOS promoters in living human cells. Adduct formation was also inhibited in the region of a putative positioned nucleosome in the c-FOS promoter. These methods should be of general use for study of in vivo protein-DNA interactions and DNA repair.
Mendeley saves you time finding and organizing research
Choose a citation style from the tabs below