Mapping psoralen cross-links at the nucleotide level in mammalian cells: Suppression of cross-linking at transcription factor- or nucleosome-binding sites

We have developed a new genomic sequencing method for detecting, with resol ution at the nucleotide level, the interstrand DNA cross-links induced by 4 ,5',8-trimethylpsoralen along single-copy genes in mammalian cells. The cro ss-links (diadducts) initially formed are converted into monoadducts by alk ali reversal prior to the use of terminal transferase-dependent PCR (TD-PCR ). After alkali reversal but not before, the DNA strands can be separated a nd 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 inv estigated by use of TD-PCR. Comparison of the adduct distribution patterns of in vivo-treated DNA with those of in vine-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 h uman cells. Adduct formation was also inhibited in the region of a putative positioned nucleosome in the c-FOS promoter. These methods should be of ge neral use for study of in vivo protein-DNA interactions and DNA repair.