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 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.
Komura
J
J
Department of Cell Biology, Graduate School of Medicine, Tohoku University, Sendai 980-8575, Japan. junkom@mail.cc.tohoku.ac.jp
Ikehata
H
H
Hosoi
Y
Y
Riggs
A D
AD
Ono
T
T
D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002460:Cell Line; D003432:Cross-Linking Reagents / Q000737:chemistry / Q000378:metabolism*; D018736:DNA Adducts / Q000037:antagonists & inhibitors / Q000378:metabolism*; D018983:DNA Footprinting; D004253:DNA Nucleotidylexotransferase / Q000378:metabolism; D011564:Furocoumarins / Q000378:metabolism; D006801:Humans; D007364:Intercalating Agents / Q000378:metabolism; D008969:Molecular Sequence Data; D009707:Nucleosomes / Q000235:genetics / Q000378:metabolism*; D016133:Polymerase Chain Reaction / Q000379:methods; D011401:Promoter Regions, Genetic; D016760:Proto-Oncogene Proteins c-fos / Q000235:genetics; D016755:Proto-Oncogene Proteins c-jun / Q000235:genetics; D014157:Transcription Factors / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D014307:Trioxsalen / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism*
D003432:Cross-Linking Reagents; D018736:DNA Adducts; D011564:Furocoumarins; D007364:Intercalating Agents; D009707:Nucleosomes; D016760:Proto-Oncogene Proteins c-fos; D016755:Proto-Oncogene Proteins c-jun; D014157:Transcription Factors; C016507:hydroxymethyltrioxsalen; C011659:angelicin; D004253:DNA Nucleotidylexotransferase; D014307:Trioxsalen