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

J. I. Komura, H. Ikehata, Y. Hosoi, A. D. Riggs, T. Ono

Research output: Contribution to journalArticlepeer-review

17 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)4096-4105
Number of pages10
JournalBiochemistry
Volume40
Issue number13
DOIs
Publication statusPublished - 2001 Apr 3

ASJC Scopus subject areas

  • Biochemistry

Fingerprint Dive into the research topics of 'Mapping psoralen cross-links at the nucleotide level in mammalian cells: Suppression of cross-linking at transcription factor- or nucleosome-binding sites'. Together they form a unique fingerprint.

Cite this