Preferential modification of GC boxes by benzo[a]pyrene-7,8-diol-9,10-epoxide

The distribution of binding sites for the ultimate carcinogen anti-benzo[a]pyrene-7,8-diol-9,10-epoxide (BPDE-l) in the 5' region of the Chinese hamster ovary aprt gene has been determined. A plasmid (pGAL) containing the entire hamster aprt gene including the 3' and 5' flanking regions was inserted into the BamHI site of the multiple cloning site of pGEM so that the T7 promoter was 5' to the aprt gene. In vitro transcription of BPDE-I-modified pGAL, using the T7 RNA polymerase, revealed two prominent transcriptional stop sites. One of these sites was located in the first exon of the aprt gene, whereas the second transcriptional stop was located approximately 150 bp upstream from the translational start site. This latter region contains two perfect GC-box consensus sequences that are potential Sp1 binding sites. Using a specific laser cutting technique to map BPDE-I DNA binding sites in the 5' flanking region of the aprt gene, we found that the DNA region containing the GC-box consensus sequences was indeed a hot spot for BPDE-I modification.