INHIBITORY EFFECTS ON THE DNA-BINDING OF AP-1 TRANSCRIPTION FACTOR TOAN AP-1 BINDING-SITE MODIFIED BY BENZO[A]PYRENE 7,8-DIHYDRODIOL 9,10-EPOXIDE DIASTEREOMERS

Benzo[a]pyrene 7,8-dihydrodiol 9,10-epoxide is an established carcinog en, known to covalently bind to DNA, in particular to the exocyclic am inogroup of dG, and thereby cause conformational changes to the double helix, AP-1 is a well-studied transcription factor that specifically binds to the DNA sequence 5'-d(TGAGTCA), The effects of more or less r andomly distributed BPDE adducts on DNA have been studied in different contexts, as well as the effects of different stimuli on transcriptio n factor binding affinity and expression, but so far no investigation has been made concerning the effect of specific modification of a tran scription factor binding site, In this study we have specifically modi fied the binding site of the transcription factor AP-1 with the (+)-an ti- or (-)-syn-enantiomers of BPDE, and have studied how this affects the binding of the Fos-Jun proteins, Both (-)-syn- and (+)-anti-BPDE, giving rise to a cis- and a trans-adduct, respectively, have been used and, in both cases, the binding of AP-1 like proteins from HeLa cell nuclear extracts to the modified binding site decreased by approximate ly 50% as compared to controls, There was no apparent difference in re sponse between the different diastereomers, so it seems that the bindi ng geometry of the adduct (either intercalated or pointing towards the 5'-end in the minor groove, respectively) is of less importance, An i nteresting feature was the apparent yield of three differently shifted bands using the modified binding site, This can be due to conformatio nal changes of the complex and/or the presence of less specific comple xes as an effect of the adduct, Recombinant, truncated Fos-Jun protein s completely failed to bind to modified binding sites when performing the same experiments as detailed above and their binding to unmodified oligonucleotide was 50% less than for native proteins from the nuclea r extract, Supershift assays, using antibodies specific for c-Fos and c-Jun proteins, and competition experiments with various unlabelled ol igonucleotides, were performed in order to check the specificity of bi nding in the observed bands, The results using the oligonucleotide con taining the unmodified binding sequence and HeLa cell nuclear extract were fully consistent with binding of c-Pos and c-Jun, whereas the bin ding to oligonucleotides containing BPDE-modified binding sequences wa s not, This implies involvement of other proteins in this event.