Site-Specific Benzo[a]pyrene Diol Epoxide-DNA Adducts Inhibit Transcription Elongation by Bacteriophage T7 RNA Polymerase

Benzo[a]pyrene, an extremely potent procarcinogen and mutagen, is metabolized to a variety of products, including the ultimate carcinogen 7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene. This product of biotransformation reacts with DNA, forming a series of adducts principally at the N² position of guanine that differ in their stereochemistry and exhibit unique biological properties. In order to gain a better understanding of the effects on RNA synthesis of these adducts, we used purified bacteriophage T7 RNA polymerase to transcribe a series of templates containing one of four stereoisomerically pure BPDE-guanine lesions—(+)-trans-, (−)-trans-, (+)-cis-, or (−)-cis-anti-N²-BPDE-guanine—or no damaged bases. To construct suitable double-stranded oligodeoxynucleotides for these studies, we annealed an 11-mer containing a site-specific stereoisomerically pure N²-BPDE-guanine adduct, a 37-mer, and a 10-mer to a complementary 58-base sequence of single-stranded DNA. The oligomers were ligated, purified, and reannealed. The resulting DNA template contained the promoter for T7 RNA polymerase and a BPDE adduct at position +16 following the transcription initiation site. The results of the transcription assays clearly demonstrate that each of the adducts inhibits elongation by T7 RNA polymerase, but they do so to significantly different extents, depending on the stereochemical characteristics of the BPDE-modified guanine. The order of inhibition is (+)-trans > (−)-trans > (+)-cis > (−)-cis, when the amount of full-length transcript for each is compared to that obtained for an unmodified template. Furthermore, premature termination of RNA synthesis occurs at or near the site of the BPDE lesion as evidenced by the formation of discrete, truncated transcripts. These results might be related to the fact that the pyrenyl moiety of the trans-BPDE adducts is situated in the minor groove of double-stranded DNA, but is quasi-intercalated into the double helix in the case of the cis stereoisomers. Our results are in agreement with previous data showing that DNA randomly damaged with BPDE is poorly transcribed; they also add a new level of complexity to understanding the influence of these adducts on DNA-dependent enzymatic RNA synthesis by showing a strong effect of lesion stereochemistry on the inhibition of elongation.