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 meta bolized to a variety of products, including the ultimate carcinogen ro xy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene. This product of biotr ansformation 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 under standing of the effects on RNA synthesis of these adducts, we used pur ified bacteriophage T7 RNA polymerase to transcribe a series of templa tes containing one of four stereoisomerically pure BPDE-guanine lesion s-(+)-trans-, (-)-trans-, (+)-cis-, or (-)-cis-anti-N2-BPDE-guanine-or no damaged bases. To construct suitable double-stranded oligodeoxynuc leotides for these studies, we annealed an 11-mer containing a site-sp ecific stereoisomerically pure N2-BPDE-guanine adduct, a 37-mer, and a 10-mer to a complementary 58-base sequence of single-stranded DNA. Th e oligomers were ligated, purified, and reannealed. The resulting DNA template contained the promoter for T7 RNA polymerase and a BPDE adduc t at position +16 following the transcription initiation site. The res ults of the transcription assays clearly demonstrate that each of the adducts inhibits elongation by T7 RNA polymerase, but they do so to si gnificantly different extents, depending on the stereochemical charact eristics 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 tem plate. Furthermore, premature termination of RNA synthesis occurs at o r near the site of the BPDE lesion as evidenced by the formation of di screte, truncated transcripts. These results might be related to the f act that the pyrenyl moiety of the trans-BPDE adducts is situated in t he minor groove of double-stranded DNA, but is quasi-intercalated into the double helix in the case of the cis stereoisomers. Our results ar e in agreement with previous data showing that DNA randomly damaged wi th BPDE is poorly transcribed; they also add a new level of complexity to understanding the influence of these adducts on DNA-dependent enzy matic RNA synthesis by showing a strong effect of lesion stereochemist ry on the inhibition of elongation.