SPECTRUM OF DNA PLATINUM ADDUCT RECOGNITION BY PROKARYOTIC AND EUKARYOTIC DNA-DEPENDENT RNA-POLYMERASES

Double-stranded DNA oligomers were constructed to evaluate the effect of bifunctional and monofunctional platinum(II) complexes at the level of DNA transcription. They contained a single lesion, which is either a cis-[Pt(NH3)2{d(GpTpG)}] intrastrand cross-link, a trans-[Pt(NH3)2{ d(GpTpG)}] intrastrand cross-link, a cis-[Pt(NH3)2{d(GpC/GpC)}] inters trand cross-link, or a (diethylenetriamine)-platinum(II)-dG adduct. Th e synthetic duplexes were multimerized and then used as templates in d inucleotide-primed reactions catalyzed by prokaryotic or eukaryotic RN A polymerases. Reactions were conducted in the presence of a single tr iphosphate substrate (single-step addition reaction) or of a combinati on of triphosphate substrates, permitting elongation of the trinucleot ide products to longer RNA chains (productive elongation reaction), re spectively. In transcription of the platinated strands, none of the DN A adducts provided an absolute block to formation of a single phosphod iester bond by either Escherichia coli RNA polymerase or wheat germ RN A polymerase II. However, the single-step addition reactions were much more impeded from transcription of bifunctional adduct-containing tem plates as compared to those containing monofunctional lesions. Product ive elongation was irreversibly blocked in transcription of the platin ated strand of templates containing a cis-d(GpTpG*) intrastrand cross -link or a cis-d(GpC/G*pC) interstrand cross-link. In both cases tran scription stopped at the level of the lesion. Termination occurred als o several nucleotides before the elongation complexes reached the inte rstrand cross-link. A substantial amount of the RNA polymerase molecul es was able of bypassing the trans-d(GpTpG*) cross-links. In all the cases single-step addition reactions were enhanced on the template str and complementary to that containing the intrastrand cross-links. The enhancement was dependent upon the nature of the lesions, being much l arger in the case of trans- than in the case of cis-d(GpTpG*)-contain ing polymer template. In transcription of the platinated strand of the template containing the monofunctional adduct, considerable bypass of the adduct was observed.