TRANSLESIONAL SYNTHESIS ON DNA TEMPLATES CONTAINING SITE-SPECIFICALLYPLACED DEOXYADENOSINE AND DEOXYGUANOSINE ADDUCTS FORMED BY THE PLANT CARCINOGEN ARISTOLOCHIC ACID

Synthetic oligonucleotides (18-mers) containing either a single deoxya denosine residue or a single deoxyguanosine residue were treated with aristolochic acid I (AAI) or aristolochic acid II (AAII), the main com ponents of the plant carcinogen aristolochic acid (AA). These reaction s resulted in the formation of site-specifically adducted oligonucleot ides containing the two known AAI-DNA adducts (dA-AAI, dG-AAI) or the two known AAII-DNA adducts (dA-AAII, dG-AAII) at position 15 from the 3' end. Using HPLC chromatography, the oligonucleotides were purified and subsequently shown to contain the adducts of interest by P-32-post labelling. The adducted oligonucleotides were used as templates in pri mer (11-mer) extension reactions catalysed by modified bacteriophage T 7 DNA polymerase (Sequenase). Regardless of the type of DNA adduct exa mined, DNA synthesis was blocked predominantly (80-90%) at the nucleot ide 3' to each adduct, although primer extension to the full length of the template was noted with unmodified control templates. However, 15 nucleotide products, indicating blocking of DNA synthesis after incor poration of a nucleotide opposite tire adduct and translesional synthe sis products were formed in all cases in different amounts, depending on the adduct structure. When a 14-mer primer together with high dNTP concentrations was used to examine nucleotide incorporation directly a cross from the four different purine adducts we found that the deoxyad enosine adducts (dA-AAI and dA-AAII) allowed incorporation of dAMP and dTMP equally well, whereas the deoxyguanosine adducts (dG-AAI and dG- AAII) allowed preferential incorporation of dCMP. Molecular dynamic si mulations showed that the aristolactam moiety of all adducts exhibit a strong stacking, with the adenine residue at the 3' end of the 14-mer primer. These studies demonstrate that all AA purine adducts provide severe blocks to DNA replication and that the guanine adducts may not be very efficient mutagenic lesions. In contrast, the translesional by pass past adenine adducts of the aristolochic acids suggests a mutagen ic potential resulting from dAMP incorporation by polymerase. AT --> T A transversion mutations would be the mutagenic consequences of AA ade nine adducts, which are consistent with the activating mutations of c- ras genes found in AA-induced tumours of rodents.