Translesional synthesis on DNA templates containing site-specifically placed deoxyadenosine and deoxyguanosine adducts formed by the plant carcinogen aristolochic acid.
Synthetic oligonucleotides (18-mers) containing either a single deoxyadenosine residue or a single deoxyguanosine residue were treated with aristolochic acid I (AAI) or aristolochic acid II (AAII), the main components of the plant carcinogen aristolochic acid (AA). These reactions resulted in the formation of site-specifically adducted oligonucleotides 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 32P-postlabelling. The adducted oligonucleotides were used as templates in primer (11-mer) extension reactions catalysed by modified bacteriophage T7 DNA polymerase (Sequenase). Regardless of the type of DNA adduct examined, DNA synthesis was blocked predominantly (80-90%) at the nucleotide 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 incorporation of a nucleotide opposite the adduct and translesional synthesis 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 across from the four different purine adducts we found that the deoxyadenosine 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 simulations 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 bypass past adenine adducts of the aristolochic acids suggests a mutagenic potential resulting from dAMP incorporation by polymerase. AT-->TA transversion mutations would be the mutagenic consequences of AA adenine adducts, which are consistent with the activating mutations of c-ras genes found in AA-induced tumours of rodents.
Division of Molecular Toxicology, German Cancer Research Center, Heidelberg.
T H
TH
Broschard
M
M
Wiessler
C W
CW
von der Lieth
H H
HH
Schmeiser
D034341:Aristolochic Acids; D001483:Base Sequence; D001665:Binding Sites; D002273:Carcinogens; D004247:DNA / Q000096:biosynthesis*; D018736:DNA Adducts / Q000096:biosynthesis* / Q000378:metabolism; D004249:DNA Damage; D004261:DNA Replication / Q000187:drug effects; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D003839:Deoxyadenosines / Q000096:biosynthesis* / Q000378:metabolism; D003849:Deoxyguanosine / Q000096:biosynthesis* / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009374:Neoplasms, Experimental / Q000378:metabolism; D009841:Oligonucleotides / Q000096:biosynthesis*; D010616:Phenanthrenes / Q000633:toxicity*; D012680:Sensitivity and Specificity; D013698:Templates, Genetic
D034341:Aristolochic Acids; D002273:Carcinogens; D018736:DNA Adducts; D003839:Deoxyadenosines; D009841:Oligonucleotides; D010616:Phenanthrenes; D004247:DNA; C000228:aristolochic acid I; D004259:DNA-Directed DNA Polymerase; D003849:Deoxyguanosine