Effect of site-specifically located aristolochic acid DNA adducts on in vitro DNA synthesis by human DNA polymerase alpha.
In order to examine the effect of purine adducts of the plant carcinogen aristolochic acid (AA) on DNA replication four 30-mer templates were prepared which contained single site-specific AA lesions. The oligonucleotides were isolated by HPLC and shown to contain the two known aristolochic acid I-DNA adducts (dA-AAI, dG-AAI) or the two known aristolochic acid II-DNA adducts (dA-AAII, dG-AAII) at position 27 from the 3' end by 32P-postlabeling. These adducts templates were replicated in primer (23-mer) extension reactions catalysed by human DNA polymerase alpha. Both AAI-DNA adducts (dA-AAI, dG-AAI) blocked DNA synthesis predominantly (80-95%) at the nucleotide 3' to the adduct, although primer extension to the full length of the template was found with unmodified control templates. Increasing dNTP concentrations had only a small effect on the DNA synthesis and translesional synthesis was negligible. In contrast, both AAII-DNA adducts showed marked differences in primer extension reactions. Blocking of DNA synthesis by the dA-AAII adduct was strongly dNTP dependent. With increasing dNTP concentrations 27 and 28 nucleotide products, indicating termination of DNA synthesis after incorporation of a nucleotide opposite this adduct and incorporation of an additional nucleotide accumulated. Only the dG-AAII adducted template allowed substantial translesional synthesis to the full length of the template (up to 25%). When a 26-mer primer was used to examine nucleotide incorporation directly across from the four purine adducts, we found no detectable incorporation of nucleotides for the dA-AAI adduct, whereas the dG-AAI adduct and both AAII-adducts (dA-AAII and dG-AAII) allowed preferential incorporation of the correct nucleotide. These results indicate that for human polymerase alpha three AA purine adducts (dA-AAI, dG-AAI and dA-AAII) provide severe blocks to DNA replication and that dG-AAII, which allows translesional synthesis, may not be a very efficient mutagenic lesion.
Broschard
T H
TH
Division of Molecular Toxicology, German Cancer Research Center, Heidelberg, Germany.
Wiessler
M
M
Schmeiser
H H
HH
D034341:Aristolochic Acids; D001483:Base Sequence; D002273:Carcinogens / Q000633:toxicity; D004247:DNA / Q000096:biosynthesis; D018736:DNA Adducts; D004257:DNA Polymerase II / Q000037:antagonists & inhibitors*; D017931:DNA Primers; D004261:DNA Replication / Q000187:drug effects*; D003854:Deoxyribonucleotides / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009153:Mutagens / Q000378:metabolism / Q000633:toxicity*; D015345:Oligonucleotide Probes; D010616:Phenanthrenes / Q000378:metabolism / Q000633:toxicity*; D013698:Templates, Genetic
D034341:Aristolochic Acids; D002273:Carcinogens; D018736:DNA Adducts; D017931:DNA Primers; D003854:Deoxyribonucleotides; D009153:Mutagens; D015345:Oligonucleotide Probes; D010616:Phenanthrenes; D004247:DNA; C000228:aristolochic acid I; D004257:DNA Polymerase II