DNA polymerase action on bulky deoxyguanosine and deoxyadenosine adducts

In order to determine how individual hydrocarbon-DNA adducts give rise to specific mutations, a single-stranded oligonucleotide, 5'-T8GT10AT8C2T4CT3CT-3', was reacted with the carcinogen 7-bromomethylbenz[a]anthracene which generates both deoxyguanosine and deoxyadenosine adducts in DNA. The products were separated by HPLC to yield unmodified oligonucleotide and oligonucleotide modified either at the single guanine, or at the single adenine, residue. Incubation of these products with 32P-5'-end-labeled primer, 5'-AGA3GA4G2-3', modified T7 DNA polymerase (Sequenase) and deoxyribonucleoside-5'-triphosphates followed by gel electrophoretic analysis indicated that unmodified oligonucleotide template allowed the primer to be rapidly extended to give species of the same length as the template (40 nucleotides) and of 41 nucleotides in length. However, primer extension for the templates containing the guanine and adenine adducts was held up initially (1 min) at the nucleotide preceding the adduct. At longer times (up to 15 min) a nucleotide was added opposite the adduct and, to a lesser extent, another nucleotide was added beyond this. Some full-length oligonucleotide was also synthesized with these carcinogen-modified templates. When synthesis was allowed to proceed only to the nucleotide preceding the adduct, and this template-extended primer complex incubated with individual nucleotide triphosphates plus Sequenase, it was found that deoxyadenosine residues were most readily incorporated opposite the adduct irrespective of whether it was a deoxyguanosine or deoxyadenosine adduct. These results, which suggest that G.C----T.A and A.T----T.A transversions would be the mutagenic consequences of formation of bulky hydrocarbon adducts at guanines and adenines respectively, are consistent with the most frequent hydrocarbon-induced mutational changes reported thus far.