Miscoding properties of model estrogen-DNA adducts in reactions catalyzed by mammalian and Escherichia coli DNA polymerases.

Abstract

The miscoding properties of the model estrogen-derived DNA adducts, N2-[3-methoxyestra-1,3,5(10)-trien-6-yl]-2'-deoxyguanosine (dG-N2-3MeE) and N6-[3-methoxyestra-1,3,5(10)-trien-6-yl]-2'- deoxyadenosine (dA-N6-3MeE), have been explored, using an in vitro experimental system to quantify base substitutions and deletions. Site-specifically modified oligodeoxynucleotides containing a single dG-N2-3MeE or dA-N6-3MeE were prepared postsynthetically and used as templates in primer extension reactions catalyzed by Escherichia coli and mammalian DNA polymerases. When the 3'-->5' exonuclease free (exo-) Klenow fragment of DNA polymerase I was used, dG-N2-3MeE promoted mostly one- and two-base deletions, along with small amounts of incorporation of dAMP, dGMP, and dCMP opposite the lesion. dA-N6-3MeE promoted the incorporation of dTMP opposite the lesion as well as two-base deletions, accompanied by the incorporation of dAMP. Using pol alpha, primer extension reactions were blocked at dG-N2-3MeE; however, dA-N6-3MeE promoted preferential incorporation of dTMP opposite the lesion with small amounts of incorporation of dCMP and deletions. Primer extension reactions catalyzed by pol delta were blocked at these lesions. When pol beta was used, dG-N2-3MeE produced small amounts of incorporation of dAMP and deletions. dA-N6-3MeE promoted preferential incorporation of dTMP, along with incorporation of dCMP and two-base deletions. The miscoding specificities and frequencies varied depending on the DNA polymerase used. These results indicate that estrogen-DNA adducts have miscoding potential.