Translesion Synthesis Past Equine Estrogen-Derived 2'-Deoxyadenosine DNA Adducts by Human DNA Polymerases eta and kappa

摘要

Hormone replacement therapy(HRT)increases the risk of developing breast,ovarian,and endometrial cancers.Equilin and equilenin are the major components of the widely prescribed drug used for HRT.4-Hydroxyequilenin(4-OHEN),a major metabolite of equilin and equilenin,promotes 4-OHEN-modified dC,dA,and dG DNA adducts.These DNA adducts were detected in breast tumor and adjacent normal tissues of several patients receiving HRT.We have recently found that the 4-OHEN-dC DNA adduct is a highly miscoding lesion generating C - T transitions and C -> G transversions.To explore the mutagenic potential of another major 4-OHEN-dA adduct,site-specifically modified oligodeoxy-nucleotides containing a single diastereoisomer of 4-OHEN-dA(Pk-1,Pk-2,and Pk-3)were prepared by a postsynthetic method and used as DNA templates for primer extension reactions catalyzed by human DNA polymerase(pol)eta and kappa that are highly expressed in the reproductive organs.Primer extension catalyzed by pol eta or pol kappa occurred rapidly on the unmodified template to form fully extended products.With the major 4-OHEN-dA-modified templates(Pk-2 and Pk-3),primer extension was retarded prior to the lesion and opposite the lesion;a fraction of the primers was extended past the lesion.Steady-state kinetic studies with pol eta and pol kappa indicated that dTMP,the correct base,was preferentially incorporated opposite the 4-OHEN-dA lesion.In addition,pol eta and pol kappa bypassed the lesion by incorporating dAMP and dCMP,respectively,opposite the lesion and extended past the lesion.The relative bypass frequency past the 4-OHEN-dA lesion with pol eta was at least 2 orders of magnitude higher than that observed with pol kappa.The bypass frequency past Pk-2 was more efficient than that past Pk-3.Thus,4-OHEN-dA is a miscoding lesion generating A -> T transversions and A -> G transitions.The miscoding frequency and specificity of 4-OHEN-dA varied depending on the stereoisomer of the 4-OHEN-dA adduct and DNA polymerase used.