Miscoding Properties of 6α- and 6β-Diastereoisomers of the N2-(Estradiol-6-yl)-2′-deoxyguanosine DNA Adduct by Y-Family Human DNA Polymerases

Abstract

Treatment with estrogen increases the risk of breast, ovary, and endometrial cancers in women. DNA damage induced by estrogen is thought to be involved in estrogen carcinogenesis. In fact, Y-family human DNA polymerases (pol) eta and kappa, which are highly expressed in the reproductive organs, miscode model estrogen-derived DNA adducts during DNA synthesis. Since the estrogen-DNA adducts are a mixture of 6alpha- and 6beta-diastereoisomers of dG-N(2)-6-estrogen or dA-N(6)-6-estrogen, the stereochemistry of each isomeric adduct on translesion synthesis catalyzed by DNA pols has not been investigated. We have recently established a phosphoramidite chemical procedure to insert 6alpha- or 6beta-isomeric N(2)-(estradiol-6-yl)-2'-deoxyguanosine (dG-N(2)-6-E(2)) into oligodeoxynucleotides. Using such site-specific modified oligomer as a template, the specificity and frequency of miscoding by dG-N(2)-6alpha-E(2) or dG-N(2)-6beta-E(2) were explored using pol eta and a truncated form of pol kappa (pol kappaDeltaC). Translesion synthesis catalyzed by pol eta bypassed both the 6alpha- and 6beta-isomers of dG-N(2)-6-E(2), with a weak blockage at the adduct site, while translesion synthesis catalyzed by pol kappaDeltaC readily bypassed both isomeric adducts. Quantitative analysis of base substitutions and deletions occurring at the adduct site showed that pol kappaDeltaC was more efficient than pol eta by incorporating dCMP opposite both 6alpha- and 6beta-isomeric dG-N(2)-6-E(2) adducts. The miscoding events occurred more frequently with pol eta, but not with pol kappaDeltaC. Pol eta promoted incorporation of dAMP and dTMP at both the 6alpha- and 6beta-isomeric adducts, generating G --> T transversions and G --> A transitions. One- and two-base deletions were also formed. The 6alpha-isomeric adduct promoted slightly lower frequency of dCMP incorporation and higher frequency of dTMP incorporation and one-base deletions, compared with the 6beta-isomeric adduct. These observations were supported by steady-state kinetic studies. Taken together, the miscoding property of the 6alpha-isomeric dG-N(2)-6-E(2) is likely to be similar to that of the 6beta-isomeric adduct.