Miscoding properties of 6α- and 6β-diastereoisomers of the N²-(estradiol-6-yl)-2′-deoxyguanosine DNA adduct by Y-family human DNA polymerases

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) η and κ, 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 6α- and 6β-diastereoisomers of dG-N²-6-estrogen or dA-N⁶-6-estrogen, the stereochemistry of each isomeric adduct on translesion synthesis catalyzed by DNA pois has not been investigated. We have recently established a phosphoramidite chemical procedure to insert 6α- or 6β-isomeric N²-(estradiol-6-yl) -2′-deoxyguanosine (dG-N²-6-E₂) into oligodeoxynucleotides. Using such site-specific modified oligomer as a template, the specificity and frequency of miscoding by dG-N²-6α-E ₂ or dG-N²-6β-E₂ were explored using pol η and a truncated form of pol κ (pol κΔC). Translesion synthesis catalyzed by pol η bypassed both the 6α- and 6β-isomers of dG-N²-6-E₂, with a weak blockage at the adduct site, while translesion synthesis catalyzed by pol κΔC readily bypassed both isomeric adducts. Quantitative analysis of base substitutions and deletions occurring at the adduct site showed that pol κΔC was more efficient than pol η by incorporating dCMP opposite both 6α- and 6β-isomeric dG-N²-6-E₂ adducts. The miscoding events occurred more frequently with pol η, but not with pol κΔC. Pol η promoted incorporation of dAMP and dTMP at both the 6α- and 6β-isomeric adducts, generating G → T transversions and G → A transitions. One- and two-base deletions were also formed. The 6α-isomeric adduct promoted slightly lower frequency of dCMP incorporation and higher frequency of dTMP incorporation and one-base deletions, compared with the 6β-isomeric adduct. These observations were supported by steady-state kinetic studies. Taken together, the miscoding property of the 6α-isomeric dG-N²-6-E₂ is likely to be similar to that of the 6β-isomeric adduct.