Translesion Synthesis Past Equine Estrogen-Derived 2‘-Deoxyadenosine DNA Adducts by Human DNA Polymerases η and κ

Abstract

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 4-OHENdC 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 oligodeoxynucleotides containing a single diastereoisomer of 4-OHEN-dA (Pk-1, Pk-2, and Pk-3) were prepared by post-synthetic method and used as DNA templates for primer extension reactions catalyzed by human DNA polymerase (pol) η and κ that are highly expressed in the reproductive organs. Primer extension catalyzed by pol η or κ 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 η and κ indicated that dTMP, the correct base, was preferentially incorporated opposite the 4-OHEN-dA lesion. In addition, pol η and κ bypassed the lesion by incorporating dAMP and dCMP, respectively, opposite the lesion and extended past the lesion. The relative bypass frequency past 4-OHEN-dA lesion with pol η was at least two orders of magnitude higher than that observed with pol κ. 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 4-OHEN-dA adduct and DNA polymerase used.

Equine estrogen-induced mammary tumors in rats

“…The mechanism of forming DNA damage induced by equine estrogens (Bolton et al, 1998) and their mutagenic properties (Yasui et al, 2003; Suzuki et al, 2004; Yasui et al, 2006 & 2007) have been explored. However, the tumorigenic potential of equine estrogens has not yet been determined.…”

“…Mutagenic events induced by 4-OHEQ were observed in a supF shuttle vector plasmid propagated in human cells (Yasui et al, 2003). We have reported that the dC and dA adducts are frequently miscoded during DNA synthesis catalyzed by human DNA polymerases expressed highly in mammary tissue and reproductive organs (Suzuki et al, 2004; Yasui et al, 2006 & 2007). …”

4-Hydroxyequilenin-Adenine Lesions in DNA Duplexes: Stereochemistry, Damage Site, and Structure

“…Specifically, the bypass frequency in pol η differed by two orders of magnitude in the members of a pair of 4-OHEN-dC stereoisomers with opposite sign CD spectra (27). For 4-OHEN-dA adducts, the bypass frequency past one stereoisomer was approximately 3 times higher than for another (28). For a pair of 4-OHENdC major adducts whose CD spectra were opposite sign, with pol κ mismatched dCMP and dAMP were inserted opposite both stereoisomers, and chain extension with partner dC was much higher than with dA (27).…”

Chemistry and Structural Biology of DNA Damage and Biological Consequences

“…Polymerases bypass the 4-OHEN-dC and dA lesions with efficiencies depending upon configuration and the identity of the damaged base [127–129]. Primer extension conducted with the polymerases Dpo4, Pol η , and Pol κ indicate that 4-OHEN-dC is bypassed with insertion of an incorrect dNTP or by strand slippage [128][130].…”