Replication past the Butadiene Diepoxide-Derived DNA Adduct <i>S</i>‑[4‑(<i>N</i>⁶‑Deoxyadenosinyl)-2,3-dihydroxybutyl]glutathione by DNA Polymerases

Abstract

1,2,3,4-Diepoxybutane (DEB), a metabolite of the carcinogen butadiene, has been shown to cause glutathione (GSH)-dependent base substitution mutations, especially A:T to G:C mutations in <i>Salmonella typhimurium</i> TA1535 [Cho, S. H., et al. (2010) <i>Chem. Res. Toxicol. 23</i>, 1544] and <i>Escherichia coli</i> TRG8 cells [Cho, S. H., and Guengerich, F. P. (2012) <i>Chem. Res. Toxicol. 25</i>, 1522]. We previously identified <i>S</i>-[4-(<i>N</i>⁶-deoxyadenosinyl)-2,3-dihydroxybutyl]­GSH [<i>N</i>⁶dA-(OH)₂butyl-GSH] as a major adduct in the reaction of <i>S</i>-(2-hydroxy-3,4-epoxybutyl)­glutathione (DEB-GSH conjugate) with nucleosides and calf thymus DNA and <i>in vivo</i> in livers of mice and rats treated with DEB [Cho, S. H., and Guengerich, F. P. (2012) <i>Chem. Res. Toxicol. 25</i>, 706]. For investigation of the miscoding potential of the major DEB-GSH conjugate-derived DNA adduct [<i>N</i>⁶dA-(OH)₂butyl-GSH] and the effect of GSH conjugation on replication of DEB, extension studies were performed in duplex DNA substrates containing the site-specifically incorporated <i>N</i>⁶dA-(OH)₂butyl-GSH adduct, <i>N</i>⁶-(2,3,4-trihydroxybutyl)­deoxyadenosine adduct (<i>N</i>⁶dA-butanetriol), or unmodified deoxyadenosine (dA) by human DNA polymerases (Pol) η, ι, and κ, bacteriophage polymerase T7, and <i>Sulfolobus solfataricus</i> polymerase Dpo4. Although dTTP incorporation was the most preferred addition opposite the <i>N</i>⁶dA-(OH)₂butyl-GSH adduct, <i>N</i>⁶dA-butanetriol adduct, or unmodified dA for all polymerases, the dCTP misincorporation frequency opposite <i>N</i>⁶dA-(OH)₂butyl-GSH was significantly higher than that opposite the <i>N</i>⁶dA-butanetriol adduct or unmodified dA with Pol κ or Pol T7. LC–MS/MS analysis of full-length primer extension products confirmed that Pol κ or Pol T7 incorporated the incorrect base C opposite the <i>N</i>⁶dA-(OH)₂butyl-GSH lesion. These results indicate the relevance of GSH-containing adducts for the A:T to G:C mutations produced by DEB