Formation of <i>S</i>‑[2‑(<i>N</i>⁶‑Deoxyadenosinyl)ethyl]glutathione in DNA and Replication Past the Adduct by Translesion DNA Polymerases

1,2-Dibromoethane (DBE, ethylene dibromide) is a potent carcinogen due at least in part to its DNA cross-linking effects. DBE cross-links glutathione (GSH) to DNA, notably to sites on 2′-deoxyadenosine and 2′-deoxyguanosine (Cmarik, J. L., et al. (1991) J. Biol. Chem. 267, 6672−6679). Adduction at the N6 position of 2′-deoxyadenosine (dA) had not been detected, but this is a site for the linkage of <i>O</i>⁶-alkylguanine DNA alkyltransferase (Chowdhury, G., et al. (2013) Angew. Chem. Int. Ed. 52, 12879−12882). We identified and quantified a new adduct, <i>S</i>-[2-(<i>N</i>⁶-deoxyadenosinyl)­ethyl]­GSH, in calf thymus DNA using LC-MS/MS. Replication studies were performed in duplex oligonucleotides containing this adduct with human DNA polymerases (hPols) η, ι, and κ, as well as with <i>Sulfolobus solfataricus</i> Dpo4, <i>Escherichia coli</i> polymerase I Klenow fragment, and bacteriophage T7 polymerase. hPols η and ι, Dpo4, and Klenow fragment were able to bypass the adduct with only slight impedance; hPol η and ι showed increased misincorporation opposite the adduct compared to that of unmodified 2′-deoxyadenosine. LC-MS/MS analysis of full-length primer extension products by hPol η confirmed the incorporation of dC opposite <i>S</i>-[2-(<i>N</i>⁶-deoxyadenosinyl)­ethyl]­GSH and also showed the production of a −1 frameshift. These results reveal the significance of <i>N</i>⁶-dA GSH-DBE adducts in blocking replication, as well as producing mutations, by human translesion synthesis DNA polymerases