Formation Of S-[2-(N-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 O-6-alkylguanine DNA alkyltransferase (Chowdhury, G., et al. (2013) Angew. Chem. Int. Ed.52, 12879-12882). We identified and quantified a new adduct, S-[2-(N-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) eta, i, and kappa, as well as with Sulfolobus solfataricus Dpo4, Escherichia coli polymerase I Klenow fragment, and bacteriophage T7 polymerase. hPols eta and i, Dpo4, and Klenow fragment were able to bypass the adduct with only slight impedance; hPol eta and i 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 eta confirmed the incorporation of dC opposite S-[2-(N-6-deoxyadenosinyl)ethyl]GSH and also showed the production of a -1 frameshift. These results reveal the significance of N-6-dA GSH-DBE adducts in blocking replication, as well as producing mutations, by human translesion synthesis DNA polymerases.