title: Formation of S-[2-(N6-Deoxyadenosinyl)ethyl]glutathione in DNA and Replication Past the Adduct by Translesion DNA Polymerases.
abstract: 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 O6-alkylguanine DNA alkyltransferase ( Chowdhury , G. , et al. ( 2013 ) Angew. Chem. Int. Ed. 52 , 12879 - 12882 ). We identified and quantified a new adduct, S-[2-(N6-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 Sulfolobus solfataricus Dpo4, Escherichia coli 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 S-[2-(N6-deoxyadenosinyl)ethyl]GSH and also showed the production of a -1 frameshift. These results reveal the significance of N6-dA GSH-DBE adducts in blocking replication, as well as producing mutations, by human translesion synthesis DNA polymerases.
authors.affiliation: Department of Biochemistry, Vanderbilt University School of Medicine , Nashville, Tennessee 37232-0146, United States.
authors.forename: Carl A
authors.initials: CA
authors.lastname: Sedgeman
authors.affiliation: Department of Biochemistry, Vanderbilt University School of Medicine , Nashville, Tennessee 37232-0146, United States.
authors.forename: Yan
authors.initials: Y
authors.lastname: Su
authors.affiliation: Department of Biochemistry, Vanderbilt University School of Medicine , Nashville, Tennessee 37232-0146, United States.
authors.forename: F Peter
authors.identifier: 0000-0002-7458-3048
authors.initials: FP
authors.lastname: Guengerich
mesh_terms: D000818:Animals; D002417:Cattle; D002853:Chromatography, Liquid; D018736:DNA Adducts; D004261:DNA Replication; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D015946:Ethylene Dibromide / Q000633:toxicity; D005978:Glutathione / Q000031:analogs & derivatives*; D053719:Tandem Mass Spectrometry
chemical_list: D018736:DNA Adducts; D015946:Ethylene Dibromide; D004259:DNA-Directed DNA Polymerase; D005978:Glutathione