Recent evidence indicates that guanine N2 adducts form in the presence of ethanol or carcinogens that alkylate DNA with >3 carbon alkyl groups. It is anticipated that N2-alkylguanine adducts will significantly perturb DNA replication because the alkyl group is in position to alter Watson-Crick base pairing; however, at present there are no data addressing replication of DNA containing N2-alkylguanine, nor is it known if or how these adducts are repaired. This proposal will close that gap in our understanding of DNA alkylator-induced mutagenesis by: 1) Oligodeoxyribonucleotides containing single N2-ethylguanine, N2-isopropylguanine, O6-ethyl guanine, and O6-isopropylguanine bases will be synthesized. 2) Oligonucleotides containing these four guanine adducts will be used as templates for DNA polymerization catalyzed in vitro by purified human DNA polymerase a, q, e, and n. Polymerase m is a recently discovered """"""""bypass"""""""" human DNA polymerase thought to be involved in replication across blocking lesions. These experiments will compare the coding and extension properties of the N2 vs. O6 alkylguanine adducts. 3) Oligonucleotides containing these four alkylguanine adducts will be incorporated site specifically into the double stranded mutation reporting shuttle plasmid pLS189. The mutation frequency and spectra induced in vivo by the N2 vs. O6 alkylguanine adducts will be determined after replication of the plasmid in normal and nucleotide excision repair (NER)-deficient human fibroblasts. The role of NER in modulating mutagenesis by these adducts can thereby be determined. 4) The question of whether the N2-alkylguanine adducts are repaired by short patch base excision repair will be addressed by incubating extracts of NER-deficient human fibroblasts with alkylguanine-containing double-stranded oligonucleotides and determining incorporation of (32P)-dGTP at the adducted position. The data provided by this proposal will gauge the biologic impact of previously unexplored N2-alkylguanine adducts in comparison to the better known mutagenic O6-alkylguanine adducts.
| Upton, Dana C; Wang, Xueying; Blans, Patrick et al. (2006) Mutagenesis by exocyclic alkylamino purine adducts in Escherichia coli. Mutat Res 599:1-10 |
| Upton, Dana C; Wang, Xueying; Blans, Patrick et al. (2006) Replication of N2-ethyldeoxyguanosine DNA adducts in the human embryonic kidney cell line 293. Chem Res Toxicol 19:960-7 |
| Perrino, Fred W; Blans, Patrick; Harvey, Scott et al. (2003) The N2-ethylguanine and the O6-ethyl- and O6-methylguanine lesions in DNA: contrasting responses from the ""bypass"" DNA polymerase eta and the replicative DNA polymerase alpha. Chem Res Toxicol 16:1616-23 |
