Studies on the Replication of the Ring Opened Formamidopyrimidine, Fapy·dG in Escherichia coli†
journal contributionposted on 04.09.2007, 00:00 by Jennifer N. Patro, Carissa J. Wiederholt, Yu Lin Jiang, James C. Delaney, John M. Essigmann, Marc M. Greenberg
Fapy·dG is produced in DNA as a result of oxidative stress from a precursor that also forms OxodG. Bypass of Fapy·dG in a shuttle vector in COS-7 cells produces G → T transversions slightly more frequently than does OxodG (Kalam, M. A., et al. (2006) Nucleic Acids Res. 34, 2305). The effect of Fapy·dG on replication in Escherichia coli was studied by transfecting M13mp7(L2) bacteriophage DNA containing the lesion within the lacZ gene in 4 local sequence contexts. For comparison, experiments were carried out side-by-side on OxodG. The efficiency of lesion bypass was determined relative to that of a genome containing native nucleotides. Fapy·dG was bypassed less efficiently than OxodG. Bypass efficiency of Fapy·dG and OxodG increased modestly in SOS-induced cells. Mutation frequencies at the site of the lesions in the originally transfected genomes were determined using the REAP assay (Delaney, J. C., Essigmann, J. M. (2006) Methods Enzymol. 408, 1). G → T transversions were the only mutations observed above background when either Fapy·dG or OxodG was bypassed. OxodG mutation frequencies ranged from 3.1% to 9.8%, whereas the G → T transversion frequencies observed upon Fapy·dG bypass were ≤1.9% in wild-type E. coli. In contrast to OxodG bypass, Fapy·dG mutation frequencies were unaffected by carrying out experiments in mutM/mutY cells. Overall, these experiments suggest that Fapy·dG is at most weakly mutagenic in E. coli. Steady-state kinetic experiments using the Klenow fragment of DNA polymerase I from E. coli suggest that a low dA misincorporation frequency opposite Fapy·dG and inefficient extension of a Fapy·dG:dA base pair work synergistically to minimize the levels of G → T transversions.
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Klenow fragmentsequence contextstransversiondA misincorporation frequencyoxidative stressFapyRing Opened Formamidopyrimidineforms OxodGexperimentDNA polymeraseCOSBypass efficiencyEscherichia coliOxodG mutation frequenciesshuttle vectorMutation frequenciestransfecting M 13mp bacteriophage DNAlacZ genetransfected genomes