DNA polymerase bypass in vitro and in E. coli of a C-nucleotide analogue of Fapy-dG.
Bypass of the configurationally stable analogue (beta-C-Fapy x dG) of the formamidopyrimidine lesion derived from 2'-deoxyguanosine oxidation (Fapy x dG) was studied in vitro and in Escherichia coli. The exonuclease deficient Klenow fragment of E. coli DNA polymerase I (Klenow exo(-)) misincorporated dA most frequently opposite beta-C-Fapy x dG, but its efficiency was <0.2% of dC insertion. Klenow exo(-) fidelity was enhanced by the enzyme's high selectivity for extending duplexes only when dC was opposite beta-C-Fapy x dG. The expectations raised by these in vitro data were realized when beta-C-Fapy x dG replication was studied in E. coli by transfecting M13mp7(L2) bacteriophage DNA containing the nucleotide analogue within the lacZ gene in 4 local sequence contexts. The bypass efficiency of beta-C-Fapy x dG varied between 45% and 70% compared to a genome containing only native nucleotides. 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. Methods Enzymol.2006, 408, 1]. The levels of mutations could not be distinguished between those observed when genomes containing native nucleotides were replicated, indicating that the mutagenicity of beta-C-Fapy x dG was <1%. These data and previous reports indicate that beta-C-Fapy x dG is a good model of Fapy x dG in E. coli. In addition, these results and the previous report of beta-C-Fapy x dG binding to the base excision repair protein formamidopyrimidine glycosylase suggest that this analogue could be useful as a DNA repair inhibitor.
Weledji
Yvonne N
YN
Department of Chemistry, Johns Hopkins University, 3400 North Charles Street, Baltimore, MD 21218, USA.
Wiederholt
Carissa J
CJ
Delaney
Michael O
MO
Greenberg
Marc M
MM
D004256:DNA Polymerase I / Q000378:metabolism*; D004926:Escherichia coli / Q000187:drug effects* / Q000201:enzymology* / Q000235:genetics; D005559:Formamides / Q000138:chemical synthesis* / Q000737:chemistry / Q000494:pharmacology*; D005663:Furans / Q000138:chemical synthesis* / Q000737:chemistry / Q000494:pharmacology*; D016680:Genome, Bacterial / Q000235:genetics; D007700:Kinetics; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009711:Nucleotides / Q000737:chemistry*; D011743:Pyrimidines / Q000138:chemical synthesis* / Q000737:chemistry / Q000494:pharmacology*
D005559:Formamides; D005663:Furans; C435881:N6-(2-deoxy-erythro-pentofuranosyl)-2,6-diamino-4-hydroxy-5-formamidopyrimidine; D009711:Nucleotides; D011743:Pyrimidines; D004256:DNA Polymerase I