PMID:11735400
| Citation |
Suzuki, N, Ohashi, E, Hayashi, K, Ohmori, H, Grollman, AP and Shibutani, S (2001) Translesional synthesis past acetylaminofluorene-derived DNA adducts catalyzed by human DNA polymerase kappa and Escherichia coli DNA polymerase IV. Biochemistry 40:15176-83 |
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| Abstract |
Human DNA polymerase kappa (pol kappa) has a sequence significantly homologous with that of Escherichia coli DNA polymerase IV (pol IV). We used a truncated form of human pol kappa (pol kappaDeltaC) and full-length pol IV to explore the miscoding properties of these enzymes. Oligodeoxynucleotides, modified site-specifically with N-(deoxyguanosin-8-yl)-2-acetylaminofluorene (dG-AAF) and N-(deoxyguanosin-8-yl)-2-aminofluorene (dG-AF), were used as DNA templates in primer extension reactions that included all four dNTPs. Reactions catalyzed by pol kappaDeltaC were partially blocked one base prior to dG-AAF or dG-AF, and also opposite both lesions. At higher enzyme concentrations, a significant fraction of primer was extended. Analysis of the fully extended reaction product revealed incorporation of dTMP opposite dG-AAF, accompanied by much smaller amounts of dCMP, dAMP, and dGMP and some one- and two-base deletions. The product terminating 3' to the adduct site contained AMP misincorporated opposite dC. On templates containing dG-AF, dAMP, dTMP, and dCMP were incorporated opposite the lesion in approximately equal amounts, together with some one-base and two-base deletions. Steady-state kinetics analysis confirmed the results obtained from primer extension reactions catalyzed by pol kappa. In contract, primer extension reactions catalyzed by pol IV were blocked effectively by dG-AAF and dG-AF. At high concentrations of pol IV, full-length products were formed containing primarily one- or two-base deletions with dCMP, the correct base, incorporated opposite dG-AF. The miscoding properties of pol kappa observed in this study are consistent with mutational spectra observed when plasmid vectors containing dG-AAF or dG-AF are introduced into simian kidney cells [Shibutani, S., et al. (2001) Biochemistry 40, 3717-3722], supporting a model in which pol kappa plays a role in translesion synthesis past acetylaminofluorene-derived lesions in mammalian cells. |
| Links | |
| Keywords |
2-Acetylaminofluorene/analogs & derivatives; 2-Acetylaminofluorene/chemistry; 2-Acetylaminofluorene/metabolism; Animals; Base Sequence; COS Cells; DNA Adducts/chemistry; DNA Adducts/genetics; DNA Adducts/metabolism; DNA Polymerase beta/metabolism; DNA-Directed DNA Polymerase; Deoxyguanosine/analogs & derivatives; Deoxyguanosine/chemistry; Deoxyguanosine/metabolism; Escherichia coli/enzymology; Fluorenes/chemistry; Fluorenes/metabolism; Humans; Kinetics; Mutagenesis, Site-Directed; Proteins/metabolism; Recombinant Proteins/metabolism; Sequence Deletion |
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