Translesion al Synthesis Past Acetylaminofluorene-Derived DNA Adducts Catalyzed by Human DNA Polymerase #kappa# and Escherichia coli DNA Polymerase IV

摘要

Human DNA polymerase #kappa# (pol #kappa#) has a sequence signficnatly homologous with that of Escherichia coli DNA polymerase IV (pol IV). We used a truncated form of human pol #kappa# (pol #kappa##DELTA#C) and full-length pol IV to explore the miscoding properties of these enzymes. Oligodeoxynucleotides, modified site-specificatlly 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 #kappa##DELTA#C 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 mosincorporated 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 extensio reactions calatlyzed 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 consistetent 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.