TY - JOUR
T1 - Translesional synthesis past acetylaminofluorene-derived DNA adducts catalyzed by human DNA polymerase κ and Escherichia coli DNA polymerase IV
AU - Suzuki, N.
AU - Ohashi, E.
AU - Hayashi, K.
AU - Ohmori, H.
AU - Grollman, A. P.
AU - Shibutani, S.
PY - 2001/12/18
Y1 - 2001/12/18
N2 - Human DNA polymerase κ (pol κ) has a sequence significantly homologous with that of Escherichia coli DNA polymerase IV (pol IV). We used a truncated form of human pol κ (pol κΔC) 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 κΔC 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 κ. 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 κ 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 κ plays a role in translesion synthesis past acetylaminofluorene-derived lesions in mammalian cells.
AB - Human DNA polymerase κ (pol κ) has a sequence significantly homologous with that of Escherichia coli DNA polymerase IV (pol IV). We used a truncated form of human pol κ (pol κΔC) 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 κΔC 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 κ. 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 κ 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 κ plays a role in translesion synthesis past acetylaminofluorene-derived lesions in mammalian cells.
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U2 - 10.1021/bi010702g
DO - 10.1021/bi010702g
M3 - Article
C2 - 11735400
AN - SCOPUS:0035909805
VL - 40
SP - 15176
EP - 15183
JO - Biochemistry
JF - Biochemistry
SN - 0006-2960
IS - 50
ER -