Translesional synthesis past acetylaminofluorene-derived DNA adducts catalyzed by human DNA polymerase kappa and Escherichia coli DNA polymerase IV.
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.
Suzuki
N
N
Laboratory of Chemical Biology, Department of Pharmacological Sciences, State University of New York at Stony Brook, Stony Brook, New York 11794-8651, USA.
Ohashi
E
E
Hayashi
K
K
Ohmori
H
H
Grollman
A P
AP
Shibutani
S
S
D015073:2-Acetylaminofluorene / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D018736:DNA Adducts / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019951:DNA Polymerase beta / Q000378:metabolism*; D004259:DNA-Directed DNA Polymerase; D003849:Deoxyguanosine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology; D005449:Fluorenes / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D011506:Proteins / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D017384:Sequence Deletion; D000099270:Y-Family DNA Polymerases
D015073:2-Acetylaminofluorene; D018736:DNA Adducts; D019951:DNA Polymerase beta; D004259:DNA-Directed DNA Polymerase; D003849:Deoxyguanosine; D005449:Fluorenes; D011506:Proteins; D011994:Recombinant Proteins; C031838:N-(deoxyguanosin-8-yl)-2-aminofluorene; C018776:N-(deoxyguanosin-8-yl)acetylaminofluorene; C404302:POLK protein, human; D000099270:Y-Family DNA Polymerases