Induction of chromosomal gene mutations in Escherichia coli by direct incorporation of oxidatively damaged nucleotides. New evaluation method for mutagenesis by damaged DNA precursors in vivo.
We have developed a new strategy for the evaluation of the mutagenicity of a damaged DNA precursor (deoxyribonucleoside 5'-triphosphate) in Escherichia coli. 8-Hydroxydeoxyguanosine triphosphate (8-OH-dGTP) and 2-hydroxydeoxyadenosine triphosphate (2-OH-dATP) were chosen for this study because they appear to be formed abundantly by reactive oxygen species in cells. We introduced the oxidatively damaged nucleotides into competent E. coli and selected mutants of the chromosomal lacI gene. Both damaged nucleotides induced lacI gene mutations in a dose-dependent manner, whereas unmodified dATP and dGTP did not appear to elicit the mutations. The addition of 50 nmol of 8-OH-dGTP and 2-OH-dATP into an E. coli suspension induced 12- and 9-fold more substitution mutations than the spontaneous event, respectively. The 8-OH-dGTP induced A.T --> C.G transversions, and the 2-OH-dATP elicited G.C --> T.A transversions. These results indicate that the two oxidatively damaged nucleotides are mutagenic in vivo and suggest that 8-OH-dGTP and 2-OH-dATP were incorporated opposite A and G residues, respectively, in the E. coli DNA. This new method enables the evaluation and comparison of the mutagenic potentials of damaged DNA precursors in vivo.
Inoue
M
M
Department of Environmental Oncology, University of Occupational and Environmental Health, 1-1 Iseigaoka, Yahatanishi-ku, Kitakyushu 807-8555, Japan.
Kamiya
H
H
Fujikawa
K
K
Ootsuyama
Y
Y
Murata-Kamiya
N
N
Osaki
T
T
Yasumoto
K
K
Kasai
H
H
D001426:Bacterial Proteins / Q000235:genetics; D001483:Base Sequence; D002876:Chromosomes, Bacterial; D004249:DNA Damage; D004269:DNA, Bacterial / Q000187:drug effects*; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins; D005069:Evaluation Studies as Topic; D056968:Lac Repressors; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009154:Mutation; D009694:Nucleic Acid Precursors; D018384:Oxidative Stress; D017382:Reactive Oxygen Species; D012097:Repressor Proteins / Q000235:genetics
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D056968:Lac Repressors; C539064:LacI protein, E coli; D009694:Nucleic Acid Precursors; D017382:Reactive Oxygen Species; D012097:Repressor Proteins