INDUCTION OF CHROMOSOMAL GENE-MUTATIONS IN ESCHERICHIA-COLI BY DIRECTINCORPORATION 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 mutagenicit y of a damaged DNA precursor (deoxyribonucleoside 5'-triphosphate) in Escherichia coil, 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 in to competent E. coli and selected mutants of the chromosomal loci gene , Both damaged nucleotides induced loci gene mutations in a dose-depen dent 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 int o an E. coli suspension induced 12- and 9-fold more substitution mutat ions 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 trans versions. These results indicate that the two oxidatively damaged nucl eotides 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. coil DNA, This new method enables the evaluation and comparison of the mutagenic potentials of damaged DNA precursors in vivo.