Abstract
[Purpose] Damaged DNA precursors (deoxyribonucleotides) are formed by reactive oxygen species induced by ionizing radiation. After damaged DNA precursors are incorporated into DNA, they might be removed by DNA repair enzymes. In this study, we examined whether a nucleotide excision repair enzyme, Eschericia coli UvrABC, could suppress mutations induced by oxidized deoxyribonucleotides in vivo.
[Methods] We introduced oxidized DNA precursors, 8-hydroxy-dGTP (8-OH-dGTP) and 2-hydroxy-dATP (2-OH-dATP), into uvrA, uvrB, and uvrC E. coli strains, and analyzed mutations in the chromosomal rpoB gene, as a mutagenesis target. Next, mutT, mutT/uvrA, and mutT/uvrB E. coli strains were treated with hydrogen peroxide (H2O2), and the rpoB mutant frequencies were calculated. We hypothesized that UvrABC incises the undamaged DNA strands, instead of the strands containing 8-hydroxyguanine (8-OH-Gua) and 2-hydroxyadenine (2-OH-Ade) formed by the incorporation of 8-OH-dGTP and 2-OH-dATP. Duplex oligonucleotides containing 8-OH-Gua and 2-OH-Ade were treated with the purified Thermus thermophilus HB8 UvrABC proteins.
[Results] Oxidized DNA precursors induced mutations much less efficiently in the uvrA and uvrB strains than in the wild type. In contrast, effect of the uvrC-deficiency was not observed. Frequency of the H2O2-induced mutations was increased in the all strains tested, and the increase was three- to four-fold lower in the mutT/uvrA and mutT/uvrB strains than in the mutT strain. Although duplex oligonucleotides containing 8-OH-Gua and 2-OH-Ade were treated with UvrABC proteins, the expected activity was not observed.
[Conclusions] It is suggested that UvrA and UvrB are involved in the enhancement, but not in the suppression, of mutations induced by oxidized deoxyribonucleotides. These results suggest a novel role for UvrA and UvrB in the processing of oxidative damage.
