Abstract
[Purpose]
Reactive oxygen species formed endogenously and by mutagens as ionizing radiation produce damaged DNA precursors (deoxyribonucleotides). The hydrolysis of the damaged DNA precursors before their incorporation into DNA by nucleotide pool sanitization enzymes would prevent the mutagenesis by the damaged DNA precursors. Mammalian MTH1, MTH2, and NUDT5 hydrolyze 8-hydroxy-dGTP or 8-hydroxy-dGDP in vitro, and are expected to function as nucleotide pool sanitization enzymes. However, it is unclear whether they actually prevent the mutagenesis by 8-hydroxy-dGTP/8-hydroxy-dGDP. In this study, we examined roles of MTH1, MTH2 and NUDT5 in suppression of the mutations induced by 8-hydroxy-dGTP in living cells by their knock-downs using siRNAs.
[Methods]
siRNAs against MTH1, MTH2, and NUDT5 and shuttle plasmid DNA containing the supF gene were transfected into human 293T cells. After 24 hr, 8-hydroxy-dGTP was introduced by means of osmotic pressure. After 48 hr, the plasmid amplified in the cells was recovered and transfected into E.coli KS40/pOF105 cells and supF mutant frequencies were calculated.
[Results]
Triple knock-downs of MTH1, MTH2, and NUDT5 increased frequency of the mutation induced by 8-hydroxy-dGTP by two-fold. The knock-downs promoted occurrence of A:T C:G transversion. We are analyzing effects of knock-down of each protein on the mutation.
[Conclusion]
MTH1, MTH2, and NUDT5 are involved in the suppression of the mutations induced by 8-hydroxy-dGTP as nucleotide pool sanitization enzymes in mammalian cells.
