Involvement of specialized DNA polymerases in mutagenesis by 8-hydroxy-dGTP in human cells

The mutagenicity of an oxidized form of dGTP, 8-hydroxy-2'-deoxyguanosine 5'-triphosphate (8-OH-dGTP). was examined using human 293T cells. Shuttle plasmid DNA containing the supF gene was first transfected into the cells, and then 8-OH-dGTP was introduced by means of osmotic pressure. The DNAs replicated in the cells were recovered and then transfected into Escherichia coli. 8-OH-dGTP induced A:T -> C:G substitution mutations in the cells. The knock-downs of DNA polymerases eta and zeta, and REV1 by siRNAs reduced the A:T -> C:G substitution mutations, suggesting that these DNA polymerases are involved in the misincorporation of 8-OH-dGTP opposite A in human cells. In contrast. the knock-down of DNA polymerase iota did not affect the 8-OH-dGTP-induced mutations. The decrease in the induced mutation frequency was more evident by double knock-downs of DNA pols eta plus zeta and REV1 plus DNA pol zeta (but not by that of DNA pol eta plus REV1), suggesting that REV1-DNA pol eta and DNA pol zeta work in different steps. These results indicate that specialized DNA polymerases are involved in the mutagenesis induced by the oxidized dGTP. (C) 2008 Elsevier B,V. All rights reserved.