Roles of specialized DNA polymerases in mutagenesis by 8-hydroxyguanine in human cells

Abstract

The formation of 8-hydroxyguanine (8-OH-Gua, 7,8-dihydro-8-oxoguanine) in DNA and in the nucleotide pool results in G:C-->T:A and A:T-->C:G substitution mutations, respectively, due to the ability of 8-OH-Gua to pair with both C and A. In this study, shuttle plasmid DNAs containing 8-OH-Gua paired with C and A in the supF gene were transfected into human 293T cells, in which specialized DNA polymerases were knocked-down. The DNAs replicated in the cells were recovered and then introduced into an indicator strain of Escherichia coli. Mutation analysis indicated that the knock-downs of DNA polymerases eta and zeta by siRNAs enhanced the G:C-->T:A mutations caused by 8-OH-Gua:C. The 8-OH-Gua:C-induced mutation frequency was not further increased by double knock-downs of DNA polymerases eta and zeta, suggesting that the two DNA polymerases work in the same pathway. In addition, the reduction of DNA polymerase eta slightly decreased the A:T-->C:G substitutions caused by 8-OH-Gua:A. These results suggest that DNA polymerases eta and zeta are involved in the bypass of 8-OH-Gua in human cells.