Sequence context effects of replication of Fapy•dG in three mutational hot spot sequences of the p53 gene in human cells

Abstract

Fapy•dG and 8-OxodGuo are formed in DNA from a common N7-dG radical intermediate by reaction with hydroxyl radical. Although cellular levels of Fapy•dG are often greater, its effects on replication are less well understood than those of 8-OxodGuo. In this study plasmid DNA containing Fapy•dG in three mutational hotspots of human cancers, codons 248, 249, and 273 of the p53 tumor suppressor gene, was replicated in HEK 293T cells. TLS efficiencies for the Fapy•dG containing plasmids varied from 72 to 89%, and were further reduced in polymerase-deficient cells. The mutation frequency (MF) of Fapy•dG ranged from 7.3 to 11.6%, with G→T and G→A as major mutations in codons 248 and 249 compared to primarily G→T in codon 273. Increased MF in hPol ι-, hPol κ-, and hPol ζ-deficient cells suggested that these polymerases more frequently insert the correct nucleotide dC opposite Fapy•dG, whereas decreased G→A in codons 248 and 249 and reduction of all mutations in codon 273 in hPol λ-deficient cells indicated hPol λ's involvement in Fapy•dG mutagenesis. In vitro kinetic analysis using isolated translesion synthesis polymerases and hPol λ incompletely corroborated the mutagenesis experiments, indicating codependence on other proteins in the cellular milieu. In conclusion, Fapy•dG mutagenesis is dependent on the DNA sequence context, but its bypass by the TLS polymerases is largely error-free.

Keywords: 2ʹ-deoxyguanosine lesion; Mutagenesis; Oxidative DNA damage; Polymerase knockout; Translesion synthesis.