Replication Past the γ‐Radiation‐Induced Guanine‐Thymine Cross‐Link G8,5‐MeT by Human and Yeast DNA Polymerase η

γ-Radiation-induced intrastrand guanine-thymine cross-link, G8,5-MeT, hinders replication in vitro and is mutagenic in mammalian cells. Herein we report in vitro translesion synthesis of G8,5-MeT by human and yeast DNA polymerase η (hPol η and yPol η). dAMP misincorporation opposite the cross-linked G by yPol η was preferred over correct incorporation of dCMP, but further extension was 100-fold less efficient for G(∗):A compared to G(∗):C. For hPol η, both incorporation and extension were more efficient with the correct nucleotides. To evaluate translesion synthesis in the presence of all four dNTPs, we have developed a plasmid-based DNA sequencing assay, which showed that yPol η was more error-prone. Mutational frequencies of yPol η and hPol η were 36% and 14%, respectively. Targeted G → T was the dominant mutation by both DNA polymerases. But yPol η induced targeted G → T in 23% frequency relative to 4% by hPol η. For yPol η, targeted G → T and G → C constituted 83% of the mutations. By contrast, with hPol η, semi-targeted mutations (7.2%), that is, mutations at bases near the lesion, occurred at equal frequency as the targeted mutations (6.9%). The kind of mutations detected with hPol η showed significant similarities with the mutational spectrum of G8,5-MeT in human embryonic kidney cells.