Research Article Replication Past the γ-Radiation-Induced Guanine-Thymine Cross-Link G[8,5-Me]T by Human and Yeast DNA Polymerase η

Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. γ-Radiation-induced intrastrand guanine-thymine cross-link, G[8,5-Me]T, hinders replication in vitro and is mutagenic in mammalian cells. Herein we report in vitro translesion synthesis of G[8,5-Me]T 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 G[8,5-Me]T in human embryonic kidney cells. 1