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. © 2010 Paromita Raychaudhury and Ashis K. Basu.
CITATION STYLE
Raychaudhury, P., & Basu, A. K. (2010). Replication past the γ-radiation-induced guanine-thymine cross-link G[8,5-Me]T by human and yeast DNA polymerase η. Journal of Nucleic Acids, 2010. https://doi.org/10.4061/2010/101495
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