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

• Main Authors: Paromita Raychaudhury, Ashis K. Basu
• Format: Article
• Language: English
• Published: Hindawi Limited 2010-01-01
• Series: Journal of Nucleic Acids
• Online Access: http://dx.doi.org/10.4061/2010/101495

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.