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

Abstract EN

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.