Effect of pH on the Misincorporation Rate of DNA Polymerase η.

The many known eukaryotic DNA polymerases are classified into four families; A, B, X, and Y. Among them, DNA polymerase η, a Y family polymerase, is a low fidelity enzyme that contributes to translesional synthesis and somatic hypermutation. Although a high mutation frequency is observed in immunoglobulin genes, translesional synthesis occurs with a high accuracy. We determined whether the misincorporation rate of DNA polymerase η varies with ambient conditions. It has been reported that DNA polymerase η is unable to exclude water molecules from the active site. This finding suggests that some ions affect hydrogen bond formation at the active site. We focused on the effect of pH and evaluated the misincorporation rate of deoxyguanosine triphosphate (dGTP) opposite template T by DNA polymerase η at various pH levels with a synthetic template-primer. The misincorporation rate of dGTP by DNA polymerase η drastically increased at pH 8.0-9.0 compared with that at pH 6.5-7.5. Kinetic analysis revealed that the Km value for dGTP on the misincorporation opposite template T was markedly affected by pH. However, this drastic change was not seen with the low fidelity DNA polymerase α.

The pH-dependent mismatch formation by DNA polymerase eta.

To clarify the molecular mechanism of mismatch formation by DNA polymerase eta, the pH-dependency of misincorporation of dNTP was studied with the synthetic template-primer. Incorporation of dNTP formed Watson-Crick type base pair, such as the incorporation of dATP opposite template T, was slightly affected by pH between 6.5 to 9.0. On the other hand, the misincorporation rate of dGTP opposite template T by DNA polymerase eta was drastically increased according to the increasing pH. Kinetical analysis revealed that this change might be due to the change of Km value for dGTP rather than that of Vmax value. This suggests that the affinity of dGTP on DNA polymerase eta during the mismatch formation with template T should be affected by pH.

Utilization efficiency of the oxidized purine nucleotide analogs by DNA polymerase eta.

To elucidate the behavior of DNA polymerase eta against the oxidized purine nucleotides, we determined the utilization efficiency of 2-hydroxy-dATP and 8-hydroxy-dGTP by the recombinant yeast DNA polymerase eta using the primer extension assay with the synthetic oligonucleotide template-primers, and compared those by DNA polymerase alpha. Results indicate that DNA polymerase eta incorporates 2-hydroxy-dATP opposite template G in addition to template T and 8-hydroxy-dGTP opposite A in addition to C, respectively. Kinetic analysis revealed that the rate of mutation caused by 2-OH-dATP and 8-OH-dGTP with DNA polymerase eta should be much higher than those with DNA polymerase alpha.