Effect of pH on the Misincorporation Rate of DNA Polymerase η

Abstract

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 K m 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.Key words misincorporation; DNA polymerase; pH; mutation DNA replication and repair must occur with a high accuracy to maintain the integrity of genomic information. Several eukaryotic DNA polymerases contribute to replication and repair, but with differing fidelity.1) Eukaryotic DNA polymerases are currently classified into four families; A, B, X, and Y.2) Among them, Y family polymerases show lower fidelity than other families, lacking the intrinsic 3′ to 5′ exonuclease activity required for proofreading. DNA polymerase η, one of the Y family polymerases, was first found as a responsible gene product of a Xeroderma pigmentosum variant.3) This polymerase contributes to translesional synthesis and somatic hypermutation of immunoglobulin variable genes. 4) Although DNA polymerase η correctly bypasses thymine-thymine dimers resulting from UV-irradiation, it shows a high misincorporation rate of 1 in 28 nucleotides on an undamaged template.1,5,6) The fidelity of a DNA polymerase depends not only on proofreading activity but also on nucleotide substrate selection at its active site. 7,8) Nucleotide selection mainly depends on the binding affinity of DNA polymerase to the incoming nucleotide substrate, with the correct binding leading to the conformational change required for the suitable positioning of the active site. Although the hydrogen bond between template and substrate bases governs nucleotide selection, 9,10) geometric selection is more important rather than the hydrogen bond. 11)Geometric selection is a checking function that determines whether the shape and size of a base pair corresponds to a correct Watson-Crick type. DNA polymerases comprise three structural domains; finger, palm and thumb; the O-helix in the finger domain governs geometric selection. However, DNA polymerase η is deficient in the O-helix structure compared with polym...

Optimization of the expression, purification and polymerase activity reaction conditions of recombinant human PrimPol

“…coli Pol I as well as S . cerevisiae and human Pol η, Pol λ and Pol α all have the pH optimum at 8–9 in Tris-HCl buffer [ 23 , 25 – 27 ]. Interestingly, PrimPol has the predicted pI at 5.07–5.18.…”