The high fidelity and unique error signature of human DNA polymerase epsilon

Abstract

Bulk replicative DNA synthesis in eukaryotes is highly accurate and efficient, primarily because of two DNA polymerases (Pols): Pols δ and ε. The high fidelity of these enzymes is due to their intrinsic base selectivity and proofreading exonuclease activity which, when coupled with post-replication mismatch repair, helps to maintain human mutation rates at less than one mutation per genome duplication. Conditions that reduce polymerase fidelity result in increased mutagenesis and can lead to cancer in mice. Whereas yeast Pol ε has been well characterized, human Pol ε remains poorly understood. Here, we present the first report on the fidelity of human Pol ε. We find that human Pol ε carries out DNA synthesis with high fidelity, even in the absence of its 3'→5' exonucleolytic proofreading and is significantly more accurate than yeast Pol ε. Though its spectrum of errors is similar to that of yeast Pol ε, there are several notable exceptions. These include a preference of the human enzyme for T→A over A→T transversions. As compared with other replicative DNA polymerases, human Pol ε is particularly accurate when copying homonucleotide runs of 4-5 bases. The base pair substitution specificity and high fidelity for frameshift errors observed for human Pol ε are distinct from the errors made by human Pol δ.

Figure 1.

Purification and polymerization and exonuclease assays of human Pol ε. (A) Purified Exo⁺and Exo⁻ human Pol ε was loaded onto a 10% SDS–polyacrylamide gel and stained with Coomassie. Molecular weight marker (MW) is shown with selected molecular weights indicated in kDa. (B) Image of DNA synthesis reaction products resolved on a 16% denaturing polyacrylamide gel. Lanes 2–5 are 1, 2, 5 and 10 min at 37°C with 1 nM Exo⁺Pol ε. Lanes 6–9 are 1, 2, 5 and 10 min at 37°C with 1 nM Exo⁻ Pol ε. Lane 1 is a control reaction with no enzyme. (C) Image of 3′–5′ exonuclease reaction products resolved on a 16% denaturing polyacrylamide gel. Lanes 2–5 are 1.4, 2.8, 5.6 and 8.6 nM Exo⁺ Pol ε. Lanes 6–9 are 1.4, 2.8, 5.6 and 8.6 nM Exo⁻ Pol ε. Reactions were performed as described in ‘Materials and Methods’ section. Template DNA sequences are shown to the side of each substrate.

Figure 2.

Base substitution and frameshift error rates for human and yeast Pol ε and human and yeast Pol δ. (A) Error rates for base pair substitutions, overall frameshifts, −1 and +1 frameshifts. Error rates were calculated as described (54). Error rates are shown for human Pol ε (black bars, this study), yeast Pol ε [dark gray bars, (59)], human Pol δ [white bars, (58)] and yeast Pol δ [light gray bars, (57)]. P-values are shown above each comparison to a human Pol ε error rate where the difference was found to be significant. The (asterisk and double asterisk) symbols indicate P-values where P ≤ 0.05 and 0.001, respectively. (B) Single nucleotide deletion dependence on homonucleotide run length. Error rates were calculated for −1 deletions in non-iterated nucleotides (1) or runs of the indicated number nucleotides (2, 3, 4/5). The legend and source of data are as in (A).

Figure 3.

Fidelity of individual base pair substitutions for human Pol ε and human Pol δ. Error rates for each of the 12 possible mispairs for the exonuclease-deficient forms of human Pol ε (black bars, this study) and human Pol δ [white bars, (58)] were calculated as described (54). P-values are shown above each comparison to a human Pol ε error rate where the difference was found to be significant. The (asterisk and double asterisk) symbols indicate P-values where P ≤ 0.05 and 0.005, respectively.

Figure 4.

Spectrum of base substitution and single-base frameshift mutations made by exonuclease-deficient human Pol ε in the lacZ gene. Base substitutions are shown above the sequence, while deletions (open triangles) and insertions (closed triangles) are shown below. The numbering is relative to the +1 at the transcription start site.