Human DNA polymerase kappa synthesizes DNA with extraordinarily low fidelity

Abstract

Escherichia coli DNA polymerase IV encoded by the dinB gene is involved in untargeted mutagenesis. Its human homologue is DNA polymerase kappa (Polkappa) encoded by the DINB1 gene. Our recent studies have indicated that human Polkappa is capable of both error-free and error-prone translesion DNA synthesis in vitro. However, it is not known whether human Polkappa also plays a role in untargeted mutagenesis. To examine this possibility, we have measured the fidelity of human Polkappa during DNA synthesis from undamaged templates. Using kinetic measurements of nucleotide incorporations and a fidelity assay with gapped M13mp2 DNA, we show that human Polkappa synthesizes DNA with extraordinarily low fidelity. At the lacZalpha target gene, human Polkappa made on average one error for every 200 nucleotides synthesized, with a predominant T-->G transversion mutation at a rate of 1/147. The overall error rate of human Polkappa is 1.7-fold lower than human Poleta, but 33-fold higher than human Polbeta, a DNA polymerase with very low fidelity. Thus, human Polkappa is one of the most inaccurate DNA polymerases known. These results support a role for human Polkappa in untargeted mutagenesis surrounding a DNA lesion and in DNA regions without damage.

Figure 1

Analysis of purified human Polκ, Polη and Polβ. (A) Purified human Polκ (200 ng), (B) Polη (80 ng) and (C) Polβ (230 ng) were analyzed by electrophoreses on 10% SDS–polyacrylamide gels and visualized by silver staining of the gels (lanes 1). Protein size markers (lanes M) are indicated on the left.

Figure 2

Proofreading nuclease assays of purified human Polκ. (A) The DNA template and four primers used for nuclease activity assays. The primers were labeled with ³²P at their 5′-ends as indicated by an asterisk and annealed individually to the template. (B) DNA substrates (50 fmol) containing annealed primer P3 were incubated without (Klenow Pol, –) or with (Klenow Pol, +) the purified Klenow fragment of E.coli DNA polymerase I (1 U) for 2 min at 30°C in the DNA polymerase assay buffer without dNTPs. Reaction products were separated by electrophoresis on a 20% denaturing polyacrylamide gel. (C) Using the indicated DNA substrates, proofreading nuclease assays were similarly performed with (Polκ, +) or without (Polκ, –) purified human Polκ (1.3 ng, 13 fmol) as in (B). However, the incubation time was extended to 30 min. DNA size markers (in nucleotides) are indicated on the right.

Figure 3

Base pairing specificity of human Polκ. (A) Each DNA template (Temp) was annealed with the 17mer primer that was labeled at its 5′-end with ³²P as indicated by an asterisk. The four template bases examined are underlined. (B and C) Polymerase assays were performed with 50 fmol of the indicated DNA templates and 7 fmol (0.7 ng) of purified human Polκ in the presence of a single dNTP or all four dNTPs (N₄) as indicated. DNA size markers (in nucleotides) are indicated on the sides. Quantitation of extended primers is shown at the bottom of the gels.

Figure 4

Kinetic analysis of C and T incorporations opposite a template G. (A) Polymerase assays were performed at 30°C for 10 min using 7 fmol (0.7 ng) of purified human Polκ, 50 fmol of the template G (Fig. 3A) and increasing concentrations of dCTP or dTTP as indicated. Primer extension products were separated from the ³²P-labeled 17mer primer by electrophoresis on a 20% denaturing polyacrylamide gel and visualized by autoradiography. DNA size markers (in nucleotides) are indicated on the sides. (B) Results in (A) were quantitated and the rate of nucleotide incorporation (primer extension) was graphed as a function of dCTP or dTTP concentrations. The scattered plots were then fitted into the Michaelis–Menton equation (see Materials and Methods). The V_max and K_m values obtained from the fitted curve were listed in Table 1.

Figure 5

Mismatch extension by human Polκ. (A) Various primers labeled at their 5′-ends with ³²P as indicated by asterisks were annealed to the 30mer template, generating 12 possible mismatches at the primer 3′-ends. (B) Mismatched substrates were incubated with (Polκ, +) or without (Polκ, –) DNA Polκ (4 ng, 40 fmol) under standard polymerase assay conditions. Lanes 25 and 26, the 30mer template was annealed with the 17mer primer 5′-*CGCGCGGCCTCCGGTTA-3′ generating a correct T–A base pair at the primer 3′-end. DNA size markers (in nucleotides) are indicated on the sides. It should be noted that the primers in the T–C (lane 2) and T–A DNA substrates (lane 26) are 4 nt longer at the 5′-end than other primers.

Figure 6

Gap filling DNA synthesis by purified human Polκ, Polη and Polβ. (A) Analysis of gapped M13mp2 DNA (Gap DNA) by a 0.8% agarose gel. Control, double-stranded M13mp2 DNA without gap. (B–D) Gapped M13mp2 DNA (8 fmol) was incubated with purified human Polκ (400 fmol), Polη (205 fmol) and Polβ (1200 fmol), respectively, for gap filling DNA syntheses (Gap filling) (see Materials and Methods). Gap filling products (6.4 fmol of M13mp2 DNA) were analyzed by electrophoreses on 0.8% agarose gels in the presence of 0.5 µg/ml ethidium bromide. NC, nicked circular DNA; GC, gapped circular DNA; CC, closed circular DNA.