Yeast DNA polymerase ϵ catalytic core and holoenzyme have comparable catalytic rates

Abstract

The holoenzyme of yeast DNA polymerase ϵ (Pol ϵ) consists of four subunits: Pol2, Dpb2, Dpb3, and Dpb4. A protease-sensitive site results in an N-terminal proteolytic fragment of Pol2, called Pol2core, that consists of the catalytic core of Pol ϵ and retains both polymerase and exonuclease activities. Pre-steady-state kinetics showed that the exonuclease rates on single-stranded, double-stranded, and mismatched DNA were comparable between Pol ϵ and Pol2core. Single-turnover pre-steady-state kinetics also showed that the kpol of Pol ϵ and Pol2core were comparable when preloading the polymerase onto the primer-template before adding Mg(2+) and dTTP. However, a global fit of the data over six sequential nucleotide incorporations revealed that the overall polymerization rate and processivity were higher for Pol ϵ than for Pol2core. The largest difference between Pol ϵ and Pol2core was observed when challenged for the formation of a ternary complex and incorporation of the first nucleotide. Pol ϵ needed less than 1 s to incorporate a nucleotide, but several seconds passed before Pol2core incorporated detectable levels of the first nucleotide. We conclude that the accessory subunits and the C terminus of Pol2 do not influence the catalytic rate of Pol ϵ but facilitate the loading and incorporation of the first nucleotide by Pol ϵ.

Keywords: DNA Polymerase; DNA Repair; DNA Replication; Enzyme Catalysis; Enzyme Kinetics.

FIGURE 1.

Protein preparations. 8 pmol of Pol ϵ and Pol2_core (amino acids 1–1228), respectively, were resolved on 8% SDS-PAGE and stained with Coomassie Brilliant Blue. Exonuclease-deficient variants, Pol ϵ exo⁻ and Pol2_core exo⁻, carry two amino acid substitutions, D290A and E292A, that are present in the previously studied pol2-4 allele (28).

FIGURE 2.

Exonuclease kinetics. Preformed enzyme-DNA complexes were rapidly mixed with Mg²⁺. The loss of primer was plotted as a function of time and fit to a biphasic decay equation to obtain rate constants for the fast phase. The rate constants obtained are given in Table 2. A and B, exonucleolytic degradation of DNA substrates by Pol2_core and Pol ϵ, respectively. Shown are single-stranded DNA (51T) (yellow, ▴), correctly primed template (50/80) (black, ▾), and single mismatch at the primer end (51T/80) (red, ■). Green lines (●), exonucleolytic degradation of double mismatch at the primer end (51TT/80). Error bars, S.E.

FIGURE 3.

Maximum rate of dTTP incorporation. Top, the amount of product formed was plotted against time using a double-exponential equation with a fast phase and a slow phase for each dTTP concentration. In the resulting plot, seven selected dTTP concentrations are shown as examples. Bottom, the observed rate constants for the fast phase were plotted as a function of dTTP concentration and fit to a hyperbola. Error bars, S.E. obtained from three independent experiments. The maximum rate constants obtained were k_pol ∼352 s⁻¹ and K_d^dTTP ∼25 μm for Pol2 exo⁻ and k_pol ∼319 s⁻¹ and K_d^dTTP ∼21 μm for Pol ϵ exo⁻.

FIGURE 4.

Elemental effect. Single-turnover nucleotide incorporation reactions were performed with Pol2_core exo⁻ (A) and Pol ϵ exo⁻ (B) either in the presence of 15 μm dTTP (black line, ●) or in the presence of 15 μm (S_p)-dTTPαS (red line, ■). The amount of product formed is plotted against time and fit in a biphasic association equation. Mean values of the two independent experiments are plotted.

FIGURE 5.

Processive polymerization rates. A, multiple nucleotide incorporations were carried out to measure the time taken by Pol2_core exo⁻ and Pol ϵ exo⁻ to incorporate 30 nucleotides under single-turnover conditions. Excess 50/80-mer (100 nm) was preincubated with enzyme (60 nm) and rapidly mixed with Mg²⁺ and physiological dNTP concentrations. B, the amount of remaining substrate (50-mer in red) and each intermediate product (51-mer in red, 52-mer in yellow, 53-mer in maroon, 54-mer in green, 55-mer in light blue, and 56-mer in black) were plotted with the KinTek global simulation software using a model defining the incorporation of the first six nucleotides and six DNA dissociation events. The polymerization rate constants obtained for Pol2_core exo⁻ and Pol ϵ exo⁻ are listed in Table 6.

FIGURE 6.

Loading of Pol ϵ onto the 3′-primer terminus. Primer extension reactions were carried out by rapidly mixing enzyme with DNA, Mg²⁺, and dTTP and then quenching the reactions at the time points indicated in A. dTTP incorporation was indicative of ternary complex formation and subsequent phosphodiester bond formation. Different lengths of DNA substrates were used to monitor the length dependence on the accessibility for loading the 3′-primer terminus into the polymerase active site and to extend the primer by one nucleotide. The relative band intensities of the incorporation of dTTP (B) were plotted against time for Pol2 _core exo⁻ and Pol ϵ exo⁻.

FIGURE 7.

Active site titration. 65 nm Pol2_core exo⁻ (●) or Pol ϵ exo⁻ (▴) was preincubated with different concentrations of DNA and rapidly mixed with dTTP and Mg²⁺ for 50 ms. The mean product formed was plotted against DNA concentration in a quadratic equation to determine the K_d^DNA of 15.65 ± 3 nm and active enzyme concentration of 45 ± 2 nm for Pol2_core exo⁻ and K_d^DNA of 11.6 ± 2 nm and active enzyme concentration of 52 ± 2 nm for Pol ϵ exo⁻. Values are S.E. obtained from three independent experiments.