Long-Range PCR Amplification of DNA by DNA Polymerase III Holoenzyme from Thermus thermophilus

Abstract

DNA replication in bacteria is accomplished by a multicomponent replicase, the DNA polymerase III holoenzyme (pol III HE). The three essential components of the pol III HE are the α polymerase, the β sliding clamp processivity factor, and the DnaX clamp-loader complex. We report here the assembly of the functional holoenzyme from Thermus thermophilus (Tth), an extreme thermophile. The minimal holoenzyme capable of DNA synthesis consists of α, β and DnaX (τ and γ), δ and δ' components of the clamp-loader complex. The proteins were each cloned and expressed in a native form. Each component of the system was purified extensively. The minimum holoenzyme from these five purified subunits reassembled is sufficient for rapid and processive DNA synthesis. In an isolated form the α polymerase was found to be unstable at temperatures above 65°C. We were able to increase the thermostability of the pol III HE to 98°C by addition and optimization of various buffers and cosolvents. In the optimized buffer system we show that a replicative polymerase apparatus, Tth pol III HE, is capable of rapid amplification of regions of DNA up to 15,000 base pairs in PCR reactions.

Figure 1

Constructs allowing Tth proteins expression by translationally coupling. Construction of plasmids expressing Tth proteins using translational coupling techniques is described under Section 2. The primers used in PCR of individual genes are listed in Table 1. The dnaE gene (α) was first cloned as a translationally coupled construct (pTAC-CCA-TE). This construct was then used as a starting point in the construction of translationally coupled vectors containing dnaB (β), DnaX (τ/γ), holA (δ) and holB (δ′), and ssb (SSB).

Figure 2

Purification of Tth DNA pol III holoenzyme subunits. α, β, τ/γ, δ, and δ′ were purified to at least 95% homogeneity as described under Section 2.

Figure 3

Requirements for individual components of the Tth pol III holoenzyme. DNA synthesis was used to monitor the activity of the subunits [7]. The pol III holoenzyme was reconstituted using native forms of each subunit. The components that were not being tested were held constant at saturating concentrations (0.05 μM δ′, 0.01 μM δ, 1.4 μM β (monomer), 0.05 μM DnaX (monomer), and 0.5 μM α) while the test subunit was titrated into the reactions as indicated. Reactions were at 55°C. The subunits titrated into the reactions were (a) α, (b) β, (c) DnaX, (d) δ, and (e) δ′. The concentrations of proteins were calculated based on the molecular mass of single subunits. “Activity” is defined as the total pmol of nucleotides incorporated.

Figure 4

Thermostability of Tth α. Gap-filling polymerase assays [31] were used to determine loss of activity of Tth α at elevated temperatures. (a) The enzyme mix was heated to 90°C for 2 min and then combined with the substrate mix and incubated for an additional 5 min at 60°C. The positive control was not subjected to the heat challenge step. (b) Components of the complete Tth holoenzyme were mixed at room temperature and incubated at 80°C (■) or 85°C (◆). Samples were removed at the indicated times.

Figure 5

Assay to optimize thermostability of the Tth pol III holoenzyme. Assays were as described under Section 2. Positive controls are reactions carried out using initial buffer at 55°C. (a) Effect of different buffers in reactions on activity at 85°C. HEPES, Tris-HCl, and TAPS-Tris buffer concentrations were 25, 50, and 20 mM, respectively. (b) Assays containing different percent of glycerol tested at 87°C. (c) Assays containing the indicated concentrations of ATP were tested at 88°C. (d) Assays containing the indicated concentrations of magnesium acetate (Mg⁺⁺) were tested at 88°C. Negative control is minus Mg⁺⁺.

Figure 6

A comparison of the activity of Tth pol III holoenzyme at 55°C versus 98°C. Assays containing the optimized buffer system were cycled 5 times at 98°C/20 s, 60°C/2 min, and 70°C/2 min prior to addition of the dNTP mix and then incubation was continued at 70°C for an additional 2 min. Positive control contained the original buffer mix and was cycled as for optimized buffer system reactions but all cycle temperatures were at 55°C. Negative controls were cycled as for the optimized buffer system reactions but contained the original buffer mix.

Figure 7

Determination of the thermostability of Tth pol III HE in cycled reactions. (a) The effect of cycle number on the amount of DNA synthesized. (b) Products from the asymmetric PCR-like reactions. The circular ssM13mp18 DNA served as the starting material. 0 cycles refers to the mix that was not cycled at any temperature. All other lanes contain products from reactions cycled for the indicated times. Each cycle consisted of steps at 94°C/30 s, 60°C/1 min, and 70°C/2 min. The noncycled reaction contains primed M13mp18 template plus Tth pol III HE incubated at 70°C for 2 min.

Figure 8

PCR reactions for amplification of short regions of DNA. PCR reactions and agarose gel analysis were as described under Section 2. PCR cycles consisted of 94°C/30 s, 55°C/1 min, and 72°C/2 min. Taq Pol indicates PCR reactions using Taq DNA polymerase (18038-018, Invitrogen) per manufacturer's instructions. Primers used are shown in Table 3. (a) PCR reactions using primers designed to yield a 200 bp PCR product. (b) PCR reactions using primers designed to yield a 512 bp PCR product. The number of PCR cycles is indicated at bottom of the figures.

Figure 9

PCR reactions to amplify intermediate and long regions of DNA. Reactions were conducted as described under Section 2. PCR reactions were allowed to proceed for 30 cycles. The same amount of primer and template was used in Tth pol III HE and Taq polymerase reactions. Primers used are shown in Table 3. PCR product sizes are as indicated. (a) The template was pET Blue-2 plasmid (Novagen) and primers were designed to yield 1500 and 2500 bp products. (b) The template was Lambda DNA-HindIII Digest and primers were designed to yield a 4650 and 7500 bp product. (c) The template was Lambda DNA-HindIII Digest and primers were designed to yield a 12500 and 15000 bp product.