Insertion of the T3 DNA polymerase thioredoxin binding domain enhances the processivity and fidelity of Taq DNA polymerase

Abstract

Insertion of the T3 DNA polymerase thioredoxin binding domain (TBD) into the distantly related thermostable Taq DNA polymerase at an analogous position in the thumb domain, converts the Taq DNA polymerase from a low processive to a highly processive enzyme. Processivity is dependent on the presence of thioredoxin. The enhancement in processivity is 20-50-fold when compared with the wild-type Taq DNA polymerase or to the recombinant polymerase in the absence of thioredoxin. The recombinant Taq DNA pol/TBD is thermostable, PCR competent and able to copy repetitive deoxynucleotide sequences six to seven times more faithfully than Taq DNA polymerase and makes 2-3-fold fewer AT-->GC transition mutations.

Figure 1

Superimposition of the thumb domains of Taq DNA polymerase (blue) with T7 DNA polymerase (pink). The arrows indicate the site of insertion of the T3 TBD (yellow). The primary amino acid sequence of Taq DNA polymerase from residue 470–507 is indicated below (blue) with the sequence of T3 TBD in yellow and the deleted region in red.

Figure 2

Polymerase activity was measured as described in the Materials and Methods following exposure to 94°C for 0, 2, 10 and 30 min. The amount of Taq DNA polymerase and Taq DNA polymerase (exo–) used in each reaction was 230 ng, while 60 ng was used for the Taq DNA pol/TBD and Taq DNA pol/TBD(exo–) enzymes. Thioredoxin was included in all reactions at 100 µM.

Figure 3

The effect of thioredoxin on processivity of the hybrid Taq DNA pol/TBD. (A) Extension assays were performed with a molar excess of template corresponding to a primer/template ratio of 470 for Taq DNA polymerase and Taq DNA polymerase (exo–) and 67 for Taq DNA pol/TBD and Taq DNA pol/TBD(exo–). Different ratios for the enzymes were used to ensure equal activity was loaded on the gel. (+) 100 µM thioredoxin, (–) no thioredoxin. No enzyme control shows the labeled primer alone. (B) Extension assay showing the effect of increasing concentrations of thioredoxin and enzyme dilution for Taq DNA pol/TBD(exo–). For each thioredoxin concentration (0.2, 2 and 20 µM), three enzyme concentrations were used (56, 28 and 5.6 pM) corresponding to a primer/template ratio of 67, 134 and 670.

Figure 4

Streptavidin processivity assay. An immobilized single-stranded DNA molecule of 2000 nt in length was incubated in a reaction containing a primer hybridized to the 5′ end, and polymerase. Extension was initiated by the addition of dNTPs including [α-³²P]dGTP, Mg²⁺ and 0.8 mg/ml activated calf thymus DNA as described in Materials and Methods. Cleavage with restriction enzymes located 18, 96, 492, 1122 and 1898 nt, respectively, from the primer terminus only occurs if primer extension results in a double-stranded DNA substrate. Full extension with 5 U Promega Taq DNA polymerase in the absence of trap DNA allowed the percentage of primers extended to be determined.

Figure 5

PCR using 1 ng of Lambda phage DNA as template. Ethidium bromide stained 0.8% agarose gel (image inverted). Lane M, 1 kb marker (GeneRuler); lanes 1–4, Taq DNA polymerase amplified 1, 5, 7 and 10 kb; lanes 5–8, Taq DNA pol/TBD no thioredoxin (–Tx) 1, 5, 7 and 10 kb; lanes 9–12, Taq DNA pol/TBD + thioredoxin (+Tx) 1, 5, 7 and 10 kb. Molecular weight marker sizes are indicated by the dark bars on the left of the gel (from top to bottom: 10, 8, 6, 5, 4, 3, 2, 1 and 0.25 kb).

Figure 6

Slippage frequency was determined by the number of frame shifted revertant colonies (Tet+ Carb+) divided by the total number of cells plated (Tet+). The result is representative of one experiment. Similar results were obtained in two similar separate experiments. The background frequency of frame shift molecules for template and vector DNA was determined to be <1 revertant per 10⁵.

Figure 7

Slippage chromatograms were obtained from PCR products amplified with either Taq DNA polymerase or Taq DNA pol/TBD. One primer was labeled with 6-FAM fluorophore and the PCR product was digested with EcoRI. The DNA was gel purified and slippage polymorphisms detected using an automated DNA sequencer (model 377; Applied Biosystems) and GENESCAN 672 software. The result is one representative of three experiments.