Inteins of Thermococcus fumicolans DNA Polymerase Are Endonucleases with Distinct Enzymatic Behaviors*

Production and Purification of the Tfu Polymerase Inteins

The coding sequences of Tfu pol-1 andTfu pol-2 inteins were amplified by PCR from Tfugenomic DNA with oligonucleotides Tfu pol-1-ATG (5′-ttagattcatatgtgtcatcctgccgacact-3′), Tfu pol-1-TAG (5′-gatgtcgacctaattgtgggtcagtatgcc-3′), Tfu pol-2-ATG (5′-ctgtacgcatatgagtgttacaggggacacag-3′), and Tfu pol-2-TAG (5′-agcgtcgaccatgttgtgaacgagtattccg-3′), respectively. These PCR products were a gift from J. Quérellou (Ifremer). They were digested by NdeI and SalI and cloned into aNdeI-SalI-digested pET26b vector (Novagen). The resulting constructs (pET26-Tfu1 and pET26-Tfu2) were sequenced by Isoprim (Toulouse, France). Escherichia coli BL21(De3)[pLysS] bacteria transformed with pET26-Tfu1 or pET26-Tfu2 were grown at 37 °C in Luria broth culture medium supplemented with 50 μg/ml kanamycin (Sigma). A 2-h induction with 0.5 mmisopropyl-β-d-thiogalactopyranoside (Sigma) was performed, and cells were lysed in 20 mm sodium phosphate, pH 7.5 (measured at 25 °C), by six cycles of freezing-thawing. The lysate was centrifuged at 10,000 × g for 15 min, and the supernatant was chromatographed on a XK26 Sepharose Q Fast Flow column (Amersham Pharmacia Biotech) at room temperature. The proteins were eluted with a 0–1 m NaCl linear gradient in 20 mm sodium phosphate, pH 7.5, at a flow rate of 5 ml/min.Tfu pol-1 and Tfu pol-2 are eluted at 0.2 and 0.7m NaCl, respectively, and are approximately 80% homogeneous. These fractions were dialyzed against 10 mmTris-HCl, pH 7.5, 50% glycerol, 0.1 mm EDTA, 1 mm dithiothreitol, 200 μg/ml bovine serum albumin, and 50 mm NaCl for storage.

The Tfu pol gene was cloned in an expression vector by Appligène-Oncor (France). BL21(De3)[pLysS] bacteria transformed with this plasmid were grown overnight at 25 °C. Extraction and purification procedures for spliced inteins were identical to those described for recombinant inteins, except that the protein extract was treated for 1 h at 70 °C before purification.

Assay of Endonuclease Activity

All the substrates were constructed by PCR as described by Weiner (16) using pUC19 as DNA matrix and different purified oligonucleotide pairs: S1–5′ (5′-tcgctatatccttcgattatagagtcgacctgcaggcatgc-3′) and S1–3′ (5′-cctaaaatctaaataagctaagaggatccccgggtaccgag-3′), S2–5′ (5′-acagacggctttttcgcaacagagtcgacctgcaggcatgc-3′) and S2–3′ (5′-atccgcgtacagcactttaaagaggatccccgggtaccgag-3′), S3–5′ (5′-agagtcgacctgcaggcatgc-3′) and S3–3′ (5′-caaaagccgtctgtatccgcgtgagaggatccccgggtaccgag-3′), S4–5′ (5′-agagtcgacctgcaggcatgc-3′) and S4–3′ (5′-atatagcgacctaaaaagaggatccccgggtaccgag-3′), and S5–5′ (5′-tcggtatatcagagtcgacctgcaggcatgc-3′) and S5–3′ (5′-cctaaaatcagaggatccccgggtaccgag-3′), for substrates 1–5, respectively (Fig. 1 b). The sequences in bold are necessary for the annealing to the pUC19 matrix at 60 °C.

The 41-bp DNA substrate 1 (S1) for Tfu pol-1 consisted of 20 bp of the 5′ end of the homing site plus 21 bp of the 3′ end, inserted in the XbaI site of pUC19. In the same way, a 43-bp substrate for Tfu pol-2 was constructed (substrate 2, S2). Substrates 3 and 4 (S3 and S4) corresponded to the minimal recognition and cleavage sites determined by the primer extension method (see below) for PI-TfuII and PI-TfuI, respectively. Substrate 5 (S5) corresponded to the minimal linear recognition site of PI-TfuI.

The resulting plasmids were either linearized by ScaI (Appligène-Oncor) and purified or the supercoiled forms of the DNA substrates were purified from a 1% agarose gel in TBE (0.09m Tris borate, 0.002 m EDTA) buffer. Finally, linear and circular forms of these substrates were diluted in water to a concentration of 100 ng/μl. Endonuclease activity assays were performed in a final volume of 10 μl, in various reaction buffers and temperatures ranging from 37 °C to 80 °C (see “Results”). The reaction mixtures were analyzed on a 1% agarose gel in TBE buffer. The amount of undigested substrates and products were quantified with the ImageQUANT program (Molecular Dynamics Inc.).

One unit of PI-TfuI or PI-TfuII endonuclease was required to digest 1 μg of ScaI-linearized DNA substrate 1 or 2, respectively, in 1 h at 70 °C, in optimal buffers. Specific activities of PI-TfuI and PI-TfuII were measured by incubating known amounts of linear DNA substrates with known amounts of purified endonucleases.

Definition of the Minimal Recognition and Cleavage Site of Endonucleases

The endonuclease recognition sites were determined by a primer extension method as described by Wenzlau et al.(17). NaOH-denatured plasmids S1 and S2 were sequenced in both orientations using the T7 polymerase sequencing kit (Amersham Pharmacia Biotech) and universal primers SeqPuc (5′-gtaacgccagggttttcc-3′) and M13Rev (5′-ggaaacagctatgaccatg-3′). After chain termination reaction, the samples were split in two. The first half of the sample was overdigested with 100 units of PI-TfuI or PI-TfuII for 1 h at 70 °C, in a 50 mmTris acetate, pH 8, buffer containing 100 mmNH₄OAc and 25 mm MnSO₄ or 75 mm Mg(OAc)₂, respectively. The cleavage reactions and the undigested half of the sequence reactions were ethanol-precipitated and resuspended in 5 μl of water. 4 μl of stop solution were added, and the samples were subjected to electrophoresis in a standard 6% denaturing polyacrylamide gel.

Inhibition of Cleavage by Reaction Products

4 μg ofScaI-linearized substrate 2 were overdigested by PI-TfuII in optimal conditions by successive additions of enzyme. Both cleavage reaction products (940 and 1790 bp) were separated and purified from each other from a 1% agarose gel in TBE buffer. These two DNA fragments were then used as competitors in standard cleavage assays of substrate 2.

Electrophoretic Mobility Shift Assay

A 119-bp DNA probe containing the 43-bp recognition and cleavage site of PI-TfuII was synthesized by PCR. The universal M13Rev oligonucleotide was first phosphorylated with [γ-³³P]ATP (NEN Life Science Products) (18) and then used together with the pUC5′-Xba oligonucleotide (5′-agctcggtacccggggat-3′) and 26 ng of substrate 2 DNA as matrix in a standard PCR amplification reaction. The 119-bp ³³P-labeled fragment was purified and diluted in water to a concentration of 25 nm. 7.5 fmol of this DNA probe were incubated in a 20 mm Tris acetate, pH 8, buffer containing 20 mmMg(OAc)₂, 20 mm NH₄OAc, 0.1%Triton X-100, and 16% glycerol, either with or without PI-TfuII, in a final volume of 20 μl, for 15 min at 37 °C or 70 °C. These reaction mixtures were then submitted to electrophoresis in a 7.5% nondenaturing polyacrylamide (30:1) gel in 0.25× TBE buffer at 220 V for 2 h. After electrophoresis, the gel was dried and radioactive bands were detected using a PhosphorImager (Molecular Dynamics Inc.). The specificity of the DNA-protein interaction was controlled using 7.5 pmol of herring sperm DNA as a nonspecific competitor in the binding reactions (data not shown).

Tfu Polymerase Inteins Were Spliced in E. coli

The completeTfu polymerase gene was expressed in E. coli, at 25 °C. The SDS-polyacrylamide gel electrophoresis separation of the crude extract shows two bands of approximately 41 and 45 kDa, which comigrated with Tfu pol inteins (Fig.2). At this temperature, a partial splicing reaction was observed since the DNA polymerase was not detected after staining the gel with Coomassie Blue, and we consistently obtained more Tfu pol-2 intein thanTfu pol-1.

Incubating this bacterial extract at 70 °C increased the yield of spliced proteins, the DNA polymerase being detected after heating for 30 min (Fig. 2). Even then, although no protein bands corresponding to the polymerase precursors were detected on the gel, splicing was probably not complete after 1 h of incubation at 70 °C because of the different amounts of each of the spliced products,i.e. the polymerase and both inteins.

Purified Tfu DNA pol is active in standard PCR reactions (Appligène-Oncor). Moreover, purified splicedTfu pol-2 has the same specific endonuclease activity as the recombinant protein. In contrast, we could never detect any endonuclease activity for the purified spliced Tfu pol-1, unlike the recombinant protein (see below). We used the recombinant proteins to investigate further the endonuclease activity of both inteins.

Endonuclease Activity of Tfu Inteins

Inteins with known endonuclease activity recognize and cut a DNA sequence spanning the junction site created when the intein coding region is deleted from the host gene (homing site). We thus constructed substrate 1 forTfu pol-1 and substrate 2 for Tfu pol-2 by cloning synthetic homing sites of 43 and 41 bp, respectively (Fig.1 b). The supercoiled and ScaI-linearized forms of these DNA substrates were purified and used to assay the endonuclease activity of both inteins.

Cleavage assays were performed in a wide range of experimental conditions, including Tris-HCl or Tris acetate buffers, pH 7–9 (measured at 25 °C), containing 0–100 mmMg(OAc)₂, 0–400 mm NH₄OAc, 0–30% glycerol, and 0–100 mm NaCl, KCl, ZnCl₂, CaCl₂, MnSO₄, or MnCl₂. The assay temperature was varied from 37 to 80 °C.

The supercoiled substrate 1 was cleaved by Tfu pol-1 intein under a large set of conditions, at an optimal temperature of 70 °C. Both Mg²⁺ and Mn²⁺ were able to stimulate the cleavage reaction (Fig. 3 a), and optimal digestion was obtained in a 50 mm Tris acetate, pH 8, buffer containing 25 mm MnSO₄ and 100 mm NH₄OAc. Open circular DNA accumulated during the reaction leading to linear DNA product, indicative of a two-step reaction. When MnSO₄ was replaced by 50 mmMg(OAc)₂, cleavage efficiency dropped approximately 5-fold and the open circular intermediate accumulated (Fig.3 a).

In contrast, Tfu pol-1 showed no endonuclease activity on linear substrate 1 under all the conditions assayed, with up to 300 ng of protein/assay, except when manganese or zinc salts were added to the reaction buffer instead of magnesium salt (Fig. 3 b). It turned out that MnSO₄ stimulated the reaction much more efficiently than MnCl₂ and ZnCl₂. Optimal cleavage of linear substrate 1 was obtained, as with circular substrate 1, in a 50 mm Tris acetate, pH 8, buffer containing 25 mm MnSO₄ and 100 mmNH₄OAc. Under these conditions, both linear and supercoiled substrates were cleaved with the same efficiency and specific activity of Tfu pol-1 (PI-TfuI) was 4,800 units/mg.

In contrast, Tfu pol-2 exhibited endonuclease activity on substrate 2 under numerous assay conditions. No difference was observed between circular and linear forms of the DNA substrate, which were cleaved with the same optimal efficiency at 70 °C (Fig.3 c), in a 50 mm Tris acetate, pH 8, buffer containing 75 mm Mg(OAc)₂, 100 mmNH₄OAc, and 10% glycerol, with a specific activity ofTfu pol-2 (PI-TfuII) of 748,000 units/mg. The cleavage efficiency was decreased 5–10-fold by replacing Mg²⁺ by Mn²⁺. Otherwise, no cleavage was observed during the endonucleasic digestion of the supercoiled DNA (Fig. 3 c). The effects of DNA topology and divalent ions used as cofactor on endonuclease activity of PI-TfuI and PI-TfuII are summarized in TableI.

Minimal Recognition Sequences

A mixture of potential double-strand DNA substrates for PI-TfuI and PI-TfuII were generated by primer extension using circular substrate 1 or substrate 2 as templates, respectively. The extension products including all the bases needed for cleavage were digested and thus disappeared from the sequence reaction, while those lacking one or more bases of the minimal recognition site were not digested. Hence, the comparison of digested and undigested sequence reactions allowed the determination of the bases necessary for cleavage (Fig.4 a). This method yielded a 16-bp non-palindromic site for PI-TfuI and a 21-bp non-palindromic site for PI-TfuII. In both cases, cleavage generated non-identical 3′ overhangs of 4 bases (Fig. 4 b). Both cleaved substrates could be religated with T4 DNA ligase. Thus, cleavage by PI-TfuI or PI-TfuII generated 5′-phosphate and 3′-hydroxyl ends. To confirm the results obtained by primer extension, plasmids containing the 21-bp site for PI-TfuII (substrate 3) or the 16-bp site for PI-TfuI (substrate 4, Fig. 1 b) were constructed and submitted to cleavage assays either as linear or supercoiled DNA.

Standard cleavage assays on linear and circular DNA substrates 3 and 4, under optimal conditions for each enzyme, highlight the different catalytic behaviors of the endonuclease inteins (Table I). While PI-TfuII cleaved with the same efficiency linear and circular substrates 2 and 3 in the magnesium-containing buffer, PI-TfuI cleaved linear substrate 1 but did not cleave linear substrate 4 even in presence of manganese (Fig.5 a). On the other hand, PI-TfuI cleaved with the same efficiency supercoiled substrates 1 and 4 (Fig. 5 b). Cleavage assays on various linear substrates for PI-TfuI (data not shown), containing synthetic homing sites of different lengths, led to a minimal linear site of 19 bp (Fig. 1 b), which is homologous to the one described for PI-PkoI (15).

Thus, PI-TfuII cleaved its homing site and made no difference between linear and supercoiled DNA containing the minimal or a larger recognition site. In contrast, PI-TfuI cleaved supercoiled DNA harboring its 16-bp recognition sequence but required a longer recognition sequence (≥19 bp) and manganese as cofactor to cleave linear DNA. Moreover, under these conditions, linear DNA was cleaved as efficiently as circular DNA (Table I).

Product Inhibition of PI-TfuII

While incubation of linear substrate 1 with PI-TfuI led to its complete cleavage, prolonged incubation of substrate 2 with PI-TfuII resulted in only partial cleavage (Fig.6 a). The same results were obtained with circular DNA substrates (data not shown). After 10 min, 80% of the cleavage was performed and the reaction stopped. Addition of fresh PI-TfuII allowed the reaction to proceed, but a loss of activity rapidly followed (data not shown). As the enzyme was stable at 70 °C, this loss of activity could result from an inhibition of PI-TfuII by one of its reaction products. To test this hypothesis, the two DNA fragments resulting from the cleavage of ScaI-linearized substrate 2 were purified and added as potential inhibitors in cleavage reactions. Under the conditions used, 50 ng of substrate 2 were digested by PI-TfuII, when no inhibitor was added to the reaction. Fig. 6 b shows that the 940-bp fragment, which includes the homing site 5′ end, inhibited weakly the cleavage reaction. On the other hand, the 1790-bp fragment, which includes the homing site 3′ end, was an efficient inhibitor of the reaction, a 1:1 ratio between substrate and DNA fragment resulting in 80% inhibition. Electrophoresis mobility shift assays confirmed the strong interaction between the 3′ cleavage fragment and PI-TfuII. Fig. 7 shows that this digestion product remained bound to the enzyme after cleavage was performed at 70 °C. Under these experimental conditions (large excess of enzyme over substrate), cleavage could also be observed at 37 °C but there was surprisingly no mobility shift for the resulting fragment.