<b>Biochemical characterization of the nuclease StoNurA from the hyperthermophilic archaeon</b> Sulfolobus tokodaii

The DNA nuclease gene ST2109 has been cloned from the hyperthermophilic archaeon Sulfolobus tokodaii and expressed in Escherichia coli. The recombinant protein StoNurA has been purified to homogeneity by affinity chromatography and gel filtration chromatography. Biochemical analyses demonstrated that StoNurA exhibited DNA binding and 5’–3’ exonuclease activities towards ssDNA and dsDNA. The temperature and pH optima of StoNurA were determined to be 65 °C and 8.0, respectively. The activity of StoNurA was found to be dependent of Mn²⁺, and its half-life of heat inactivation at 100 °C was 5 min. Gel filtration chromatography revealed that StoNurA could form dimers in solution. Pull-down assays also showed that StoNurA physically interacted with a DNA helicase (StoHerA). Our data suggest that NurA may play a key functional role in the processing of DNA recombinational repair.

Key words
DNA nuclease; StoNurA; dimer; interaction; recombinational repair

Authorship

Tao Wei

School of Food and Biological Engineering, Zhengzhou University of Light Industry, 5 Dongfeng Rd, Zhengzhou 450002, P.R. ChinaZhengzhou University of Light IndustryChinaZhengzhou, China, ChinaSchool of Food and Biological Engineering, Zhengzhou University of Light Industry, 5 Dongfeng Rd, Zhengzhou 450002, P.R. China

Kunpeng Yang

Jie Zang

Duobin Mao

Correspondence to: Duobin Mao E-mail: duobinmao@126.com

SCIMAGO INSTITUTIONS RANKINGS

Figures | Tables

Figures (6)
Tables (1)

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Figure 1
(a) SDS-PAGE of StoNurA. Lanes M, molecular weight stardards; Lane 1, crude cell extract; Lane 2, supermatant after heat-treatment for 30 minutes at 70 °C; Lane 3, the partially purified enzyme after nickel column; Lane 4, the purified enzyme after gel filtration chromatography. (b) Identification of StoNurA in S. tokodaii cells. S. tokodaii cell extracts (100 µg) and purified StoNurA (50 ng) were seperated by 12% SDS-PAGE gels. These gels were subjected to Western blot analyses with anti-StoNurA polyclonal antibodies to detect StoNurA. (see the colors in the online version).

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Figure 2
Gel filtration analysis of StoNurA using Sephacryl S-200 column. The arrows indicated the eluted positions of StoNurA and protein markers (from left to the right): alcohol dehydrogenase (150 kDa, 54.12 ml), StoNurA (60.85 ml), albumin (66 kDa, 64.58 ml) and carbonic anhydrase (29 kDa, 77.54 ml). (see the colors in the online version).

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Figure 3
Nuclease activities of StoNurA. The reaction mixtures (20 μl) contained increasing amounts (0, 20, 40, 60, 80 and 100 ng) of the StoNurA and 2 pmol ssDNA (a) or dsDNA (b). The sample were incubated at 65 °C for 30 min. The products were resolved on 15% polyacrylamide gels containing 7 M urea and visualized using a FluoImager 585. Each value was calculated based on the results of three independent reactions.

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Figure 4
Biochemical characterization of StoNurA. (a) Effect of pH on the purified StoNurA. Optimal pH of StoNurA at pHs ranging from 6.5 to 9.5 was measured for 30 min at 65 °C. (b) Effect of temperature on the purified StoNurA. Optimal temperature of StoNurA was determined with the ssDNA as substrates in 20 mM of HEPES buffer (pH 8.0) at different temperatures ranging from 45 to 90 °C. (c) Thermostability of StoNurA. The residual enzyme activity was measured after incubation of the purified enzyme at 80 °C (diamonds), 90 °C (boxes), and 100 °C (triangles), respectively. Each value was calculated based on the results of three independent reactions.

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Figure 5
(a) Effect of metal ions on the nuclease activity of the purified StoNurA. The concentration of all metal ions were adjusted to 5 mM. (b) Effect of NaCl concentration on the nuclease activity of the purified StoNurA. The nuclease activities of the treated samples were determined by the standard assay using ssDNA as the substrates. Each value was calculated based on the results of three independent reactions.

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Figure 6
(a) Physical interaction between StoNurA and StoHerA by pull-down assay. Lane 1, molecular size markers; Lane 2, purified His-tagged StoNurA;Lane 3, purified untagged StoHerA; Lane 4, flow-through of the mixture; Lane 5, wash with washing buffer; Lane 6, elution with buffer containing 500 mM imidazole. (b) Sephacryl S-200 gel filtration of purified StoHerA and StoNurA. Purified proteins were incubated alone (StoNurA: top panel, StoHerA: middle panel) or in combination (bottom panel) at 65 °C for 30 min before gel filtration, as indicated, and fractions were analyzed by 12% SDS-PAGE. Migration of protein markers are indicated. (see the colors in the online version).

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TABLE I
Purification of the nuclease StoNurA from S. tokodaii ^a.

Figure 1 (a) SDS-PAGE of StoNurA. Lanes M, molecular weight stardards; Lane 1, crude cell extract; Lane 2, supermatant after heat-treatment for 30 minutes at 70 °C; Lane 3, the partially purified enzyme after nickel column; Lane 4, the purified enzyme after gel filtration chromatography. (b) Identification of StoNurA in S. tokodaii cells. S. tokodaii cell extracts (100 µg) and purified StoNurA (50 ng) were seperated by 12% SDS-PAGE gels. These gels were subjected to Western blot analyses with anti-StoNurA polyclonal antibodies to detect StoNurA. (see the colors in the online version).

Figure 2 Gel filtration analysis of StoNurA using Sephacryl S-200 column. The arrows indicated the eluted positions of StoNurA and protein markers (from left to the right): alcohol dehydrogenase (150 kDa, 54.12 ml), StoNurA (60.85 ml), albumin (66 kDa, 64.58 ml) and carbonic anhydrase (29 kDa, 77.54 ml). (see the colors in the online version).

Figure 3 Nuclease activities of StoNurA. The reaction mixtures (20 μl) contained increasing amounts (0, 20, 40, 60, 80 and 100 ng) of the StoNurA and 2 pmol ssDNA (a) or dsDNA (b). The sample were incubated at 65 °C for 30 min. The products were resolved on 15% polyacrylamide gels containing 7 M urea and visualized using a FluoImager 585. Each value was calculated based on the results of three independent reactions.

Figure 4 Biochemical characterization of StoNurA. (a) Effect of pH on the purified StoNurA. Optimal pH of StoNurA at pHs ranging from 6.5 to 9.5 was measured for 30 min at 65 °C. (b) Effect of temperature on the purified StoNurA. Optimal temperature of StoNurA was determined with the ssDNA as substrates in 20 mM of HEPES buffer (pH 8.0) at different temperatures ranging from 45 to 90 °C. (c) Thermostability of StoNurA. The residual enzyme activity was measured after incubation of the purified enzyme at 80 °C (diamonds), 90 °C (boxes), and 100 °C (triangles), respectively. Each value was calculated based on the results of three independent reactions.

Figure 5 (a) Effect of metal ions on the nuclease activity of the purified StoNurA. The concentration of all metal ions were adjusted to 5 mM. (b) Effect of NaCl concentration on the nuclease activity of the purified StoNurA. The nuclease activities of the treated samples were determined by the standard assay using ssDNA as the substrates. Each value was calculated based on the results of three independent reactions.

Figure 6 (a) Physical interaction between StoNurA and StoHerA by pull-down assay. Lane 1, molecular size markers; Lane 2, purified His-tagged StoNurA;Lane 3, purified untagged StoHerA; Lane 4, flow-through of the mixture; Lane 5, wash with washing buffer; Lane 6, elution with buffer containing 500 mM imidazole. (b) Sephacryl S-200 gel filtration of purified StoHerA and StoNurA. Purified proteins were incubated alone (StoNurA: top panel, StoHerA: middle panel) or in combination (bottom panel) at 65 °C for 30 min before gel filtration, as indicated, and fractions were analyzed by 12% SDS-PAGE. Migration of protein markers are indicated. (see the colors in the online version).

TABLE I Purification of the nuclease StoNurA from S. tokodaii ^a.

Step		Total activity (U)	Specific activity (U/mg)	Purification fold	Yield (%)
Crude cell extract	25.8	5645	218.8	1	100
Heat treatment	15.2	4281	281.6	1.3	75.8
Ni-NTA affinity	4.6	2572	559.1	2.6	45.6
Sephacryl S-200 gel filtration	2.1	1872	891.4	4.1	33.2

^aOne unit of the nuclease activity was defined as the amount of enzyme releasing 1 µmol acid-soluble nucleotides per hour under the assay conditions. The amount of acid-soluble nucleotides was measured according to the conditions described by Curtis et al. (1966)CURTIS PJ, BURDON MG and SMELLIE RMS. 1966. The purification from rat liver of a nuclease hydrolysing ribonucleic acid and deoxyribonucleic acid. Biochem J 98: 813-817..

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<b>Biochemical characterization of the nuclease StoNurA from the hyperthermophilic archaeon</b> Sulfolobus tokodaii

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[1] Correspondence to: Duobin Mao E-mail: duobinmao@126.com