Recombinant thermostable AP exonuclease from Thermoanaerobacter tengcongensis: cloning, expression, purification, properties and PCR application

• Autorzy: Dabrowski S. , Brillowska-Dabrowska A. , Ahring B. K.
• Języki publikacji: PL

Abstrakty

Apurinic/apyrimidinic (AP) sites in DNA are considered to be highly mutagenic and must be corrected to preserve genetic integrity, especially at high temperatures. The gene encoding a homologue of AP exonuclease was cloned from the thermophilic anaerobic bacterium Thermoanaerobacter tengcongensis and transformed into Escherichia coli. The protein product showed high identity (80%) to human Ape1 nuclease, whereas to E.coli exonuclease III – 78%. This is the first prokaryotic AP nuclease that exhibits such high identity to human Ape1 nuclease. The very high expression level (57% of total soluble proteins) of fully active and soluble His₆ -tagged Tte AP enzyme with His₆ -tag on C-terminal end was obtained in Escherichia coli Rosetta (DE3) pLysS. The active enzyme was purified up to 98% homogeneity in one chromatographic step using metal-affinity chromatography on Ni²⁺-IDA-Sepharose resin. The yield was 90 mg (14 000 kU) of pure His₆ -tagged Tte AP (153 kU/mg) from 1 liter of culture. The optimal conditions of Tte AP endo-, exonuclease and 3’-nuclease activity were investigated using fluorescein labeled dsDNA with inserted AP sites and ssDNA. Optimal Tte AP endonuclease activity was observed at 70–75°C, pH 8.0 and at low Mg²⁺ concentration (0.5 mM). Higher Mg2+ concentration (> 1 mM) enhanced 3’-5’ exonuclease activity and at Mg²⁺ concentration > 2.0 mM 3’ nuclease activity was observed.Because of the endonuclease activity of Tte AP exonuclease, the enzyme was applied in PCR amplification of long DNA templates. Tte AP exonuclease eliminated AP-sites in DNA template and improved the efficiency of DNA amplification.

Słowa kluczowe

EN

exonuclease; Thermoanaerobacter tengcongensis; cloning; expression; purification; DNA damage; polymerase chain reaction; application; AP endonuclease

• Wydawca: -
• Czasopismo: Polish Journal of Microbiology
• Rocznik: 2013
• Tom: 62
• Numer: 2
• Opis fizyczny: p.121-129,fig.,ref.

Twórcy

autor

Dabrowski S.

• The Environmental Microbiology and Biotechnology Research Group, BioCentrum, Soltofts Plads, Building 227, Technical University of Denmark, DK-2800 Kgs. Lyngby, Denmark

autor

Brillowska-Dabrowska A.

• Department of Microbiology, Gdansk University of Technology, Gdansk, Poland

autor

Ahring B. K.

• The Environmental Microbiology and Biotechnology Research Group, BioCentrum, Soltofts Plads, Building 227, Technical University of Denmark, DK-2800 Kgs. Lyngby, Denmark

Bibliografia

• Bao Q., Y. Tian, W. Li, Z Xu, Z. Xuan, S. Hu, W. Dong, J. Yang, Y. Chen, Y. Xue and others. 2002. A complete sequence of the T. tengcongensis genome. Genome Res. 12: 689–700.
• Bennett R.A. 1999. The Saccharomyces cerevisiae ETH1 gene, an inducible homolog of exonuclease III that provides resistance to DNA-damaging agents and limits spontaneous mutagenesis. Mol. Cell. Biol. 19: 1800–1809.
• Bradford M.M. 1976 A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analyt. Biochem. 72: 248–254.
• Brinkmann U., R.E. Mattes and P. Buckel. 1989. High-level expression of recombinant genes in Escherichia coli is dependent on the availability of the dnaY gene product. Gene 85: 109–114.
• Carpenter E.P., Corbett A., Thomson H., Adacha J., Jensen K., Bergeron J., Kasampalidis I., Exley R., Winterbotham M., Tang C., Baldwin G.S., Freemont P. 2007. AP endonuclease paralogues with distinct activities in DNA repair and bacterial pathogenesis. The EMBO Journal 26: 1363–1372.
• Chou K.M., M. Kukhanova and Y.C. Cheng. 2000. A novel action of human apurinic/apyrimidinic endonuclease: excision of L-configuration deoxyribonucleoside analogs from the 3’ termini of DNA. J. Biol. Chem. 275: 31009–31015.
• Dabrowski S. and B.K. Ahring. 2003. Cloning, expression, and purification of the His6-tagged hyper-thermostable dUTPase from Pyrococcus woesei in Escherichia coli: application in PCR. Protein Expr. Purif. 31: 72–78.
• Dabrowski S. and J. Kur. 1998. Cloning and expression in Escherichia coli of the recombinant his-tagged DNA polymerases from Pyrococcus furiosus and Pyrococcus woesei. Protein Expr. Purif. 14: 131–138.
• Demple B. and L. Harrison. 1994. Repair of oxidative damage to DNA: enzymology and biology. Annu. Rev. Biochem. 63: 915–948.
• Dubendorff J.W. and F.W. Studier. 1991. Controlling basal expression in an inducible T7 expression system by blocking the target T7 promoter with lac repressor. J. Mol. Biol. 219: 45–59.
• Fromenty B., C. Demeilliers, A. Mansouri and D. Pessayre. 2000. Escherichia coli exonuclease III enhances long PCR amplification of damaged DNA templates. Nucleic. Acids. Res. 28: E50.
• Guo L.H. and R. Wu. 1982. New rapid methods for DNA sequencing based in exonuclease III digestion followed by repair synthesis. Nucleic. Acids. Res. 10: 2065–2084.
• Hadi M.Z., K. Ginalski, L.H. Nguyen and D.M. Wilson. 2002. Determinants in nuclease specificity of Ape1 and Ape2, human homologues of Escherichia coli exonuclease III. J. Mol. Biol. 316: 853–866.
• Hadi M.Z. and D.M. Wilson. 2000. Second human protein with homology to the Escherichia coli abasic endonuclease exonuclease III. Environ. Mol. Mutagen. 36: 312–324.
• Henikoff S. 1984. Unidirectional digestion with exonuclease III creates targeted breakpoints for DNA sequencing. Gene 28: 351–359.
• Johnson R.E., C. A. Torres-Ramos, T. Izumi, S. Mitra, S. Prakash and L. Prakash. 1998. Identification of APN2, the Saccharomyces cerevisiae homolog of the major human AP endonuclease HAP1, and its role in the repair of abasic sites. Genes Dev. 12: 3137–3143.
• Kneller D.G., F.E. Cohen. and R. Langridge. 1990. Improvements in protein secondary structure prediction by an enhanced neural network. J. Mol. Biol. 214: 171–182.
• Lebedeva N.A., S.N. Khodyreva, A. Favre and O.I. Lavrik. 2003. AP endonuclease 1 has no biologically significant 3’-5’-exonuclease activity. Biochem. Biophys. Res. Commun. 300: 182–187.
• McClelland J.L. and D.E. Rumelhart. 1988. Explorations in Parallel Distributed Processing. Vol. 3. pp. 316–362. MIT Press, Cambridge, MA.
• Moffatt B.A. and P.K. Pfaffle. 1995. Single-step purification of a thermostable DNA polymerase expressed in Escherichia coli. Biotechniques 19: 780–784.
• Novy R., D. Drott, K. Yaeger and R. Mierendorf. 2001. Overcoming the codon bias of E.coli for enhanced protein expression. inNovations 12: 1–3.
• Novy R. and B. Morris. 2001. Use of glucose to control basal expression in the pET System. inNovations 13: 8–10.
• Porath J. and B. Olin. 1985. Affinity adsorption and immobilized metal ion chromatography of biomaterials. Serum protein affinities for gel-immobilized iron and nickel ions. Biochemistry 22: 1621–1630.
• Richardson C.C. and A.A. Kornberg. 1964. DNA phosphatase-exonuclease from Escherichia coli. II. Characterization of the exonuclease activity. J. Biol. Chem. 239: 251–258.
• Saitou N. and Nei M. 1987. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4: 406–425.
• Schmiedel R., E.B. Kuettner, A. Keim, N. Sträter and T. Greiner-Stöffele. 2008. Structure and function of the abasic site specificity pocket of an AP endonuclease from Archaeoglobus fulgidus. DNA Repair (Amst). 8: 219–31.
• Seidel H.M., D.L. Pompliano and J.R. Knowes. 1992. Phosphonate biosynthesis: molecular cloning of the gene for phosphoenolpyruvate mutase from Tetrahymena pyriformis and overexpression of the gene product in Escherichia coli. Biochemistry 31: 2598–2608.
• Seki S., M. Hatsushika, S. Watanabe, K. Akiyama, K. Nagao and K. Tsutsui. 1992. cDNA cloning, sequencing, expression and possible domain structure of human APEX nuclease homologous to Esche­richia coli exonuclease III. Biochim. Biophys. Acta 1131: 287–299.
• Shida T., K. Kaneda, T. Ogawa and J. Sekiguchi. 1999. Abasic site recognition mechanism by the Escherichia coli exonuclease III. Nucleic. Acids. Symp. Ser. 42: 195–196.
• Studier F.W. and B. A. Moffatt. 1986. Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J. Mol. Biol. 189: 113–130.
• Suh D., D.M. Wilson and L.F. Povirk. 1997. 3’-phosphodiesterase activity of human apurinic/apyrimidinic endonuclease at DNA double-strand break ends. Nucleic. Acids. Res. 25: 2495–2500.
• Unk I., L. Haracska, R.E. Johnson, S. Prakash and L. Prakash. 2000. Apurinic endonuclease activity of yeast Apn2 protein. J. Biol. Chem. 275: 22427–22434.
• Unk I., L. Haracska, S. Prakash and L. Prakash. 2001. 3’-phosphodiesterase and 3’-5’ exonuclease activities of yeast Apn2 protein and requirement of these activities for repair of oxidative DNA damage. Mol Cell Biol 21: 1656–1661.
• Vandeyar M.A., M.P. Weiner, C.J. Hutton and C.A. Batt. 1988. A simple and rapid method for the selection of oligodeoxynucleotide-directed mutants. Gene 65: 129–133.
• Wilson D.M. and D. Barsky. 2001. The major human abasic endonuclease: formation, consequences and repair of abasic lesions in DNA. Mutat. Res. 485: 283–307.
• Wilson D.M., B. Engleward and L. Samson. 1998. Procariotic base excision repair. In DNA Damage and Repair pp. 29–64. Humana Press, Inc., Totowa, NJ.
• Wilson D.M., M. Takeshita, A.P. Grollman and B. Demple. 1995. Incision activity of human apurinic endonuclease (Ape) at abasic site analogs in DNA. J. Biol. Chem. 270: 16002–16007.
• Xue Y., Y. Xu, Y. Liu, Y. Ma and P. Zhou. 2001. Thermoanaerobacter tengcongensis sp. nov., a novel anaerobic, saccharolytic, thermophilic bacterium isolated from a hot spring in Tengcong, China. Int. J. Syst. Evol. Microbiol. 51: 1335–1341.