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.
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