Biochemical characterization of apurinic/apyrimidinic endonuclease Triticum aestivum

Abstract

The gene encoding putative AP-endonuclease of Triticum aestivum was identified by homology to human and Arabidopsis AP-endonucleases by application of tBLASTn NCBI database search tool. For the first time cDNA of the wheat AP-endonuclease gene was isolated by reverse transcription-PCR. Functional expression of TaAPE1L in E.coli was performed and the protein was purified with nickel-based affinity chromatography to homogenous state. Through use of 5’-[P32] – marked oligonucleotide duplexes containing single tetrahydrofuran (analogue of AP-site) AP-endonuclease activity of TaAPE1L was shown. TaApe1L-catalyzed AP endonuclease activity increases depending on incubation time and that addition of EDTA in the reaction mixture completely blocks AP endonuclease activity of TaApe1L