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|標題:||Arabidopsis ENDO2: Its Catalytic Role and Requirement of N-Glycosylation for Function||作者:||Ko, C.Y.
|關鍵字:||N-glycosylation;ENDO2;single-strand-preferring nuclease;mismatch;specific endonuclease;programmed cell death;programmed cell-death;strand-specific nucleases;penicillium-citrinum;substrate-specificity;p1 nuclease;dna;identification;endonuclease;purification;thaliana||Project:||Journal of Agricultural and Food Chemistry||期刊/報告no：:||Journal of Agricultural and Food Chemistry, Volume 60, Issue 20, Page(s) 5169-5179.||摘要:||
The Arabidopsis thaliana At1g68290 gene encoding an endonuclease was isolated and designated ENDO2, which was cloned into a binary vector to overexpress ENDO2 with a C-terminal 6 x His-tag in A. thaliana. Our Arabidopsis transgenic lines harboring 35SP::ENDO2 produced stable active enzyme with high yield. The protein was affinity purified from transgenic plants, and its identity was confirmed by liquid chromatography-mass spectrometry and automatic Edman degradation. ENDO2 enzyme digests RNA, ssDNA, and dsDNA, with a substrate preference for ssDNA and RNA. The activity toward ssDNA (361.7 U/mg) is greater than its dsDNase activity (14.1 U/mg) at neutral pH. ENDO2 effectively cleaves mismatch regions in heteroduplex DNA containing single base pair mismatches or insertion/deletion bases and can be applied to high-throughput detection of single base mutation. Our data also validated that the removal of sugar groups from ENDO2 strongly affects its enzymatic stability and activity.
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