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標題: Arabidopsis ENDO2: Its Catalytic Role and Requirement of N-Glycosylation for Function
作者: Ko, C.Y.
Lai, Y.L.
Liu, W.Y.
Lin, C.H.
Chen, Y.T.
Chen, L.F.O.
Lin, T.Y.
Shaw, J.F.
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.
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.
ISSN: 0021-8561
DOI: 10.1021/jf300945c
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