title: Arabidopsis ENDO2: its catalytic role and requirement of N-glycosylation for function.
abstract: 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 × 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.
authors.affiliation: Institute of Bioinformatics and Structural Biology,  National Tsing Hua University , Hsinchu, 30013, Taiwan.
authors.forename: Chia-Yun
authors.initials: CY
authors.lastname: Ko
authors.forename: Yi-Ling
authors.initials: YL
authors.lastname: Lai
authors.forename: Wen-Yu
authors.initials: WY
authors.lastname: Liu
authors.forename: Chia-Hui
authors.initials: CH
authors.lastname: Lin
authors.forename: Yu-Ting
authors.initials: YT
authors.lastname: Chen
authors.forename: Long-Fang O
authors.initials: LF
authors.lastname: Chen
authors.forename: Tsai-Yun
authors.initials: TY
authors.lastname: Lin
authors.forename: Jei-Fu
authors.initials: JF
authors.lastname: Shaw
mesh_terms: D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics; D029681:Arabidopsis Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D004277:DNA, Single-Stranded / Q000378:metabolism; D004720:Endonucleases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006031:Glycosylation; D008969:Molecular Sequence Data; D030821:Plants, Genetically Modified / Q000201:enzymology / Q000235:genetics; D012313:RNA / Q000378:metabolism; D016415:Sequence Alignment; D013379:Substrate Specificity
chemical_list: D029681:Arabidopsis Proteins; D004277:DNA, Single-Stranded; D012313:RNA; C574857:ENDO2 protein, Arabidopsis; D004720:Endonucleases