title: Enzymatic cleavage of type II restriction endonucleases on the 2'-O-methyl nucleotide and phosphorothioate substituted DNA.
abstract: The effects of nucleotide analogue substitution on the cleavage efficiencies of type II restriction endonucleases have been investigated. Six restriction endonucleases (EcoRV, SpeI, XbaI, XhoI, PstI and SphI) were investigated respectively regarding their cleavage when substrates were substituted by 2'-O-methyl nucleotide (2'-OMeN) and phosphorothioate (PS). Substitutions were made in the recognition sequence and the two nucleotides flanking the recognition sequence for each endonuclease. The endonuclease cleavage efficiencies were determined using FRET-based assay. Results demonstrated a position-dependent inhibitory effect of substitution on the cleavage efficiency for all the six endonucleases. In general, the 2'-OMeN substitutions had greater impact than the PS substitutions on the enzymatic activities. Nucleotides of optimal substitutions for protection against RE cleavage were identified. Experimental results and conclusions in this study facilitate our insight into the DNA-protein interactions and the enzymatic cleavage mechanism, particularly for those whose detailed structure information is not available. In addition, the information could benefit the development of bioengineering and synthetic biology.
authors.affiliation: Key Laboratory of Medical Cell Biology, Ministry of Education, Department of Biochemistry and Molecular Biology, China Medical University, Shenyang, Liaoning, China.
authors.forename: Guojie
authors.initials: G
authors.lastname: Zhao
authors.forename: Jun
authors.initials: J
authors.lastname: Li
authors.forename: Zhaoxue
authors.initials: Z
authors.lastname: Tong
authors.forename: Bin
authors.initials: B
authors.lastname: Zhao
authors.forename: Runqing
authors.initials: R
authors.lastname: Mu
authors.forename: Yifu
authors.initials: Y
authors.lastname: Guan
mesh_terms: D004247:DNA / Q000737:chemistry* / Q000378:metabolism*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism*; D009711:Nucleotides / Q000737:chemistry* / Q000378:metabolism*; D010710:Phosphates / Q000737:chemistry; D011485:Protein Binding
chemical_list: D009711:Nucleotides; D010710:Phosphates; C083120:phosphorodithioic acid; D004247:DNA; D015252:Deoxyribonucleases, Type II Site-Specific