title: 2'-O-methyl nucleotide modified DNA substrates influence the cleavage efficiencies of BamHI and BglII.
abstract: Induction of endonucleolytic DNA cleavage is an essential event that links the initiating stimuli to the final effects of cells. The cleavage efficiency and thus the final yield could be affected by many factors, including structures of DNA substrates, composite structures of enzymes-substrates or enzymes-nucleic analogs and so on. However, it is not clear whether a nucleotide derivative-substituted in DNA substrates can influence the efficiency of enzymatic cleavage. To investigate the effect of sugar pucker conformation on DNA-protein interactions, we used 2'-O-methyl modified nucleotides (OMeN) to modify DNA substrates of isocaudemers BamHI and BglII in this study, and used FRET assay as an efficient method for analysis of enzyme cleavage. Experimental results demonstrated that OMeN-substituted recognition sequences influenced the cleavage rates significantly in a position-dependent manner. OMeN substitutions can reduce the cleavage as expected. Surprisingly, OMeN substitutions can also enhance the cleavage rates. The kinetics parameters of Vmax and Km have been obtained by fitting the Michaelis-Menten kinetic equation. These 2'- OMe nucleotides could behave as a regulatory element to modulate the enzymatic activity in vitro, and this property could enrich our understanding about the endonuclease cleavage mechanism and enhance our ability to regulate the enzymatic cleavage efficiency for applications in 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 110001.
authors.forename: Zhaoxue
authors.initials: Z
authors.lastname: Tong
authors.forename: Bin
authors.initials: B
authors.lastname: Zhao
authors.forename: Guojie
authors.initials: G
authors.lastname: Zhao
authors.forename: Hong
authors.initials: H
authors.lastname: Shang
authors.forename: Yifu
authors.initials: Y
authors.lastname: Guan
mesh_terms: D000241:Adenosine / Q000031:analogs & derivatives / Q000737:chemistry; D001426:Bacterial Proteins / Q000378:metabolism*; D003562:Cytidine / Q000031:analogs & derivatives / Q000737:chemistry; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D053837:DNA Cleavage; D015245:Deoxyribonuclease BamHI / Q000378:metabolism*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D006151:Guanosine / Q000031:analogs & derivatives / Q000737:chemistry; D007700:Kinetics; D009841:Oligonucleotides / Q000235:genetics; D013936:Thymidine / Q000031:analogs & derivatives / Q000737:chemistry
chemical_list: C520288:2'-O-methylthymidine; D001426:Bacterial Proteins; D009841:Oligonucleotides; C024341:2'-O-methyladenosine; D006151:Guanosine; C052203:2'-O-methylcytidine; D003562:Cytidine; D004247:DNA; D015245:Deoxyribonuclease BamHI; C024404:BglII endonuclease; D015252:Deoxyribonucleases, Type II Site-Specific; D000241:Adenosine; D013936:Thymidine; C024900:2'-O-methylguanosine