title: Effects of 2'-O-methyl nucleotide substitution on EcoRI endonuclease cleavage activities.
abstract: To investigate the effect of sugar pucker conformation on DNA-protein interactions, we used 2'-O-methyl nucleotide (2'-OMeN) to modify the EcoRI recognition sequence -TGAATTCT-, and monitored the enzymatic cleavage process using FRET method. The 2'-O-methyl nucleotide has a C3'-endo sugar pucker conformation different from the C2'-endo sugar pucker conformation of native DNA nucleotides. The initial reaction velocities were measured and the kinetic parameters, Km and Vmax were derived using Michaelis-Menten equation. Experimental results showed that 2'-OMeN substitutions for the EcoRI recognition sequence decreased the cleavage efficiency for A2, A3 and T4 substitutions significantly, and 2'-OMeN substitution for T5 residue inhibited the enzymatic activity completely. In contrast, substitutions for G1 and C6 could maintain the original activity. 2'-fluoro nucleic acid (2'-FNA) and locked nucleic acid (LNA) having similar C3'-endo sugar pucker conformation also demonstrated similar enzymatic results. This position-dependent enzymatic cleavage property might be attributed to the phosphate backbone distortion caused by the switch from C2'-endo to C3'-endo sugar pucker conformation, and was interpreted on the basis of the DNA-EcoRI structure. These 2'-modified nucleotides could behave as a regulatory element to modulate the enzymatic activity in vitro, and this property will have potential applications in genetic engineering and biomedicine.
authors.affiliation: Key Laboratory of Medical Cell Biology (Ministry of Education), Department of Biochemistry and Molecular Biology, China Medical University, Shenyang, China.
authors.forename: Guojie
authors.initials: G
authors.lastname: Zhao
authors.forename: Bin
authors.initials: B
authors.lastname: Zhao
authors.forename: Zhaoxue
authors.initials: Z
authors.lastname: Tong
authors.forename: Runqing
authors.initials: R
authors.lastname: Mu
authors.forename: Yifu
authors.initials: Y
authors.lastname: Guan
mesh_terms: D001483:Base Sequence / Q000235:genetics; D004247:DNA / Q000378:metabolism*; D053837:DNA Cleavage; D015246:Deoxyribonuclease EcoRI / Q000235:genetics* / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D007700:Kinetics; D009841:Oligonucleotides / Q000235:genetics
chemical_list: D009841:Oligonucleotides; D004247:DNA; D015246:Deoxyribonuclease EcoRI