| 摘 要： | To further understand the ligation mechanism, effects of 2＇-O-methyl nucleotide （2＇-OMeN） on the T4 DNA ligation efficiency were investigated. Fluorescence resonance energy transfer assay was used to monitor the nick-joining process by T4 DNA ligase. Results showed that substitutions at 5＇- and 3＇-ends of the nick decreased the ligation efficiency by 48.7% ± 6.7% and 70.6% ±4.0%, respectively. Substitutions at both 5＇- and 3＇-ends decreased the ligation efficiency by 76.6%±1.3%. Corresponding kinetic para- meters, Vmax, Kin, and kcat, have been determined in each case by using the Michaelis-Menten equation. The kinetic data showed that the 2＇-OMeN substitutions reduced the maximal initial velocity and increased the Michaelis constant of T4 DNA ligase. Mismatches at 5＇- and 3＇-ends of the nick have also shown different influences on the ligation. Results here showed that the sugar pucker conformation at 3＇-end impairs the ligation efficiency more profoundly than that at 5＇-end. Different concentrations of Mg2＋, Ca2＋, K＋, Na＋, and ATP were also demonstrated to affect the T4 DNA ligase activ- ity. These results enriched our knowledge about the effects of 2＇-OMEN substitutions on the T4 DNA ligase.
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