EcoRI核酸限制性内测酶切割Ad_5-DNA的动力学研究

本文用荧光扫描的技术研究了腺病毒5一型(Ad_5)DNA被EcoRI内切酶切割的动力学。这种技术能够定量地测定在琼脂糖凝胶上经溴乙锭染色的分离DNA片段的荧光强度,而且能根据各片段的荧光强度对切割时间的依赖关系,求得相应切割位点的切割速度。我们发现同一DNA分子上各切割位点的切割速度有着很大的差异,而且总的切割速度和酶—底物复合物的解离速度对酶浓度和温度有着明显的依赖性。切点两侧的碱基成分d(G-C)/d(A-T)比率高,则要求较多的活化能。

Kinetic Studies on Cleavage of Adenovirus Type 5 DNA by Restriction Endonuclease EcoRI

The two EcoRI restriction sites on Adenovirus Type 5 DNA have been affirmed and relative lenghts of EcoRI digest products of Ad_5DNA are 77%, 7%,16%for the fragment A, C,B respectively. The kinetic of clea- vage of Ad_5 DNA by EcoRI was studied with using quantitative evalua- tion of the ethidium bromide fluorescence of DNA fragments separated on agarose gel. The overall rate constants of cleavage at different EcoRI site have been determined, The rate constants for two cleavage sites depend on the enzyme concentration and reaction temperature. The modynamic studies have shed light on the importance of the nucleotide sequences around the restriction sites, which modulate the activation barriers for the cleavage process itself.