Fish E L, Vournakis J N
Molecular Therapeutics, Inc., West Haven, CT 06516.
Nucleic Acids Res. 1987 Nov 25;15(22):9417-28. doi: 10.1093/nar/15.22.9417.
Deoxyribonuclease I digestion of the deoxyoligodecamer 5'd(ATCGTACGAT)2(3') has been examined in detail to study the kinetic and structural properties of this enzyme substrate system in solution. In addition, these studies have defined, in general, those DNase I conditions to be used in future drug-DNA footprinting experiments. Special attention has been taken of those properties of DNase I that are critical for quantitation of ligand binding to small DNA fragments, and that aid in designing oligomers to be used in footprinting experiments. Enzyme activity was observed at all phosphodiester bonds in the decamer studied with varying affinity, except for the first four bonds at the 5' end of the oligomer. The DNA substrate concentration is always in excess, in order to achieve conditions of no more than one DNase I cleavage per DNA molecule. Reactions were controlled so that 65% or more of the initial amount of decamer substrate remained after DNase I digestion. It was observed that the rate of enzyme reactivity decreases with digestion time and is sensitive to the experimental conditions.
已对脱氧核糖核酸酶I对脱氧寡聚体5'd(ATCGTACGAT)2(3')的消化作用进行了详细研究，以探讨该酶-底物系统在溶液中的动力学和结构特性。此外，这些研究总体上确定了未来药物-DNA足迹实验中要使用的脱氧核糖核酸酶I条件。特别关注了脱氧核糖核酸酶I的那些特性，这些特性对于定量配体与小DNA片段的结合至关重要，并且有助于设计用于足迹实验的寡聚物。在所研究的十聚体中，除了寡聚物5'端的前四个磷酸二酯键外，可以观察到在所有磷酸二酯键处均有不同亲和力的酶活性。DNA底物浓度始终过量，以便实现每个DNA分子不超过一次脱氧核糖核酸酶I切割的条件。控制反应，使得在脱氧核糖核酸酶I消化后，十聚体底物初始量的65%或更多得以保留。观察到酶反应速率随消化时间降低，并且对实验条件敏感。
