Windolph S, Fritz A, Oelgeschläger T, Wolfes H, Alves J
Zentrum Biochemie, Medizinische Hochschule Hannover, D-30623 Hannover, Germany.
Biochemistry. 1997 Aug 5;36(31):9478-85. doi: 10.1021/bi970076g.
We have generated several EcoRI mutants which exhibit a decreased cleavage rate on one of the five specific cleavage sites in bacteriophage lambda-DNA. To study the influence of the sequence context on the cleavage rate in more detail, we developed a site selection assay. From a complete set of 4096 plasmid substrates, differing in three bases on both sides of a recognition sequence, optimal (best cut) and unfavorable (worst cut) sequences were selected by repeated limited digestion, separation, and in vivo amplification of cleaved and uncleaved plasmids. In order to compare the sequence preferences of the inner arm mutant K130E and the wild type enzyme, the cleavage rates and sequences of individual plasmids from the resulting pools were determined. The inner arm mutant K130E selected pools with clearly defined consensus sequences and a high amount of palindromic sequences. The cleavage rates of the selected sequences are specific for the K130E mutant as is shown by their cleavage with other mutants. In contrast, wild type EcoRI does not lead to a selection in this assay. Pre-steady state kinetics show that preferences for a certain sequence context are a result of differences in the dissociation rates of the wild type enzyme. EcoRI is evolved to efficiently recognize and cleave each nonmethylated DNA invading the cell. Therefore, a fast dissociation after cleavage is not mandatory.
我们已经构建了几种EcoRI突变体，它们对噬菌体λ-DNA的五个特定切割位点之一的切割速率有所降低。为了更详细地研究序列背景对切割速率的影响，我们开发了一种位点选择测定法。从一套完整的4096个质粒底物中，这些底物在识别序列两侧的三个碱基上有所不同，通过对切割和未切割质粒进行重复的有限消化、分离和体内扩增，选择了最佳（切割效果最好）和不利（切割效果最差）的序列。为了比较内臂突变体K130E和野生型酶的序列偏好，测定了所得文库中各个质粒的切割速率和序列。内臂突变体K130E选择的文库具有明确界定的共有序列和大量的回文序列。所选序列的切割速率对K130E突变体具有特异性，这通过它们与其他突变体的切割情况得以证明。相比之下，野生型EcoRI在该测定中不会导致选择。稳态前动力学表明，对特定序列背景的偏好是野生型酶解离速率差异的结果。EcoRI经过进化以有效识别和切割侵入细胞的每个非甲基化DNA。因此，切割后快速解离并非必需。
