Pein C D, Reuter M, Meisel A, Cech D, Krüger D H
Institute of Bioorganic Chemistry, Humboldt University, Berlin, FRG.
Nucleic Acids Res. 1991 Oct 11;19(19):5139-42. doi: 10.1093/nar/19.19.5139.
The restriction endonuclease EcoRII is unable to cleave DNA molecules when recognition sites are very far apart. The enzyme, however can be activated in the presence of DNA molecules with a high frequency of EcoRII sites or by oligonucleotides containing recognition sites: Addition of the activator molecules stimulates cleavage of the refractory substrate. We now show that endonucleolysis of the stimulator molecules is not a necessary prerequisite of enzyme activation. A total EcoRII digest of pBR322 DNA or oligonucleotide duplexes with simulated EcoRII ends (containing the 5' phosphate group), as well as oligonucleotide duplexes containing modified bases within the EcoRII site, making them resistant to cleavage, are all capable of enzyme activation. For activation EcoRII requires the interaction with at least two recognition sites. The two sites may be on the same DNA molecule, on different oligonucleotide duplexes, or on one DNA molecule and one oligonucleotide duplex. The efficiency of functional intramolecular cooperation decreases with increasing distance between the sites. Intermolecular site interaction is inversely related to the size of the stimulator oligonucleotide duplex. The data are in agreement with a model whereby EcoRII simultaneously interacts with two recognition sites in the active complex, but cleavage of the site serving as an allosteric activator is not necessary.
当识别位点相距很远时，限制性内切酶EcoRII无法切割DNA分子。然而，在具有高频率EcoRII位点的DNA分子存在下或通过含有识别位点的寡核苷酸，该酶可以被激活：添加激活剂分子会刺激难切割底物的切割。我们现在表明，刺激分子的核酸内切不是酶激活的必要前提条件。pBR322 DNA或具有模拟EcoRII末端（含有5'磷酸基团）的寡核苷酸双链体的完全EcoRII消化产物，以及在EcoRII位点内含有修饰碱基从而使其抗切割的寡核苷酸双链体，都能够激活该酶。为了激活，EcoRII需要与至少两个识别位点相互作用。这两个位点可以在同一个DNA分子上、不同的寡核苷酸双链体上，或者一个DNA分子和一个寡核苷酸双链体上。功能性分子内协同作用的效率随着位点之间距离的增加而降低。分子间位点相互作用与刺激寡核苷酸双链体的大小呈负相关。这些数据与一个模型相符，即EcoRII在活性复合物中同时与两个识别位点相互作用，但作为变构激活剂的位点的切割不是必需的。
