Sutherland E, Coe L, Raleigh E A
New England Biolabs, Beverly, MA 10915.
J Mol Biol. 1992 May 20;225(2):327-48. doi: 10.1016/0022-2836(92)90925-a.
McrBC-mediated restriction of modified DNA has been studied extensively by genetic methods, but little is known of its molecular action. We have used overproducing plasmid constructs to facilitate purification of the McrBL and McrC proteins, and report preliminary characterization of the activity of the complex. Both proteins are required for cleavage of appropriately modified DNA in vitro, in a reaction absolutely dependent on GTP. ATP inhibits the reaction. The sequence and modification requirements for cleavage of the substrate reflect those seen in vivo. The position of cleavage was examined at the nucleotide level, revealing that cleavage occurs at multiple positions in a small region. Based upon these observations, and upon cleavage of model oligonucleotide substrates, it is proposed that the recognition site for this enzyme consists of the motif RmC(N40-80)RmC, with cleavage occurring at multiple positions on both strands, between the modified C residues. In subunit composition, cofactor requirement, and relation between cleavage and recognition site, McrBC does not fit into any of the classes (types I to IV) of restriction enzyme so far described.
通过遗传学方法已对McrBC介导的修饰DNA限制作用进行了广泛研究，但对其分子作用却知之甚少。我们利用过量表达的质粒构建体来促进McrBL和McrC蛋白的纯化，并报告了该复合物活性的初步表征。两种蛋白在体外切割适当修饰的DNA时都是必需的，该反应绝对依赖于GTP。ATP抑制该反应。底物切割的序列和修饰要求反映了体内观察到的情况。在核苷酸水平上检查了切割位置，发现切割发生在一个小区域的多个位置。基于这些观察结果以及对模型寡核苷酸底物的切割，提出该酶的识别位点由基序RmC(N40 - 80)RmC组成，两条链上在修饰的C残基之间的多个位置发生切割。在亚基组成、辅因子需求以及切割与识别位点之间的关系方面，McrBC不符合迄今为止所描述的任何一类（I型至IV型）限制酶。
