Bhagwat A S, Johnson B, Weule K, Roberts R J
Cold Spring Harbor Laboratory, New York 11724.
J Biol Chem. 1990 Jan 15;265(2):767-73.
The EcoRII endonuclease cleaves DNA containing the sequence CC(A/T)GG before the first cytosine. The methylation of the second cytosine in the sequence by either the EcoRII methylase or Dcm, a chromosomally coded protein in Escherichia coli, inhibits the cleavage. The gene for the EcoRII endonuclease was mapped by analysis of derivatives containing linker insertions, transposon insertions, and restriction fragment deletions. Surprisingly, plasmids carrying the wild-type endonuclease gene and the EcoRII methylase gene interrupted by transposon insertions appeared to be lethal to dcm+ strains of E. coli. We conclude that not all the EcoRII/Dcm recognition sites in the cellular DNA are methylated in dcm+ strains. The DNA sequence of a 1650-base pair fragment containing the endonuclease gene was determined. It revealed an open reading frame that could code for a 45.6-kDa protein. This predicted size is consistent with the known size of the endonuclease monomer (44 kDa). The endonuclease and methylase genes appear to be transcribed convergently from separate promoters. The reading frame of the endonuclease gene was confirmed at three points by generating random protein fusions between the endonuclease and beta-galactosidase, followed by an analysis of the sequence at the junctions. One of these fusions is missing 18 COOH-terminal amino acids of the endonuclease but still displays significant ability to restrict incoming phage in addition to beta-galactosidase activity. No striking similarity between the sequence of the endonuclease and any other protein in the PIR data base was found. The knowledge of the primary sequence of the endonuclease and the availability of the various constructs involving its gene should be helpful in the study of the interaction of the enzyme with its substrate DNA.
EcoRII核酸内切酶在第一个胞嘧啶之前切割含有序列CC(A/T)GG的DNA。EcoRII甲基化酶或Dcm（大肠杆菌中的一种染色体编码蛋白）对该序列中第二个胞嘧啶的甲基化会抑制切割。通过分析含有接头插入、转座子插入和限制性片段缺失的衍生物，对EcoRII核酸内切酶基因进行了定位。令人惊讶的是，携带野生型内切酶基因和被转座子插入中断的EcoRII甲基化酶基因的质粒对大肠杆菌的dcm+菌株似乎是致死的。我们得出结论，在dcm+菌株中，细胞DNA中的并非所有EcoRII/Dcm识别位点都被甲基化。测定了包含内切酶基因的1650个碱基对片段的DNA序列。它揭示了一个开放阅读框，可编码一个45.6 kDa的蛋白质。这个预测大小与已知的内切酶单体大小（44 kDa）一致。内切酶基因和甲基化酶基因似乎从不同的启动子反向转录。通过在核酸内切酶和β-半乳糖苷酶之间产生随机蛋白融合，然后分析接头处的序列，在三个点上证实了核酸内切酶基因的阅读框。这些融合体之一缺失了核酸内切酶18个COOH末端氨基酸，但除了β-半乳糖苷酶活性外，仍显示出显著限制进入噬菌体的能力。在PIR数据库中未发现核酸内切酶序列与任何其他蛋白质之间有明显相似性。核酸内切酶一级序列的知识以及涉及其基因的各种构建体的可得性，应有助于研究该酶与其底物DNA的相互作用。
