Gubler M, Bickle T A
Microbiology Department, Biozentrum, University of Basel, Switzerland.
EMBO J. 1991 Apr;10(4):951-7. doi: 10.1002/j.1460-2075.1991.tb08029.x.
We have investigated the role of a four amino acid element that is repeated twice and three times, respectively, in the specificity polypeptides of the two allelic restriction-modification systems EcoR124 and EcoR124/3. We had earlier shown that this difference in amino acid sequence between the two systems is solely responsible for the different DNA sequence specificities of the two systems. The effect of single amino acid substitutions and small insertion and deletion mutations on restriction activity and modification specificity was determined in vivo by phage infection assays and in vitro by methylation of DNA with purified modification methylases. Mutant restriction-modification systems with changes in the number and the length of the central amino acid repeats exhibited decreased restriction activity and in some cases relaxed substrate specificity. Our data strongly support the idea that the repetitive amino acid motif in the specificity polypeptides forms part of a flexible interdomain linker. It may be responsible for positioning on the DNA the two major specificity polypeptide domains which are thought to contact independently the half sites of the split recognition sequences typical for all type I restriction-modification systems.
我们研究了一种四氨基酸元件的作用，该元件在两种等位基因限制修饰系统EcoR124和EcoR124/3的特异性多肽中分别重复出现两次和三次。我们之前已经表明，这两种系统之间氨基酸序列的差异是造成这两种系统不同DNA序列特异性的唯一原因。通过噬菌体感染试验在体内以及用纯化的修饰甲基化酶对DNA进行甲基化反应在体外确定了单个氨基酸取代以及小的插入和缺失突变对限制活性和修饰特异性的影响。中心氨基酸重复序列的数量和长度发生变化的突变限制修饰系统表现出限制活性降低，在某些情况下底物特异性也有所放宽。我们的数据有力地支持了这样一种观点，即特异性多肽中的重复氨基酸基序构成了一个灵活的结构域间连接子的一部分。它可能负责将两个主要的特异性多肽结构域定位在DNA上，据认为这两个结构域独立地与所有I型限制修饰系统典型的分裂识别序列的半位点相接触。
