Heitman J, Model P
Rockefeller University, New York, NY 10021.
EMBO J. 1990 Oct;9(10):3369-78. doi: 10.1002/j.1460-2075.1990.tb07538.x.
The EcoRI restriction endonuclease cleaves DNA molecules at the sequence GAATTC. We devised a genetic screen to isolate EcoRI mutants with altered or broadened substrate specificity. In vitro, the purified mutant enzymes cleave both the wild-type substrate and sites which differ from this by one nucleotide (EcoRI star sites). These mutations identify four residues involved in substrate recognition and catalysis that are different from the amino acids proposed to recognize the substrate based on the EcoRI-DNA co-crystal structure. In fact, these mutations suppress EcoRI mutants altered at some of the proposed substrate binding residues (R145, R200). We argue that these mutations permit cleavage of additional DNA sequences either by perturbing or removing direct DNA-protein interactions or by facilitating conformational changes that allosterically couple substrate binding to DNA scission.
EcoRI 限制性内切酶在 GAATTC 序列处切割 DNA 分子。我们设计了一种遗传筛选方法来分离具有改变或拓宽底物特异性的 EcoRI 突变体。在体外，纯化的突变酶既能切割野生型底物，也能切割与野生型底物相差一个核苷酸的位点（EcoRI 星号位点）。这些突变鉴定出了四个参与底物识别和催化的残基，它们与基于 EcoRI-DNA 共晶体结构提出的识别底物的氨基酸不同。事实上，这些突变抑制了在一些提议的底物结合残基（R145、R200）处发生改变的 EcoRI 突变体。我们认为，这些突变通过干扰或消除直接的 DNA-蛋白质相互作用，或通过促进构象变化（这种构象变化将底物结合与 DNA 断裂进行变构偶联），从而允许切割额外的 DNA 序列。
