Flores H, Osuna J, Heitman J, Soberón X
Department of Molecular Biology, Instituto de Biotecnología/UNAM, Cuernavaca, Mor, México.
Gene. 1995 May 19;157(1-2):295-301. doi: 10.1016/0378-1119(94)00863-n.
EcoRI recognizes and cleaves DNA at GAATTC sites and is one of the best characterized sequence-specific restriction endonucleases (ENases). In previous studies, an EcoRI mutant, which exhibited relaxed substrate specificity and cleaved both canonical and EcoRI star sites, was isolated. This mutant enzyme has Tyr instead of His114. Here, we subjected residue 114 of the EcoRI ENase to saturation mutagenesis. The resulting mutant enzymes were characterized both in vivo and in vitro, resulting in the identification of mutants with canonical (H114K, Q, D, I) or relaxed (H114Y, F, S, T) specificity, as well as one mutant with severely impaired activity (H114P). In the X-ray structure of an EcoRI-substrate complex, His114 is located between the catalytic and recognition regions of EcoRI and may directly contact the DNA phosphate backbone. Based on our genetic and biochemical findings and the X-ray structure, we propose that His114 participates in substrate recognition and catalysis, either directly, via protein-DNA interactions, or indirectly, by mediating conformational changes that trigger DNA cleavage in response to substrate recognition.
EcoRI可识别GAATTC位点并在该位点切割DNA，它是特征最为明确的序列特异性限制性内切酶（ENases）之一。在之前的研究中，分离出了一种EcoRI突变体，该突变体表现出宽松的底物特异性，能够切割标准位点和EcoRI星号位点。这种突变酶的第114位氨基酸是酪氨酸而非组氨酸。在此，我们对EcoRI内切酶的第114位残基进行了饱和诱变。对产生的突变酶进行了体内和体外表征，从而鉴定出具有标准特异性（H114K、Q、D、I）或宽松特异性（H114Y、F、S、T）的突变体，以及一个活性严重受损的突变体（H114P）。在EcoRI-底物复合物X射线结构中，组氨酸114位于EcoRI的催化区域和识别区域之间，可能直接与DNA磷酸主链接触。基于我们的遗传学和生物化学研究结果以及X射线结构，我们提出组氨酸114通过蛋白质-DNA相互作用直接参与底物识别和催化，或者通过介导构象变化间接参与，该构象变化响应底物识别触发DNA切割。
