Yu X, Angov E, Camerini-Otero R D, Egelman E H
Department of Cell Biology and Neuroanatomy, University of Minnesota Medical School, Minneapolis 55455, USA.
Biophys J. 1995 Dec;69(6):2728-38. doi: 10.1016/S0006-3495(95)80144-X.
The Escherichia coli RecA protein has served as a model for understanding protein-catalyzed homologous recombination, both in vitro and in vivo. Although RecA proteins have now been sequenced from over 60 different bacteria, almost all of our structural knowledge about RecA has come from studies of the E. coli protein. We have used electron microscopy and image analysis to examine three different structures formed by the RecA protein from the thermophilic bacterium Thermus aquaticus. This protein has previously been shown to catalyze an in vitro strand exchange reaction at an optimal temperature of about 60 degrees C. We show that the active filament formed by the T. aquaticus RecA on DNA in the presence of a nucleotide cofactor is extremely similar to the filament formed by the E. coli protein, including the extension of DNA to a 5.1-A rise per base pair within this filament. This parameter appears highly conserved through evolution, as it has been observed for the eukaryotic RecA analogs as well. We have also characterized bundles of filaments formed by the T. aquaticus RecA in the absence of both DNA and nucleotide cofactor, as well as hexameric rings of the protein formed under all conditions examined. The bundles display a very large plasticity of mass within the RecA filament, as well as showing a polymorphism in filament-filament contacts that may be important to understanding mutations that affect surface residues on the RecA filament.
大肠杆菌RecA蛋白已成为在体外和体内理解蛋白质催化的同源重组的模型。尽管现在已对60多种不同细菌的RecA蛋白进行了测序，但我们关于RecA的几乎所有结构知识都来自对大肠杆菌蛋白的研究。我们利用电子显微镜和图像分析来研究嗜热细菌嗜热水生栖热菌的RecA蛋白形成的三种不同结构。该蛋白先前已被证明在约60摄氏度的最佳温度下催化体外链交换反应。我们表明，在核苷酸辅因子存在下，嗜热水生栖热菌RecA在DNA上形成的活性细丝与大肠杆菌蛋白形成的细丝极其相似，包括该细丝内DNA延伸至每碱基对5.1埃的上升。通过进化，这个参数似乎高度保守，因为在真核RecA类似物中也观察到了这一点。我们还对在没有DNA和核苷酸辅因子的情况下嗜热水生栖热菌RecA形成的细丝束以及在所研究的所有条件下形成的该蛋白的六聚体环进行了表征。这些细丝束在RecA细丝内显示出非常大的质量可塑性，并且在细丝与细丝的接触中表现出多态性，这可能对理解影响RecA细丝表面残基的突变很重要。
