Yu X, Shibata T, Egelman E H
Department of Cell Biology and Neuroanatomy, University of Minnesota Medical School, Minneapolis, MN, 55455, USA.
J Mol Biol. 1998 Nov 13;283(5):985-92. doi: 10.1006/jmbi.1998.2141.
Studies of the Escherichia coli RecA protein are expected to illuminate mechanisms of DNA recombination and repair in bacteria, and in all higher organisms as well, due to the functional and structural homology with the eukaryotic Rad51 protein. The active form of the RecA protein is a helical filament formed on DNA in the presence of ATP or ATP analogs, and this has been studied at low-resolution by electron microscopy. An atomic model of the protein comes from an X-ray crystallographic study of a filament formed in the absence of DNA and ATP. This filament is believed to be an inactive, storage form of the protein. A key step in generating an atomic model of the active filament, and a detailed model for function, is to understand the large conformational changes that occur between these two states. Towards this end, we have decorated active RecA-DNA filaments with monoclonal antibodies (ARM191) against a known epitope (residues 285 to 320) to determine the position of this epitope in the low-resolution structure. Electron microscopy and three-dimensional reconstruction of the RecA-antibody complex reveal that the lobe containing the epitope is very disordered on the surface of the filament, but in a position similar to that in the inactive crystal filament. The antibody binding also induces a significant conformational change in the RecA filament. This study shows that the basic orientation of the subunit is likely to be similar within the inactive and active filaments, and that the large movement of mass that occurs between these two states must involve other residues than the 285-320 region.
由于大肠杆菌RecA蛋白与真核生物Rad51蛋白在功能和结构上具有同源性，对其研究有望阐明细菌以及所有高等生物中DNA重组和修复的机制。RecA蛋白的活性形式是在ATP或ATP类似物存在下在DNA上形成的螺旋丝，这已通过电子显微镜在低分辨率下进行了研究。该蛋白的原子模型来自对在没有DNA和ATP的情况下形成的丝的X射线晶体学研究。这种丝被认为是该蛋白的无活性储存形式。生成活性丝原子模型以及详细功能模型的关键一步是了解这两种状态之间发生的巨大构象变化。为此，我们用针对已知表位（残基285至320）的单克隆抗体（ARM191）修饰活性RecA-DNA丝，以确定该表位在低分辨率结构中的位置。RecA-抗体复合物的电子显微镜和三维重建显示，含有该表位的叶在丝表面非常无序，但处于与无活性晶体丝中相似的位置。抗体结合还会在RecA丝中诱导显著的构象变化。这项研究表明，亚基的基本取向在无活性丝和活性丝中可能相似，并且这两种状态之间发生的大量质量移动必定涉及285-320区域以外的其他残基。
