Frykholm Karolin, Morimatsu Katsumi, Nordén Bengt
Department of Chemical and Biological Engineering, Chalmers University of Technology, SE-412 96 Göteborg, Sweden.
Biochemistry. 2006 Sep 19;45(37):11172-8. doi: 10.1021/bi060621q.
RecA protein and its eukaryotic homologue Rad51 protein catalyzes the DNA strand exchange, which is a key reaction of homologous recombination. At the initial step of the reaction, RecA proteins form a helical filament on a single-stranded DNA (ssDNA). Binding of double-stranded DNA (dsDNA) to the filament triggers the homology search; as homology is found, the exchange of strands occurs, and the displaced DNA is released. These are the principal steps of genetic recombination; however, despite many years of extensive study of RecA activities, the details of the mechanism are still obscure. A high-resolution structure of the active nucleoprotein filament could provide information to help understand this process. Using a linear dichroism polarized-light spectroscopy technique, in combination with protein engineering (the site-specific linear dichroism method), we have previously studied the arrangement of RecA in complex with ssDNA. In the present study, we have used this approach to search for structural variations of RecA at the atomic level as the DNA in the complex is changed from ssDNA to dsDNA. The structural data of the RecA-dsDNA filament are found to be very similar to the data previously obtained for the RecA-ssDNA complex, indicating that the overall orientation and also the internal structure of RecA in the active filament are not markedly altered when the bound DNA changes from single- to double-stranded. The implications of the structural similarities as well as the significance of some conformational variations observed for a few amino acid residues that may be involved in interactions with DNA are discussed.
RecA蛋白及其真核同源物Rad51蛋白催化DNA链交换，这是同源重组的关键反应。在反应的初始阶段，RecA蛋白在单链DNA（ssDNA）上形成螺旋丝。双链DNA（dsDNA）与该丝的结合引发同源性搜索；当发现同源性时，发生链交换，被置换的DNA被释放。这些是基因重组的主要步骤；然而，尽管对RecA活性进行了多年的广泛研究，其机制的细节仍然不清楚。活性核蛋白丝的高分辨率结构可以提供有助于理解这一过程的信息。我们之前使用线性二色性偏振光光谱技术，结合蛋白质工程（位点特异性线性二色性方法），研究了RecA与ssDNA复合物中的排列。在本研究中，我们使用这种方法来寻找当复合物中的DNA从ssDNA变为dsDNA时，RecA在原子水平上的结构变化。发现RecA-dsDNA丝的结构数据与之前获得的RecA-ssDNA复合物的数据非常相似，这表明当结合的DNA从单链变为双链时，活性丝中RecA的整体取向以及内部结构没有明显改变。讨论了结构相似性的含义以及观察到的一些可能参与与DNA相互作用的氨基酸残基的构象变化的意义。
