Mazin Alexander V, Alexeev Andrei A, Kowalczykowski Stephen C
Department of Biochemistry, Drexel University College of Medicine, Philadelphia, Pennsylvania 19102-1192, USA.
J Biol Chem. 2003 Apr 18;278(16):14029-36. doi: 10.1074/jbc.M212779200. Epub 2003 Feb 3.
Homologous recombination is important for the repair of double-stranded DNA breaks in all organisms. Rad51 and Rad54 proteins are two key components of the homologous recombination machinery in eukaryotes. In vitro, Rad51 protein assembles with single-stranded DNA to form the helical nucleoprotein filament that promotes DNA strand exchange, a basic step of homologous recombination. Rad54 protein interacts with this Rad51 nucleoprotein filament and stimulates its DNA pairing activity, suggesting that Rad54 protein is a component of the nucleoprotein complex involved in the DNA homology search. Here, using physical criteria, we demonstrate directly the formation of Rad54-Rad51-DNA nucleoprotein co-complexes that contain equimolar amounts of each protein. The binding of Rad54 protein significantly stabilizes the Rad51 nucleoprotein filament formed on either single-stranded DNA or double-stranded DNA. The Rad54-stabilized nucleoprotein filament is more competent in DNA strand exchange and acts over a broader range of solution conditions. Thus, the co-assembly of an interacting partner with the Rad51 nucleoprotein filament represents a novel means of stabilizing the biochemical entity central to homologous recombination, and reveals a new function of Rad54 protein.
同源重组对于所有生物体中双链DNA断裂的修复都很重要。Rad51和Rad54蛋白是真核生物同源重组机制的两个关键组成部分。在体外，Rad51蛋白与单链DNA组装形成促进DNA链交换的螺旋核蛋白丝，这是同源重组的一个基本步骤。Rad54蛋白与这种Rad51核蛋白丝相互作用并刺激其DNA配对活性，表明Rad54蛋白是参与DNA同源性搜索的核蛋白复合物的一个组成部分。在此，我们利用物理标准直接证明了含有等摩尔量每种蛋白质的Rad54-Rad51-DNA核蛋白共复合物的形成。Rad54蛋白的结合显著稳定了在单链DNA或双链DNA上形成的Rad51核蛋白丝。Rad54稳定的核蛋白丝在DNA链交换方面更具活性，并且在更广泛的溶液条件下起作用。因此，与Rad51核蛋白丝共同组装相互作用伙伴代表了一种稳定同源重组核心生化实体的新方法，并揭示了Rad54蛋白的新功能。
