Bugreev Dmitry V, Golub Efim I, Stasiak Alicja Z, Stasiak Andrzej, Mazin Alexander V
Department of Biochemistry and Molecular Biology, Drexel University College of Medicine, Philadelphia, Pennsylvania 19102, USA.
J Biol Chem. 2005 Jul 22;280(29):26886-95. doi: 10.1074/jbc.M502248200. Epub 2005 May 25.
Rad51 and its meiotic homolog Dmc1 are key proteins of homologous recombination in eukaryotes. These proteins form nucleoprotein complexes on single-stranded DNA that promote a search for homology and that perform DNA strand exchange, the two essential steps of genetic recombination. Previously, we demonstrated that Ca2+ greatly stimulates the DNA strand exchange activity of human (h) Rad51 protein (Bugreev, D. V., and Mazin, A. V. (2004) Proc. Natl. Acad. Sci. U. S. A. 101, 9988-9993). Here, we show that the DNA strand exchange activity of hDmc1 protein is also stimulated by Ca2+. However, the mechanism of stimulation of hDmc1 protein appears to be different from that of hRad51 protein. In the case of hRad51 protein, Ca2+ acts primarily by inhibiting its ATPase activity, thereby preventing self-conversion into an inactive ADP-bound complex. In contrast, we demonstrate that hDmc1 protein does not self-convert into a stable ADP-bound complex. The results indicate that activation of hDmc1 is mediated through conformational changes induced by free Ca2+ ion binding to a protein site that is distinct from the Mg2+.ATP-binding center. These conformational changes are manifested by formation of more stable filamentous hDmc1.single-stranded DNA complexes. Our results demonstrate a universal role of Ca2+ in stimulation of mammalian DNA strand exchange proteins and reveal diversity in the mechanisms of this stimulation.
Rad51及其减数分裂同源物Dmc1是真核生物中同源重组的关键蛋白。这些蛋白在单链DNA上形成核蛋白复合物，促进同源性搜索并进行DNA链交换，这是基因重组的两个基本步骤。此前，我们证明Ca2+能极大地刺激人（h）Rad51蛋白的DNA链交换活性（Bugreev，D. V.，和Mazin，A. V.（2004年）《美国国家科学院院刊》101，9988 - 9993）。在此，我们表明hDmc1蛋白的DNA链交换活性也受到Ca2+的刺激。然而，hDmc1蛋白的刺激机制似乎与hRad51蛋白不同。就hRad51蛋白而言，Ca2+主要通过抑制其ATP酶活性起作用，从而防止自身转化为无活性的ADP结合复合物。相比之下，我们证明hDmc1蛋白不会自身转化为稳定的ADP结合复合物。结果表明，hDmc1的激活是通过游离Ca2+离子与一个不同于Mg2+.ATP结合中心的蛋白位点结合诱导的构象变化介导的。这些构象变化表现为形成更稳定的丝状hDmc1.单链DNA复合物。我们的结果证明了Ca2+在刺激哺乳动物DNA链交换蛋白方面的普遍作用，并揭示了这种刺激机制的多样性。
