Aihara H, Ito Y, Kurumizaka H, Terada T, Yokoyama S, Shibata T
The Institute of Physical and Chemical Research (RIKEN), Wako-shi, Japan.
J Mol Biol. 1997 Nov 28;274(2):213-21. doi: 10.1006/jmbi.1997.1403.
RecA protein and its homologs catalyze homologous pairing of dsDNA and ssDNA, a critical reaction in homologous genetic recombination in various organisms from a virus, microbes to higher eukaryotes. In this reaction, RecA protein forms a nucleoprotein filament on ssDNA, which in turn binds to naked dsDNA for homology search. We suggested that the C-terminal domain of RecA protein plays a role in capturing the dsDNA. Here, we isolated the C-terminal domain as a soluble form and determined the solution structure by NMR spectroscopy. The overall folding of the NMR structure agrees with that of the corresponding part of the reported crystal structure, but a remarkable difference was found in a solvent-exposed region due to intermolecular contacts in the crystal. Then, we studied the interaction between the C-terminal domain and DNA, and found that significant chemical shift changes were induced in a specific region by titration with dsDNA. SsDNA induced a much smaller chemical shift perturbation. The difference of DNA concentrations to give the half-saturation of the chemical shift change showed a higher affinity of the C-terminal region toward dsDNA. Combined with our previous results, these provide direct evidence that the defined region in the C-terminal domain furnishes a binding surface for DNA.
RecA蛋白及其同源物催化双链DNA（dsDNA）和单链DNA（ssDNA）的同源配对，这是从病毒、微生物到高等真核生物等各种生物体中同源基因重组的关键反应。在这个反应中，RecA蛋白在ssDNA上形成核蛋白丝，进而与裸露的dsDNA结合以进行同源性搜索。我们认为RecA蛋白的C末端结构域在捕获dsDNA中发挥作用。在此，我们以可溶形式分离出C末端结构域，并通过核磁共振光谱法确定其溶液结构。核磁共振结构的整体折叠与报道的晶体结构的相应部分一致，但由于晶体中的分子间接触，在溶剂暴露区域发现了显著差异。然后，我们研究了C末端结构域与DNA之间的相互作用，发现通过用dsDNA滴定，在特定区域诱导了显著的化学位移变化。ssDNA诱导的化学位移扰动要小得多。产生化学位移变化半饱和的DNA浓度差异表明C末端区域对dsDNA具有更高的亲和力。结合我们之前的结果，这些提供了直接证据，表明C末端结构域中的特定区域为DNA提供了一个结合表面。
