Takahashi Masayuki, Maraboeuf Fabrice, Morimatsu Katsumi, Selmane Tassadite, Fleury Fabrice, Norden Bengt
UMR 216, Centre National de la Recherche Scientifique and Institut Curie, F-91405 Orsay, France.
J Mol Biol. 2007 Jan 19;365(3):603-11. doi: 10.1016/j.jmb.2006.10.042. Epub 2006 Oct 17.
RecA protein recognises two complementary DNA strands for homologous recombination. To gain insight into the molecular mechanism, the thermodynamic parameters of the DNA binding have been characterised by isothermal calorimetry. Specifically, conformational changes of protein and DNA were searched for by measuring variations in enthalpy change (DeltaH) with temperature (heat capacity change, DeltaC(p)). In the presence of the ATP analogue ATPgammaS, the DeltaH for the binding of the first DNA strand depends upon temperature (large DeltaC(p)) and the type of buffer, in a way that is consistent with the organisation of disordered parts and the protonation of RecA upon complex formation. In contrast, the binding of the second DNA strand occurs without any pronounced DeltaC(p), indicating the absence of further reorganisation of the RecA-DNA filament. In agreement with these findings, a significant change in the CD spectrum of RecA was observed only upon the binding of the first DNA strand. In the absence of nucleotide cofactor, the DeltaH of DNA binding is almost independent of temperature, indicating a requirement for ATP in the reorganisation of RecA. When the second DNA strand is complementary to the first, the DeltaH is larger than that for non-complementary DNA strand, but less than the DeltaH of the annealing of the complementary DNA without RecA. This small DeltaH could reflect a weak binding that may facilitate the dissociation of only partly complementary DNA and thus speed the search for complementary DNA. The DeltaH of binding DNA sequences displaying strong base-base stacking is small for both the first and second binding DNA strand, suggesting that the second is also stretched upon interaction with RecA. These results support the proposal that the RecA protein restructures DNA, preparing it for the recognition of a complementary second DNA strand, and that the recognition is due mainly to direct base-base contacts between DNA strands.
RecA蛋白识别两条互补的DNA链以进行同源重组。为深入了解其分子机制，已通过等温滴定量热法对DNA结合的热力学参数进行了表征。具体而言，通过测量焓变（ΔH）随温度的变化（热容变化，ΔC(p)）来寻找蛋白质和DNA的构象变化。在ATP类似物ATPγS存在的情况下，第一条DNA链结合的ΔH取决于温度（大的ΔC(p)）和缓冲液类型，其方式与无序部分的组织以及复合物形成时RecA的质子化一致。相比之下，第二条DNA链的结合没有任何明显的ΔC(p)，表明RecA-DNA细丝没有进一步的重组。与这些发现一致，仅在第一条DNA链结合时才观察到RecA的圆二色光谱有显著变化。在没有核苷酸辅因子的情况下，DNA结合的ΔH几乎与温度无关，表明RecA的重组需要ATP。当第二条DNA链与第一条互补时，ΔH大于非互补DNA链的ΔH，但小于没有RecA时互补DNA退火的ΔH。这个小的ΔH可能反映了一种弱结合，它可能仅促进部分互补DNA的解离，从而加速对互补DNA的搜索。对于第一条和第二条结合DNA链，显示出强碱基堆积的DNA序列结合的ΔH都很小，这表明第二条链在与RecA相互作用时也被拉伸。这些结果支持了这样的提议，即RecA蛋白重组DNA，使其为识别互补的第二条DNA链做好准备，并且这种识别主要是由于DNA链之间直接的碱基接触。
