DiCapua E, Schnarr M, Timmins P A
Institut Laue-Langevin, Grenoble, France.
Biochemistry. 1989 Apr 18;28(8):3287-92. doi: 10.1021/bi00434a025.
Purified recA protein is found as rodlike homopolymers, and it forms filamentous complexes with double-stranded DNA that are stable in the presence of ATP gamma S, a nonhydrolyzable analogue of ATP. The structure of these filaments has been described in some detail by electron microscopy. Here we confirm the mass per length of 6.5 recA/100 A in solution by small-angle neutron scattering and extend the analysis to homopolymers of recA protein, finding a mass per length of about 7 recA/100 A and a radial mass distribution (cross-sectional radius of gyration) significantly different for the two filaments. The models proposed so far for the structure of the complex have placed the DNA in the center of the filament. Here we verify this assumption using small-angle neutron scattering to locate the DNA in the complexes, exploiting the contrast variation method in D2O/H2O mixtures. Model calculations show that the natural contrast difference between DNA and protein is not sufficient to locate the DNA (which accounts for only 4.7% of the mass in the complex). When deuterated DNA is used, the contrast difference is enhanced, and model calculations and experiment then converge, indicating that the DNA is indeed near the axis of the complex.
纯化的RecA蛋白以棒状同聚物形式存在，并且它与双链DNA形成丝状复合物，在ATPγS（一种ATP的不可水解类似物）存在下这种复合物是稳定的。这些丝状结构已通过电子显微镜进行了较为详细的描述。在这里，我们通过小角中子散射证实了溶液中每100埃长度有6.5个RecA的质量，并将分析扩展到RecA蛋白的同聚物，发现每100埃长度约有7个RecA的质量，且两种丝状结构的径向质量分布（回转半径）显著不同。到目前为止提出的复合物结构模型将DNA置于丝状结构的中心。在这里，我们利用D2O/H2O混合物中的对比变化方法，通过小角中子散射来确定复合物中DNA的位置，从而验证这一假设。模型计算表明，DNA和蛋白质之间的天然对比差异不足以确定DNA的位置（DNA仅占复合物质量的4.7%）。当使用氘代DNA时，对比差异增强，模型计算和实验结果趋于一致，表明DNA确实靠近复合物的轴。
