Chiu S K, Wong B C, Chow S A
Department of Biochemistry, University of Hong Kong.
J Biol Chem. 1990 Dec 5;265(34):21262-8.
RecA protein catalyzes homologous pairing of partially single-stranded duplex DNA and fully duplex DNA to form stable joint molecules. We constructed circular duplex DNA with various defined gap lengths and studied the pairing reaction between the gapped substrate with fully double-stranded DNA. The reaction required a stoichiometric amount of RecA protein, and the optimal reaction was achieved at a ratio of 1 RecA monomer per 4 base pairs. The length of the gap, ranging from 141 to 1158 nucleotides, had little effect on the efficiency of homologous pairing. By using a circular gapped duplex DNA prepared from the chimeric phage M13Gori1, we were able to show the formation of nonintertwined or paranemic joints in duplex regions between the gapped and fully duplex molecules. The formation of such paranemic joints occurred efficiently and included nearly all of the DNA in the reaction mixture. The reaction required negative superhelicity, and pairing was greatly reduced with linear or nicked circular DNA. We conclude that one functional role of the single-stranded gap is for facilitating the binding of RecA protein to the duplex region of the gapped DNA. Once the nucleoprotein filament is formed, homologous pairing between the gapped and fully duplex DNA can take place anywhere along the length of the nucleoprotein complex.
RecA蛋白催化部分单链双链DNA与完全双链DNA的同源配对，形成稳定的连接分子。我们构建了具有各种确定缺口长度的环状双链DNA，并研究了有缺口的底物与完全双链DNA之间的配对反应。该反应需要化学计量的RecA蛋白，最佳反应在每4个碱基对1个RecA单体的比例下实现。缺口长度在141至1158个核苷酸之间，对同源配对效率影响不大。通过使用从嵌合噬菌体M13Gori1制备的环状有缺口双链DNA，我们能够证明在有缺口分子和完全双链分子之间的双链区域形成了非缠绕或平行排列的连接。这种平行排列连接的形成高效发生，并且几乎包括反应混合物中的所有DNA。该反应需要负超螺旋，线性或带切口的环状DNA会大大降低配对。我们得出结论，单链缺口的一个功能作用是促进RecA蛋白与有缺口DNA的双链区域结合。一旦形成核蛋白丝，有缺口的DNA与完全双链DNA之间的同源配对可以在核蛋白复合物长度的任何位置发生。
