Chow S A, Chiu S K, Wong B C
Department of Biochemistry, University of Honk Kong.
Biochimie. 1991 Feb-Mar;73(2-3):157-61. doi: 10.1016/0300-9084(91)90198-a.
RecA protein promotes homologous pairing and symmetrical strand exchange between partially single-stranded duplex DNA and fully duplex molecules. We constructed circular gapped DNA with a defined gap length and studied the pairing reaction between the gapped substrate and fully duplex DNA. RecA protein polymerizes onto the single-stranded and duplex regions of the gapped DNA to form a nucleoprotein filament. The formation of such filaments requires a stoichiometric amount of RecA protein. Both the rate and yield of joint molecule formation were reduced when the pairing reaction was carried out in the presence of a sub-saturating amount of RecA protein. The amount of RecA protein required for optimal pairing corresponds to the binding site size of RecA protein at saturation on duplex DNA. The result suggests that in the 4-stranded system the single-stranded as well as the duplex regions are involved in pairing. By using fully duplex DNA that shares different lengths and regions of homology with the gapped molecule, we directly showed that the duplex region of the gapped DNA increased both the rate and yield of joint molecule formation. The present study indicates that even though strand exchange in the 4-stranded system must require the presence of a single-stranded region, the pairing that occurs in duplex regions between DNA molecules is functionally significant and contributes to the overall activity of the gapped DNA.
RecA蛋白促进部分单链双链DNA与完全双链分子之间的同源配对和对称链交换。我们构建了具有确定缺口长度的环状缺口DNA，并研究了缺口底物与完全双链DNA之间的配对反应。RecA蛋白在缺口DNA的单链和双链区域聚合形成核蛋白丝。这种丝的形成需要化学计量的RecA蛋白。当在亚饱和量的RecA蛋白存在下进行配对反应时，接头分子形成的速率和产率都会降低。最佳配对所需的RecA蛋白量与双链DNA上饱和时RecA蛋白的结合位点大小相对应。结果表明，在四链系统中，单链区域和双链区域都参与配对。通过使用与缺口分子具有不同长度和同源区域的完全双链DNA，我们直接表明缺口DNA的双链区域提高了接头分子形成的速率和产率。本研究表明，尽管四链系统中的链交换必须需要单链区域的存在，但DNA分子双链区域之间发生的配对在功能上具有重要意义，并有助于缺口DNA的整体活性。
