Jayasena V K, Johnston B H
Cell and Molecular Biology Laboratory, SRI International, Menlo Park, CA 94025-3493.
J Mol Biol. 1993 Apr 5;230(3):1015-24. doi: 10.1006/jmbi.1993.1216.
The RecA protein (RecA) of Escherichia coli has the ability to pair a single-stranded DNA to a homologous sequence in a duplex DNA without requiring denaturation of the duplex. This ability has stimulated interest in the use of RecA for targeting probes to genomic DNA. However, because pairing generally requires that the double-stranded DNA either have a homologous end or be negatively supercoiled, the application of RecA to targeting has been very limited. Here, we show that if the sequence complementary to the probe is also included in the reaction, RecA can pair the two single strands to sites distant from any ends on linear DNA. The resulting structure, termed a complement-stabilized D-loop (csD-loop), is cleavable by restriction endonucleases and, upon removal of RecA, remains stable to temperatures up to the tm of the double-stranded probe. These results indicate that the csD-loop probably consists of two side-by-side Watson-Crick duplexes, much like a replication bubble. This novel reaction of RecA may be useful in gene mapping and isolation, as well as in sequence-specific cleavage of genomic DNA, and might have functions in vivo.
大肠杆菌的RecA蛋白（RecA）能够将单链DNA与双链DNA中的同源序列配对，而无需双链解链。这种能力激发了人们对利用RecA将探针靶向基因组DNA的兴趣。然而，由于配对通常要求双链DNA要么有同源末端，要么呈负超螺旋，RecA在靶向方面的应用非常有限。在此，我们表明，如果反应中也包含与探针互补的序列，RecA可以将两条单链与线性DNA上远离任何末端的位点配对。所形成的结构称为互补稳定D环（csD环），可被限制性内切酶切割，并且在去除RecA后，在高达双链探针解链温度（tm）的温度下仍保持稳定。这些结果表明，csD环可能由两个并排的沃森-克里克双链体组成，很像一个复制泡。RecA的这种新反应可能在基因图谱绘制和分离以及基因组DNA的序列特异性切割中有用，并且可能在体内具有功能。
