Gamper H B, Hou Y M, Kmiec E B
Department of Biological Sciences, University of Delaware, Newark, Delaware 19716, USA.
Biochemistry. 2000 Dec 12;39(49):15272-81. doi: 10.1021/bi001704o.
Strand exchange between two duplexes is usually initiated as a three-strand event that requires the presence of a single-stranded overhang or gap in one of the two molecules. Here we show that the RecA protein can catalyze a four-strand exchange. Specifically, it can recombine short hairpin substrates with homologous stems provided that one of the hairpins possesses a chimeric DNA/RNA backbone. This four-strand exchange reaction goes to completion in the presence of ATPgammaS and releases a stable heteroduplex upon removal of the RecA protein. Under identical conditions, strand exchange between two DNA hairpins is incomplete and generates a nascent heteroduplex that rapidly dissociates when the RecA protein is denatured. Since presynaptic filament formation does not appear to melt either type of hairpin, we propose that exchange occurs between homologously aligned duplexes that are extended and unwound within a RecA filament. The first reaction provides a mechanism for gene targeting by chimeric double-hairpin oligonucleotides while the second reaction explains the ability of the RecA protein to transiently align double-stranded DNA molecules.
两条双链之间的链交换通常以三链事件启动，这需要两个分子之一中存在单链突出端或缺口。在这里，我们表明RecA蛋白可以催化四链交换。具体而言，它可以将短发夹底物与同源茎进行重组，前提是其中一个发夹具有嵌合的DNA/RNA主链。这种四链交换反应在ATPγS存在下完成，并在去除RecA蛋白后释放出稳定的异源双链体。在相同条件下，两个DNA发夹之间的链交换是不完全的，并产生一个新生的异源双链体，当RecA蛋白变性时，该异源双链体会迅速解离。由于突触前细丝的形成似乎不会使任何一种发夹解链，我们提出交换发生在RecA细丝内延伸并解旋的同源排列的双链体之间。第一个反应为嵌合双发夹寡核苷酸进行基因靶向提供了一种机制，而第二个反应解释了RecA蛋白瞬时排列双链DNA分子的能力。
