Reconstitution of DNA strand exchange mediated by Rhp51 recombinase and two mediators
Reconstitution of DNA strand exchange mediated by Rhp51 recombinase and two mediators
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DOI：
10.1371/journal.pbio.0060088
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发表时间：
2008-04-01
期刊：
影响因子：
9.8
通讯作者：
Iwasaki, Hiroshi
中科院分区：
文献类型：
--
作者：
Kurokawa, Yumiko;Murayama, Yasuto;Iwasaki, Hiroshi
在裂殖酵母粟酒裂殖酵母中，遗传证据表明，两种介质 Rad22（粟酒裂殖酵母 Rad52 同源物）和 Swi5-Sfr1 复合体参与 Rhp51（粟酒裂殖酵母 Rad51 同源物）的共同途径 - 介导同源重组 (HR) 和 HR 修复。在这里，我们展示了 HR 期间 DNA 链交换核心步骤的体外重建。我们的系统完全由均质纯化的蛋白质组成，包括 Rhp51（两种介质）和复制蛋白 A (RPA)，它反映了体内遗传要求。使用该系统，我们首次提供了强有力的生化证据，证明两种介质的协同作用将 Rhp51 加载到预涂有 RPA 的单链 DNA (ssDNA) 上。对反应的剖析表明，Rad22 克服了 RPA 对 Rhp51-Swi5-Sfr1 介导的链交换的抑制作用。此外，Rad22 消除了体外系统中严格添加蛋白质顺序的要求。然而，尽管存在 Rad22，Swi5-Sfr1 仍然对于链交换至关重要。重要的是，Rhp51（但 Rad22 和 Swi5-Sfr1 介体）都不是从 ssDNA 中取代 RPA 的因素。 Swi5-Sfr1 以 ATP 依赖性方式稳定 Rhp51-ssDNA 丝，这种稳定与 Rhp51 链交换反应的激活相关。 Rad22 单独不能激活 Rhp51 突触前丝。 AMP-PNP 是一种不可水解的 ATP 类似物，可诱导 Rhp51 类似的稳定作用，但这种稳定作用独立于 Swi5-Sfr1。然而，进行性链转移需要 ATP 的水解，从而形成长异源双链体。我们的体外重建系统表明，这两种介质对于介导 Rhp51 加载到 RPA 预涂的 ssDNA 上具有不可或缺但又不同的作用。
In the fission yeast Schizosaccharomyces pombe, genetic evidence suggests that two mediators, Rad22 ( the S. pombe Rad52 homolog) and the Swi5-Sfr1 complex, participate in a common pathway of Rhp51 ( the S. pombe Rad51 homolog) - mediated homologous recombination ( HR) and HR repair. Here, we have demonstrated an in vitro reconstitution of the central step of DNA strand exchange during HR. Our system consists entirely of homogeneously purified proteins, including Rhp51, the two mediators, and replication protein A ( RPA), which reflects genetic requirements in vivo. Using this system, we present the first robust biochemical evidence that concerted action of the two mediators directs the loading of Rhp51 onto single-stranded DNA ( ssDNA) precoated with RPA. Dissection of the reaction reveals that Rad22 overcomes the inhibitory effect of RPA on Rhp51-Swi5-Sfr1-mediated strand exchange. In addition, Rad22 negates the requirement for a strict order of protein addition to the in vitro system. However, despite the presence of Rad22, Swi5-Sfr1 is still essential for strand exchange. Importantly, Rhp51, but neither Rad22 nor the Swi5-Sfr1 mediator, is the factor that displaces RPA from ssDNA. Swi5-Sfr1 stabilizes Rhp51-ssDNA filaments in an ATP-dependent manner, and this stabilization is correlated with activation of Rhp51 for the strand exchange reaction. Rad22 alone cannot activate the Rhp51 presynaptic filament. AMP-PNP, a nonhydrolyzable ATP analog, induces a similar stabilization of Rhp51, but this stabilization is independent of Swi5-Sfr1. However, hydrolysis of ATP is required for processive strand transfer, which results in the formation of a long heteroduplex. Our in vitro reconstitution system has revealed that the two mediators have indispensable, but distinct, roles for mediating Rhp51 loading onto RPA-precoated ssDNA.