Direct observation of individual RecA filaments assembling on single DNA molecules
Direct observation of individual RecA filaments assembling on single DNA molecules
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DOI：
10.1038/nature05197
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发表时间：
2006-10-19
期刊：
影响因子：
64.8
通讯作者：
Kowalczykowski, Stephen C.
中科院分区：
文献类型：
--
作者：
Galletto, Roberto;Amitani, Ichiro;Kowalczykowski, Stephen C.
大肠杆菌RecA对于通过同源重组修复DNA双链断裂至关重要（1）。修复需要RecA核蛋白丝的形成。先前的研究已经表明了细丝组装的机制，其中RecA蛋白在DNA上的缓慢成核之后是快速生长（2-7）。然而，这一过程的许多方面仍然不清楚，包括成核和生长的速率以及ATP水解的参与，主要是因为缺乏单丝水平的可视化。在这里，我们报告的直接观察使用荧光修饰的RecA的个别双链DNA分子上的细丝组装。核蛋白丝使DNA饱和并将其延长1.6倍。在早期的时间点，看到离散的RecA簇，允许从单个细胞核的单丝生长的分析。新生RecA细丝的形成不依赖于ATP水解，但依赖于核苷酸辅因子的类型和RecA浓度，表明成核涉及4 - 5个ATP-RecA单体与DNA的结合。单个RecA细丝以3 - 10 nm s（-1）的速率生长。生长是双向的，与成核相反，不依赖于核苷酸辅因子，表明添加了2 - 7个单体s（-1）。这些结果与广泛的遗传和生化研究结果雅阁，表明在体内的组装是在成核步骤控制。我们预期我们的方法和结论可以扩展到相关的真核对应物Rad 51（参考文献8）和组装介体的调节（9-11）。
Escherichia coli RecA is essential for the repair of DNA double-strand breaks by homologous recombination(1). Repair requires the formation of a RecA nucleoprotein filament. Previous studies have indicated a mechanism of filament assembly whereby slow nucleation of RecA protein on DNA is followed by rapid growth(2-7). However, many aspects of this process remain unclear, including the rates of nucleation and growth and the involvement of ATP hydrolysis, largely because visualization at the single-filament level is lacking. Here we report the direct observation of filament assembly on individual double-stranded DNA molecules using fluorescently modified RecA. The nucleoprotein filaments saturate the DNA and extend it 1.6-fold. At early time points, discrete RecA clusters are seen, permitting analysis of single-filament growth from individual nuclei. Formation of nascent RecA filaments is independent of ATP hydrolysis but is dependent on the type of nucleotide cofactor and the RecA concentration, suggesting that nucleation involves binding of 4 - 5 ATP - RecA monomers to DNA. Individual RecA filaments grow at rates of 3 - 10 nm s(-1). Growth is bidirectional and, in contrast to nucleation, independent of nucleotide cofactor, suggesting addition of 2 - 7 monomers s(-1). These results are in accord with extensive genetic and biochemical studies, and indicate that assembly in vivo is controlled at the nucleation step. We anticipate that our approach and conclusions can be extended to the related eukaryotic counterpart, Rad51 (ref. 8), and to regulation by assembly mediators(9-11).