Stephen Kowalczykowski及其同事通过单分子显微镜对这种细丝的成核作用和伸展进行了定性。他们获得的数据说明了为什么所谓的“mediator蛋白”对于促进加载和生长可能是必需的,同时那些预测也通过向反应中添加细菌RecFOR复合物而得到证实。

-2012年11月8日《自然》

中文翻译


【题目】单链DNA成像有望揭示乳腺癌起源

【译文】大肠杆菌RecA是一类对于同源重组至关重要的DNA链交换蛋白普遍类型中正在界定的成员,而同源重组是通过修复被破坏的DNA维持基因组完整性的途径。 为了发挥功能,丝状的RecA必须成核并在单链DNA(ssDNA)上直接与ssDNA结合蛋白(SSB)相互竞争而生长,SSB可以快速结合并持续隔绝ssDNA,动态地阻断了RecA的组装。 这种在DNA晶格上动态的自组装(与另一种蛋白相互竞争)相比与其他丝状形式蛋白,如肌动蛋白和微管蛋白是RecA家族所特有的。该过程的复杂性妨碍了我们理解RecA丝状组装,因为总体测量不能可靠地区分核化期和生长期,尽管人们已经做了大量和多种尝试。先前单分子分析已经测量了RecA及其真核同源物RAD51的核化以及在裸露的双链DNA和ssDNA上的生长;然而,体内RecA自组装的模板是SSB包裹的ssDNA。利用单分子显微技术,本研究直接在SSB包裹的ssDNA单分子上可视化RecA丝状组装,同时测量了核化和生长过程。我们证实RecA二聚体是核化所必需的,并紧随通过单体添加的丝状生长过程,这与发现核化,而非生长,是由核苷酸和镁离子辅因子所调控的相一致。丝状生长是双向的,虽然在5′ 3′方向更快速。核化和生长均在生理条件下被抑制,表明重组调控因子在体内自组装中发挥至关重要的作用。我们定义了一个两步运动机制,即SSB滑行和/或部分分离(DNA打开)期间RecA在短暂暴露的ssDNA上核化,随后RecA丝状生长。进一步研究证实了重组介导因子蛋白对——RecOR (RecO和RecR)促进了RecA核化和丝状生长,而且RecF的引入更进一步刺激RecA核化。

英文原稿


[Title]: Direct imaging of RecA nucleation and growth on single molecules of SSB-coated ssDNA

[Authors]:Jason C. Bell,1, 2, 3 Jody L. Plank,1, 2, 4 Christopher C. Dombrowski1, 2, 4 & Stephen C. Kowalczykowski1, 2, 3

[Abstract]:Escherichia coli RecA is the defining member of a ubiquitous class of DNA strand-exchange proteins that are essential for homologous recombination, a pathway that maintains genomic integrity by repairing broken DNA. To function, filaments of RecA must nucleate and grow on single-stranded DNA (ssDNA) in direct competition with ssDNA-binding protein (SSB), which rapidly binds and continuously sequesters ssDNA, kinetically blocking RecA assembly. This dynamic self-assembly on a DNA lattice, in competition with another protein, is unique for the RecA family compared to other filament-forming proteins such as actin and tubulin. The complexity of this process has hindered our understanding of RecA filament assembly because ensemble measurements cannot reliably distinguish between the nucleation and growth phases, despite extensive and diverse attempts. Previous single-molecule assays have measured the nucleation and growth of RecA—and its eukaryotic homologue RAD51—on naked double-stranded DNA and ssDNA; however, the template for RecA self-assembly in vivo is SSB-coated ssDNA. Using single-molecule microscopy, here we directly visualize RecA filament assembly on single molecules of SSB-coated ssDNA, simultaneously measuring nucleation and growth. We establish that a dimer of RecA is required for nucleation, followed by growth of the filament through monomer addition, consistent with the finding that nucleation, but not growth, is modulated by nucleotide and magnesium ion cofactors. Filament growth is bidirectional, albeit faster in the 5′ 3′direction. Both nucleation and growth are repressed at physiological conditions, highlighting the essential role of recombination mediators in potentiating assembly in vivo. We define a two-step kinetic mechanism in which RecA nucleates on transiently exposed ssDNA during SSB sliding and/or partial dissociation (DNA unwrapping) and then the RecA filament grows. We further demonstrate that the recombination mediator protein pair, RecOR (RecO and RecR), accelerates both RecA nucleation and filament growth, and that the introduction of RecF further stimulates RecA nucleation.

原文地址

http://www.nature.com/nature/journal/v491/n7423/full/nature11598.html

 

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