Robertson Ragan B, Moses Dana N, Kwon YoungHo, Chan Pamela, Zhao Weixing, Chi Peter, Klein Hannah, Sung Patrick, Greene Eric C
Department of Biological Sciences, Columbia University, New York, NY 10032, USA.
J Mol Biol. 2009 May 15;388(4):703-20. doi: 10.1016/j.jmb.2009.03.049. Epub 2009 Mar 24.
Rad51 is the core component of the eukaryotic homologous recombination machinery and assembles into elongated nucleoprotein filaments on DNA. We have used total internal reflection fluorescence microscopy and a DNA curtain assay to investigate the dynamics of individual Saccharomyces cerevisiae Rad51 nucleoprotein filaments. For these experiments the DNA molecules were end-labeled with single fluorescent semiconducting nanocrystals. The assembly and disassembly of the Rad51 nucleoprotein filaments were visualized by tracking the location of the labeled DNA end in real time. Using this approach, we have analyzed yeast Rad51 under a variety of different reaction conditions to assess parameters that impact the stability of the nucleoprotein filament. We show that Rad51 readily dissociates from DNA in the presence of ADP or in the absence of nucleotide cofactor, but that free ATP in solution confers a fivefold increase in the stability of the nucleoprotein filaments. We also probe how protein dissociation is coupled to ATP binding and hydrolysis by examining the effects of ATP concentration, and by the use of the nonhydrolyzable ATP analogue adenosine 5'-(beta, gamma-imido) triphosphate and ATPase active-site mutants. Finally, we demonstrate that the Rad51 gain-of-function mutant I345T dissociates from DNA with kinetics nearly identical to that of wild-type Rad51, but assembles 30% more rapidly. Together, these results provide a framework for studying the biochemical behaviors of S. cerevisiae Rad51 nucleoprotein filaments at the single-molecule level.
Rad51是真核生物同源重组机制的核心组成部分，可在DNA上组装成细长的核蛋白丝。我们使用全内反射荧光显微镜和DNA帘试验来研究单个酿酒酵母Rad51核蛋白丝的动力学。在这些实验中，DNA分子用单个荧光半导体纳米晶体进行末端标记。通过实时跟踪标记的DNA末端的位置来观察Rad51核蛋白丝的组装和解聚。使用这种方法，我们在各种不同的反应条件下分析了酵母Rad51，以评估影响核蛋白丝稳定性的参数。我们发现，在存在ADP或不存在核苷酸辅因子的情况下，Rad51很容易从DNA上解离，但溶液中的游离ATP可使核蛋白丝的稳定性提高五倍。我们还通过检查ATP浓度的影响以及使用不可水解的ATP类似物腺苷5'-（β，γ-亚氨基）三磷酸和ATP酶活性位点突变体，来探究蛋白质解离如何与ATP结合和水解偶联。最后，我们证明功能获得型突变体I345T的Rad51从DNA上解离的动力学与野生型Rad51几乎相同，但组装速度快30%。总之，这些结果为在单分子水平上研究酿酒酵母Rad51核蛋白丝的生化行为提供了一个框架。
