Prasad Tekkatte Krishnamurthy, Yeykal Caitlyn C, Greene Eric C
Department of Biochemistry and Molecular Biophysics, Columbia University, College of Physicians and Surgeons, Black Building Room 536, 650 West 168th Street, New York, NY 10032, USA.
J Mol Biol. 2006 Oct 27;363(3):713-28. doi: 10.1016/j.jmb.2006.08.046. Epub 2006 Aug 22.
Rad51 is the core component of the eukaryotic homologous recombination machinery and assembles into extended nucleoprotein filaments on DNA. To study the dynamic behavior of Rad51 we have developed a single-molecule assay that relies on a combination of hydrodynamic force and microscale diffusion barriers to align individual DNA molecules on the surface of a microfluidic sample chamber that is coated with a lipid bilayer. When visualized with total internal reflection fluorescence microscopy (TIRFM), these "molecular curtains" allow for the direct visualization of hundreds of individual DNA molecules. Using this approach, we have analyzed the binding of human Rad51 to single molecules of double-stranded DNA under a variety of different reaction conditions by monitoring the extension of the fluorescently labeled DNA, which coincides with assembly of the nucleoprotein filament. We have also generated several mutants in conserved regions of Rad51 implicated in DNA binding, and tested them for their ability to assemble into extended filaments. We show that proteins with mutations within the DNA-binding surface located on the N-terminal domain still retain the ability to form extended nucleoprotein filaments. Mutations in the L1 loop, which projects towards the central axis of the filament, completely abolish assembly of extended filaments. In contrast, most mutations within or near the L2 DNA-binding loop, which is also located near the central axis of the filament, do not affect the ability of the protein to assemble into extended filaments on double-stranded (ds)DNA. Taken together, these results demonstrate that the L1-loop plays a crucial role in the assembly of extended nucleoprotein filaments on dsDNA, but the N-terminal domain and the L2 DNA-binding loop have significantly less impact on this process. The results presented here also provide an important initial framework for beginning to study the biochemical behaviors of Rad51 nucleoprotein filaments using our novel experimental system.
Rad51是真核生物同源重组机制的核心组成部分，可在DNA上组装成延伸的核蛋白丝。为了研究Rad51的动态行为，我们开发了一种单分子检测方法，该方法依赖于流体动力和微尺度扩散屏障的组合，以将单个DNA分子排列在涂有脂质双层的微流控样品室表面上。当用全内反射荧光显微镜（TIRFM）观察时，这些“分子帘”允许直接观察数百个单个DNA分子。使用这种方法，我们通过监测荧光标记DNA的延伸来分析人Rad51在各种不同反应条件下与双链DNA单分子的结合，这与核蛋白丝的组装一致。我们还在Rad51的保守区域中产生了几个与DNA结合有关的突变体，并测试了它们组装成延伸丝的能力。我们表明，位于N端结构域的DNA结合表面内有突变的蛋白质仍保留形成延伸核蛋白丝的能力。向丝的中心轴突出的L1环中的突变完全消除了延伸丝的组装。相比之下，也位于丝的中心轴附近的L2 DNA结合环内或附近的大多数突变不会影响蛋白质在双链（ds）DNA上组装成延伸丝的能力。综上所述，这些结果表明L1环在dsDNA上延伸核蛋白丝的组装中起关键作用，但N端结构域和L2 DNA结合环对这一过程的影响要小得多。本文给出的结果也为使用我们的新型实验系统开始研究Rad51核蛋白丝的生化行为提供了一个重要的初始框架。
