Visualization and quantification of nascent RAD51 filament formation at single-monomer resolution
Visualization and quantification of nascent RAD51 filament formation at single-monomer resolution
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DOI：
10.1073/pnas.1307824111
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发表时间：
2014-10-21
影响因子：
11.1
通讯作者：
Peterman, Erwin J. G.
中科院分区：
文献类型：
--
作者：
Candelli, Andrea;Holthausen, Jan Thomas;Peterman, Erwin J. G.
在双链DNA断裂的重组修复过程中，RAD51重组酶以核蛋白细丝的形式聚集在单链DNA周围，形成能够促进同源识别和链交换的催化熟练结构。介体和辅助因子指导和控制RAD51细丝的动作和动力学。要阐明这些控制机制，就必须开发定量探测RAD51灯丝动力学的瞬变方面的方法。在这里，我们结合荧光显微镜、光钳和微流控技术来可视化RAD51细丝在裸露的单链DNA上的组装，并用单单体灵敏度来量化这一过程。我们表明，细丝是从大小不一的RAD51核中播种的。这种异质性似乎是由于RAD51在溶液中的自组装和核与DNA的大小依赖的相互作用时间之间的能量平衡引起的。我们发现成核本质上是底物选择性的，强烈地有利于在裸露的单链DNA上形成细丝。此外，我们设计了一种光漂白后的单分子荧光恢复实验来独立观察细丝的成核和生长，从而允许直接测量它们对细丝形成的贡献。我们的发现对RAD51丝在裸露的单链DNA上的形成有了全面的、定量的理解，这将作为阐明介体如何帮助RAD51丝组装和辅助因素控制丝动态的基础。
During recombinational repair of double-stranded DNA breaks, RAD51 recombinase assembles as a nucleoprotein filament around single-stranded DNA to form a catalytically proficient structure able to promote homology recognition and strand exchange. Mediators and accessory factors guide the action and control the dynamics of RAD51 filaments. Elucidation of these control mechanisms necessitates development of approaches to quantitatively probe transient aspects of RAD51 filament dynamics. Here, we combine fluorescence microscopy, optical tweezers, and microfluidics to visualize the assembly of RAD51 filaments on bare single-stranded DNA and quantify the process with single-monomer sensitivity. We show that filaments are seeded from RAD51 nuclei that are heterogeneous in size. This heterogeneity appears to arise from the energetic balance between RAD51 self-assembly in solution and the size-dependent interaction time of the nuclei with DNA. We show that nucleation intrinsically is substrate selective, strongly favoring filament formation on bare single-stranded DNA. Furthermore, we devised a single-molecule fluorescence recovery after photobleaching assay to independently observe filament nucleation and growth, permitting direct measurement of their contributions to filament formation. Our findings yield a comprehensive, quantitative understanding of RAD51 filament formation on bare single-stranded DNA that will serve as a basis to elucidate how mediators help RAD51 filament assembly and accessory factors control filament dynamics.