Direct Single-Molecule Observation of Mode and Geometry of RecA-Mediated Homology Search
Direct Single-Molecule Observation of Mode and Geometry of RecA-Mediated Homology Search
复制标题
DOI：
10.1021/acsnano.7b06208
复制
发表时间：
2018-01-01
期刊：
影响因子：
17.1
通讯作者：
Walti, Christoph
中科院分区：
文献类型：
--
作者：
Lee, Andrew J.;Endo, Masayuki;Walti, Christoph
基因组的完整性，当受到损害的累积DNA损伤，是通过有效的修复，通过同源重组。对于该过程，普遍存在的重组酶A（RecA）及其同源物如人Rad 51具有核心重要性，能够在单链和双链DNA内比对和交换同源序列，以便交换出缺陷区域。在这里，我们直接观察RecA同源性搜索在单分子水平上使用高速原子力显微镜（HS-AFM）结合定制的DNA折纸框架，以适合AFM成像的方式呈现反应靶点的广泛争论的机制。我们发现，RecA核蛋白丝移动沿着DNA基板通过短距离的促进扩散，或幻灯片，穿插较长距离的随机移动，或跳。重要的是，从特定的相互作用的几何形状，我们发现，双链底物DNA驻留在二级DNA结合位点内的RecA核蛋白丝螺旋槽在同源搜索。这项工作表明，定制的DNA折纸，结合HS-AFM，可以用来直接揭示动态蛋白质-DNA相互作用的构象和几何信息，这是以前无法在单个单分子水平。
Genomic integrity, when compromised by accrued DNA lesions, is maintained through efficient repair via homologous recombination. For this process the ubiquitous recombinase A (RecA), and its homologues such as the human Rad51, are of central importance, able to align and exchange homologous sequences within single-stranded and double-stranded DNA in order to swap out defective regions. Here, we directly observe the widely debated mechanism of RecA homology searching at a single-molecule level using high-speed atomic force microscopy (HS-AFM) in combination with tailored DNA origami frames to present the reaction targets in a way suitable for AFM-imaging. We show that RecA nucleoprotein filaments move along DNA substrates via short-distance facilitated diffusions, or slides, interspersed with longer-distance random moves, or hops. Importantly, from the specific interaction geometry, we find that the double-stranded substrate DNA resides in the secondary DNA binding-site within the RecA nucleoprotein filament helical groove during the homology search. This work demonstrates that tailored DNA origami, in conjunction with HS-AFM, can be employed to reveal directly conformational and geometrical information on dynamic protein-DNA interactions which was previously inaccessible at an individual single-molecule level.