Micro-homology intermediates: RecA's transient sampling revealed at the single molecule level.
Micro-homology intermediates: RecA's transient sampling revealed at the single molecule level.
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DOI：
10.1093/nar/gkaa1258
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发表时间：
2021-02-22
影响因子：
14.9
通讯作者：
Wälti C
中科院分区：
文献类型：
--
作者：
Lee AJ;Endo M;Hobbs JK;Davies AG;Wälti C
重组酶A（RecA）是同源重组的核心。然而，尽管取得了重大进展，RecA能够协调同源性搜索的机制仍然难以捉摸。DNA纳米结构增强的高速AFM提供了直接和在单分子水平上研究RecA重组机制所需的空间和时间分辨率。我们提出了直接在原位观察RecA协调的同源DNA链的对齐，形成一个稳定的重组产物内的支持DNA纳米结构。我们显示存在的微妙和短暂的状态的相互作用景观，这表明，RecA瞬时采样微同源性在整个搜索序列比对的单个RecA单体水平。这些瞬时相互作用形成了在>8个核苷酸种子处形成稳定配对之前寻找序列同源性的早期步骤。序列微同源性的去除导致在该位置处的相关瞬时取样的损失。描述DNA折纸框架内同源重组反应方案的示意图和相关的HS-AFM显微照片。从左至右：内部悬浮DNA分子（对照、NPF和反应）的布局参照内置极性标记（黑色三角形）显示。RecA NPF（核蛋白丝）形成于NPF DNA分子的30 nt ssDNA区域上，并通过UV光可切割接头（黄色星星）连接到DNA框架中。暴露于UV使NPF从其系链释放，引发同源重组。这最终在反应DNA上序列同源性区域（绿色）形成突触接头。HS-AFM图像比例尺= 40 nm。
Recombinase A (RecA) is central to homologous recombination. However, despite significant advances, the mechanism with which RecA is able to orchestrate a search for homology remains elusive. DNA nanostructure-augmented high-speed AFM offers the spatial and temporal resolutions required to study the RecA recombination mechanism directly and at the single molecule level. We present the direct in situ observation of RecA-orchestrated alignment of homologous DNA strands to form a stable recombination product within a supporting DNA nanostructure. We show the existence of subtle and short-lived states in the interaction landscape, which suggests that RecA transiently samples micro-homology at the single RecA monomer-level throughout the search for sequence alignment. These transient interactions form the early steps in the search for sequence homology, prior to the formation of stable pairings at >8 nucleotide seeds. The removal of sequence micro-homology results in the loss of the associated transient sampling at that location. A schematic diagram depicting the homologous recombination reaction scheme within the DNA origami frame and associated HS-AFM micrographs. Left to right: the layout of the internal suspended DNA molecules (control, NPF and reaction) is shown in reference to an inbuilt polarity marker (black triangle). The RecA NPF (nucleoprotein filament) is formed on the 30 nt ssDNA region of the NPF DNA molecule and is connected into the DNA frame via a UV photocleavable linker (yellow star). Exposure to UV releases the NPF from its tether initiating homologous recombination. This culminates in the formation of a synaptic joint at the region of sequence homology (green) on the reaction DNA. HS-AFM image scale bar = 40 nm.
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影响因子：
14.9
作者：
Lee AJ;Sharma R;Hobbs JK;Wälti C
通讯作者：
Wälti C
影响因子：
56.9
作者：
Keren, K;Krueger, M;Braun, E
通讯作者：
Braun, E
影响因子：
14.9
作者：
Douglas SM;Marblestone AH;Teerapittayanon S;Vazquez A;Church GM;Shih WM
通讯作者：
Shih WM
影响因子：
13.8
作者：
Bell JC;Kowalczykowski SC
通讯作者：
Kowalczykowski SC
影响因子：
5.6
作者：
EGELMAN, EH;STASIAK, A
通讯作者：
STASIAK, A