Visualization of direct and diffusion-assisted RAD51 nucleation by full-length human BRCA2 protein
Visualization of direct and diffusion-assisted RAD51 nucleation by full-length human BRCA2 protein
复制标题
DOI：
10.1016/j.molcel.2023.06.031
复制
发表时间：
2023-07
期刊：
影响因子：
16
通讯作者：
O. Beláň;Luke A. Greenhough;L. Kuhlen;Roopesh Anand;Artur Kaczmarczyk;D. Gruszka;H. Yardimci;Xiaodong Zhang;David S. Rueda;S. West;S. Boulton
中科院分区：
文献类型：
--
作者：
O. Beláň;Luke A. Greenhough;L. Kuhlen;Roopesh Anand;Artur Kaczmarczyk;D. Gruszka;H. Yardimci;Xiaodong Zhang;David S. Rueda;S. West;S. Boulton
同源重组（HR）对于DNA双链断裂、干扰的复制叉（RF）和复制后单链DNA（ssDNA）缺口的无错误修复是必不可少的。为了启动HR，重组介体和肿瘤抑制蛋白BRCA 2在RAD51旁系同源物刺激RAD51细丝生长之前促进RAD51在ssDNA上成核。虽然ssDNA与BRCA2的结合与RAD51成核有关，但BRCA2与双链DNA（dsDNA）结合的功能仍不清楚。在这里，我们利用单分子（SM）成像可视化BRCA 2介导的RAD 51成核在真实的时间使用纯化的蛋白质。我们报告说，BRCA2核和稳定RAD51的ssDNA直接或通过一个不受欢迎的扩散辅助传递机制，涉及绑定和沿着滑动dsDNA，这需要多个dsDNA结合模块BRCA2的合作行动。总的来说，我们的工作揭示了BRCA2依赖性RAD51加载到ssDNA上的两种不同机制，我们认为这对维持基因组稳定性和癌症抑制的多种功能至关重要。
Homologous recombination (HR) is essential for error-free repair of DNA double-strand breaks, perturbed replication forks (RFs), and post-replicative single-stranded DNA (ssDNA) gaps. To initiate HR, the recombination mediator and tumor suppressor protein BRCA2 facilitates nucleation of RAD51 on ssDNA prior to stimulation of RAD51 filament growth by RAD51 paralogs. Although ssDNA binding by BRCA2 has been implicated in RAD51 nucleation, the function of double-stranded DNA (dsDNA) binding by BRCA2 remains unclear. Here, we exploit single-molecule (SM) imaging to visualize BRCA2-mediated RAD51 nucleation in real time using purified proteins. We report that BRCA2 nucleates and stabilizes RAD51 on ssDNA either directly or through an unappreciated diffusion-assisted delivery mechanism involving binding to and sliding along dsDNA, which requires the cooperative action of multiple dsDNA-binding modules in BRCA2. Collectively, our work reveals two distinct mechanisms of BRCA2-dependent RAD51 loading onto ssDNA, which we propose are critical for its diverse functions in maintaining genome stability and cancer suppression.