BRCA2 chaperones RAD51 to single molecules of RPA-coated ssDNA.
BRCA2 chaperones RAD51 to single molecules of RPA-coated ssDNA.
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BRCA2 将 RAD51 陪伴到 RPA 包被的单链 DNA 的单分子上。
DOI：
10.1073/pnas.2221971120
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发表时间：
2023-04-04
影响因子：
11.1
通讯作者：
Kowalczykowski, Stephen C.
中科院分区：
文献类型：
--
作者：
Bell, Jason C.;Dombrowski, Christopher C.;Plank, Jody L.;Jensen, Ryan B.;Kowalczykowski, Stephen C.
尽管进行了数十年的遗传和细胞生物学研究，但只有在全长BRCA 2纯化后，才有可能对人类BRCA 2在重组DNA修复中的功能进行机械生化分析。这些机制研究对于BRCA 2在基因组维持中的分子功能至关重要。在这里，我们使用单分子方法来可视化RAD 51在涂覆有复制蛋白A（RPA）的单链DNA（ssDNA）的单个分子上的组装，并观察这一过程如何受到肿瘤抑制蛋白BRCA 2的调节。我们表明，BRCA 2作为一个伴侣成核RAD 51，并将其提供给RPA包被的ssDNA。这项工作推进了对BRCA 2分子功能的理解，从而以一种重要的方式促进了对乳腺癌分子病因学的理解。乳腺癌易感基因BRCA 2的突变会大大增加个体一生中患乳腺癌和卵巢癌的风险。BRCA 2通过同源重组增强DNA修复抑制肿瘤形成。重组的核心是RAD 51核蛋白丝的组装，其在染色体损伤位点处或附近产生的单链DNA（ssDNA）上形成。然而，复制蛋白-A（RPA）快速结合并持续螯合这种ssDNA，对抑制不受调节的重组的RAD 51细丝组装施加动力学屏障。其中BRCA 2是人类中的定义成员，它缓解了催化RAD 51细丝形成的动力学障碍。我们结合微流控，显微镜和显微操作，直接测量全长BRCA 2的结合和组装的RAD 51丝上的RPA包被的ssDNA区域内的单个DNA分子，旨在模拟切除的DNA损伤常见的复制偶联重组修复。我们证明，RAD 51的二聚体是自发成核所需的最低限度，然而，生长自终止衍射极限以下。BRCA 2加速RAD 51的成核至接近RAD 51与裸ssDNA的快速缔合的速率，从而克服RPA施加的动力学阻断。此外，BRCA 2通过将短的预组装的RAD 51细丝陪伴到与RPA复合的ssDNA上而消除了对RAD 51的限速成核的需要。因此，BRCA 2通过启动RAD 51细丝形成来调节重组。
Despite decades of genetic and cell biological studies, mechanistic biochemical analyses of human BRCA2 function in recombinational DNA repair have only been possible since the purification of full-length BRCA2. These mechanistic studies crucially inform with respect to the molecular function of BRCA2 in genome maintenance. Here, we use single-molecule methods to visualize the assembly of RAD51 on individual molecules of single-stranded DNA (ssDNA) coated with replication protein-A (RPA) and to see how this process is regulated by the tumor suppressor protein, BRCA2. We show that BRCA2 serves as a chaperone to nucleate RAD51 and deliver it to RPA-coated ssDNA. This work advances understanding of the molecular functions of BRCA2 and, consequently, the molecular etiology of breast cancer in an important way. Mutations in the breast cancer susceptibility gene, BRCA2, greatly increase an individual’s lifetime risk of developing breast and ovarian cancers. BRCA2 suppresses tumor formation by potentiating DNA repair via homologous recombination. Central to recombination is the assembly of a RAD51 nucleoprotein filament, which forms on single-stranded DNA (ssDNA) generated at or near the site of chromosomal damage. However, replication protein-A (RPA) rapidly binds to and continuously sequesters this ssDNA, imposing a kinetic barrier to RAD51 filament assembly that suppresses unregulated recombination. Recombination mediator proteins—of which BRCA2 is the defining member in humans—alleviate this kinetic barrier to catalyze RAD51 filament formation. We combined microfluidics, microscopy, and micromanipulation to directly measure both the binding of full-length BRCA2 to—and the assembly of RAD51 filaments on—a region of RPA-coated ssDNA within individual DNA molecules designed to mimic a resected DNA lesion common in replication-coupled recombinational repair. We demonstrate that a dimer of RAD51 is minimally required for spontaneous nucleation; however, growth self-terminates below the diffraction limit. BRCA2 accelerates nucleation of RAD51 to a rate that approaches the rapid association of RAD51 to naked ssDNA, thereby overcoming the kinetic block imposed by RPA. Furthermore, BRCA2 eliminates the need for the rate-limiting nucleation of RAD51 by chaperoning a short preassembled RAD51 filament onto the ssDNA complexed with RPA. Therefore, BRCA2 regulates recombination by initiating RAD51 filament formation.
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影响因子：
7.2
作者：
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通讯作者：
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影响因子：
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通讯作者：
Kowalczykowski, Stephen C.
影响因子：
7.2
作者：
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通讯作者：
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通讯作者：
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