Department of Microbiology and Molecular Genetics, University of California, Davis, CA 95616.
Department of Molecular and Cellular Biology, University of California, Davis, CA 95616.
Proc Natl Acad Sci U S A. 2023 Apr 4;120(14):e2221971120. doi: 10.1073/pnas.2221971120. Epub 2023 Mar 28.
Mutations in the breast cancer susceptibility gene, , greatly increase an individual's lifetime risk of developing breast and ovarian cancers. 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.
乳腺癌易感基因 的突变极大地增加了个体一生中罹患乳腺癌和卵巢癌的风险。 通过同源重组增强 DNA 修复来抑制肿瘤形成。 重组的核心是 RAD51 核蛋白丝的组装，该丝在染色体损伤部位或其附近产生的单链 DNA（ssDNA）上形成。 然而，复制蛋白 A（RPA）迅速结合并不断隔离这种 ssDNA，对 RAD51 丝组装施加动力学障碍，从而抑制不受调节的重组。 重组介体蛋白 - 其中 BRCA2 是人类中的定义成员 - 缓解了这种动力学障碍，以催化 RAD51 丝的形成。 我们结合了微流控、显微镜和微操作技术，直接测量全长 BRCA2 与 RAD51 丝在单个 DNA 分子上组装的结合情况，这些 DNA 分子设计用于模拟复制偶联重组修复中常见的切除 DNA 损伤。 我们证明，RAD51 的二聚体对于自发成核是最小必需的； 然而，生长在衍射极限以下自动终止。 BRCA2 加速 RAD51 的成核速度，接近 RAD51 与裸露 ssDNA 的快速结合，从而克服了 RPA 施加的动力学障碍。 此外，BRCA2 通过将短的预组装 RAD51 丝引导到与 RPA 结合的 ssDNA 复合物上，消除了 RAD51 成核的限速步骤。 因此，BRCA2 通过启动 RAD51 丝的形成来调节重组。
