Genome Damage and Stability Centre, University of Sussex, Brighton, UK.
EMBO J. 2013 Jan 9;32(1):112-25. doi: 10.1038/emboj.2012.304. Epub 2012 Nov 23.
Non-homologous end joining (NHEJ) is critical for the maintenance of genetic integrity and DNA double-strand break (DSB) repair. NHEJ is regulated by a series of interactions between core components of the pathway, including Ku heterodimer, XLF/Cernunnos, and XRCC4/DNA Ligase 4 (Lig4). However, the mechanisms by which these proteins assemble into functional protein-DNA complexes are not fully understood. Here, we show that the von Willebrand (vWA) domain of Ku80 fulfills a critical role in this process by recruiting Aprataxin-and-PNK-Like Factor (APLF) into Ku-DNA complexes. APLF, in turn, functions as a scaffold protein and promotes the recruitment and/or retention of XRCC4-Lig4 and XLF, thereby assembling multi-protein Ku complexes capable of efficient DNA ligation in vitro and in cells. Disruption of the interactions between APLF and either Ku80 or XRCC4-Lig4 disrupts the assembly and activity of Ku complexes, and confers cellular hypersensitivity and reduced rates of chromosomal DSB repair in avian and human cells, respectively. Collectively, these data identify a role for the vWA domain of Ku80 and a molecular mechanism by which DNA ligase proficient complexes are assembled during NHEJ in mammalian cells, and reveal APLF to be a structural component of this critical DSB repair pathway.
非同源末端连接（NHEJ）对于维持遗传完整性和修复 DNA 双链断裂（DSB）至关重要。NHEJ 受到途径核心成分之间一系列相互作用的调节，包括 Ku 异二聚体、XLF/Cernunnos 和 XRCC4/DNA 连接酶 4（Lig4）。然而，这些蛋白质组装成功能性蛋白-DNA 复合物的机制尚不完全清楚。在这里，我们表明 Ku80 的 von Willebrand (vWA) 结构域通过将 Aprataxin-and-PNK-Like Factor (APLF) 招募到 Ku-DNA 复合物中，在这个过程中起着关键作用。APLF 反过来又作为支架蛋白，促进 XRCC4-Lig4 和 XLF 的募集和/或保留，从而组装能够在体外和细胞中有效进行 DNA 连接的多蛋白 Ku 复合物。破坏 APLF 与 Ku80 或 XRCC4-Lig4 之间的相互作用会破坏 Ku 复合物的组装和活性，并分别导致禽类和人类细胞的细胞敏感性增加和染色体 DSB 修复率降低。总之，这些数据确定了 Ku80 的 vWA 结构域的作用以及在哺乳动物细胞中进行 NHEJ 时 DNA 连接酶功能复合物组装的分子机制，并揭示了 APLF 是这个关键 DSB 修复途径的结构组成部分。
