53BP1 activates nonhomologous end joining (NHEJ) and inhibits homologous recombination (HR) repair of DNA double-strand breaks (DSBs). Dissociation of 53BP1 from DSBs and consequent activation of HR, a less error-prone pathway than NHEJ, helps maintain genome integrity during DNA replication; however, the underlying mechanisms are not fully understood. Here, we demonstrate that E3 ubiquitin ligase SPOP promotes HR during S phase of the cell cycle by excluding 53BP1 from DSBs. In response to DNA damage, ATM kinase–catalyzed phosphorylation of SPOP causes a conformational change in SPOP, revealed by x-ray crystal structures, that stabilizes its interaction with 53BP1. 53BP1-bound SPOP induces polyubiquitination of 53BP1, eliciting 53BP1 extraction from chromatin by a valosin-containing protein/p97 segregase complex. Our work shows that SPOP facilitates HR repair over NHEJ during DNA replication by contributing to 53BP1 removal from chromatin. Cancer-derived SPOP mutations block SPOP interaction with 53BP1, inducing HR defects and chromosomal instability.

53BP1 激活非同源末端连接 (NHEJ) 并抑制 DNA 双链断裂 (DSB) 的同源重组 (HR) 修复。 53BP1 从 DSB 解离并随后激活 HR，这是一种比 NHEJ 更不易出错的途径，有助于在 DNA 复制过程中保持基因组完整性；然而，其根本机制尚未完全了解。在这里，我们证明 E3 泛素连接酶 SPOP 通过从 DSB 中排除 53BP1 来促进细胞周期 S 期的 HR。 X 射线晶体结构显示，为了响应 DNA 损伤，ATM 激酶催化的 SPOP 磷酸化会导致 SPOP 发生构象变化，从而稳定其与 53BP1 的相互作用。 53BP1 结合的 SPOP 诱导 53BP1 多泛素化，从而通过含有缬洛新的蛋白/p97 分离酶复合物从染色质中提取 53BP1。我们的工作表明，在 DNA 复制过程中，SPOP 通过促进 53BP1 从染色质上去除，促进了 NHEJ 上的 HR 修复。癌症衍生的 SPOP 突变会阻断 SPOP 与 53BP1 的相互作用，从而诱导 HR 缺陷和染色体不稳定。