The DNA-dependent protein kinase (DNA-PK), which is composed of the KU heterodimer and the large catalytic subunit (DNA-PKcs), is a classical nonhomologous end-joining (cNHEJ) factor. Naïve B cells undergo class switch recombination (CSR) to generate antibodies with different isotypes by joining two DNA double-strand breaks at different switching regions via the cNHEJ pathway. DNA-PK and the cNHEJ pathway play important roles in the DNA repair phase of CSR. To initiate cNHEJ, KU binds to DNA ends and recruits and activates DNA-PK. Activated DNA-PK phosphorylates DNA-PKcs at the S2056 and T2609 clusters. Loss of T2609 cluster phosphorylation increases radiation sensitivity but whether T2609 phosphorylation has a role in physiological DNA repair remains elusive. Using the DNA-PKcs^5A mouse model carrying alanine substitutions at the T2609 cluster, here we show that loss of T2609 phosphorylation of DNA-PKcs does not affect the CSR efficiency. Yet, the CSR junctions recovered from DNA-PKcs^5A/5A B cells reveal increased chromosomal translocations, extensive use of distal switch regions (consistent with end resection), and preferential usage of microhomology—all signs of the alternative end-joining pathway. Thus, these results uncover a role of DNA-PKcs T2609 phosphorylation in promoting cNHEJ repair pathway choice during CSR.

由 KU 异二聚体和大催化亚基 (DNA-PKcs) 组成的 DNA 依赖性蛋白激酶 (DNA-PK) 是一种经典的非同源末端连接 (cNHEJ) 因子。幼稚 B 细胞通过 cNHEJ 途径在不同转换区域连接两条 DNA 双链断裂，进行类别转换重组 (CSR) 以产生具有不同同种型的抗体。DNA-PK 和 cNHEJ 通路在 CSR 的 DNA 修复阶段发挥重要作用。为了启动 cNHEJ，KU 与 DNA 末端结合并招募和激活 DNA-PK。活化的 DNA-PK 在 S2056 和 T2609 簇处磷酸化 DNA-PKcs。T2609 簇磷酸化的缺失增加了辐射敏感性，但 T2609 磷酸化是否在生理 DNA 修复中起作用仍然难以捉摸。使用DNA-PKcs ^5A在 T2609 簇上携带丙氨酸取代的小鼠模型，在这里我们表明 DNA-PKcs 的 T2609 磷酸化的丧失不会影响 CSR 效率。然而，从DNA-PKcs ^5A/5A B 细胞中恢复的 CSR 连接显示染色体易位增加、远端开关区域的广泛使用（与末端切除一致）和微同源的优先使用——所有这些迹象表明替代末端连接途径。因此，这些结果揭示了 DNA-PKcs T2609 磷酸化在促进 CSR 期间 cNHEJ 修复途径选择中的作用。