Björkman Andrea, Du Likun, Felgentreff Kerstin, Rosner Cornelia, Pankaj Kamdar Radhika, Kokaraki Georgia, Matsumoto Yoshihisa, Davies E Graham, van der Burg Mirjam, Notarangelo Luigi D, Hammarström Lennart, Pan-Hammarström Qiang
Department of Laboratory Medicine, Karolinska Institutet, 141 86 Stockholm, Sweden;
Division of Immunology, Harvard Medical School, Boston Children's Hospital, Boston, MA 02115;
J Immunol. 2015 Dec 15;195(12):5608-15. doi: 10.4049/jimmunol.1501633. Epub 2015 Nov 6.
Nonhomologous end-joining (NHEJ) is one of the major DNA double-strand break repair pathways in mammalian cells and is required for both V(D)J recombination and class switch recombination (CSR), two Ig gene-diversification processes occurring during B cell development. DNA-dependent protein kinase, catalytic subunit (DNA-PKcs) is a component of the classical NHEJ machinery and has a critical function during V(D)J recombination. However, its role in CSR has been controversial. In this study, we examined the pattern of recombination junctions from in vivo-switched B cells from two DNA-PKcs-deficient patients. One of them harbored mutations that did not affect DNA-PKcs kinase activity but caused impaired Artemis activation; the second patient had mutations resulting in diminished DNA-PKcs protein expression and kinase activity. These results were compared with those from DNA-PKcs-deficient mouse B cells. A shift toward the microhomology-based alternative end-joining at the recombination junctions was observed in both human and mouse B cells, suggesting that the classical NHEJ pathway is impaired during CSR when DNA-PKcs is defective. Furthermore, cells from the second patient showed additional or more severe alterations in CSR and/or NHEJ, which may suggest that DNA-PKcs and/or its kinase activity have additional, Artemis-independent functions during these processes.
非同源末端连接（NHEJ）是哺乳动物细胞中主要的DNA双链断裂修复途径之一，是B细胞发育过程中发生的两种Ig基因多样化过程，即V（D）J重组和类别转换重组（CSR）所必需的。DNA依赖性蛋白激酶催化亚基（DNA-PKcs）是经典NHEJ机制的一个组成部分，在V（D）J重组过程中具有关键作用。然而，其在CSR中的作用一直存在争议。在本研究中，我们检查了两名DNA-PKcs缺陷患者体内转换B细胞的重组连接模式。其中一名患者携带的突变不影响DNA-PKcs激酶活性，但导致Artemis激活受损；第二名患者的突变导致DNA-PKcs蛋白表达和激酶活性降低。将这些结果与DNA-PKcs缺陷小鼠B细胞的结果进行了比较。在人和小鼠B细胞的重组连接处均观察到向基于微同源性的替代末端连接的转变，这表明当DNA-PKcs有缺陷时，经典NHEJ途径在CSR过程中受损。此外，第二名患者的细胞在CSR和/或NHEJ中表现出额外的或更严重的改变，这可能表明DNA-PKcs和/或其激酶活性在这些过程中具有额外的、不依赖于Artemis的功能。
