Howard Hughes Medical Institute, Program in Cellular and Molecular Medicine, Boston Children's Hospital, and Department of Genetics, Harvard Medical School, Boston, MA 02115, USA.
Proc Natl Acad Sci U S A. 2013 Feb 5;110(6):2234-9. doi: 10.1073/pnas.1222573110. Epub 2013 Jan 23.
Classical nonhomologous end joining (C-NHEJ) is a major mammalian DNA double-strand break (DSB) repair pathway that is required for assembly of antigen receptor variable region gene segments by V(D)J recombination. Recombination activating gene endonuclease initiates V(D)J recombination by generating DSBs between two V(D)J coding gene segments and flanking recombination signal sequences (RS), with the two coding ends and two RS ends joined by C-NHEJ to form coding joins and signal joins, respectively. During C-NHEJ, recombination activating gene factor generates two coding ends as covalently sealed hairpins and RS ends as blunt 5'-phosphorylated DSBs. Opening and processing of coding end hairpins before joining by C-NHEJ requires the DNA-dependent protein kinase catalytic subunit (DNA-PKcs). However, C-NHEJ of RS ends, which do not require processing, occurs relatively normally in the absence of DNA-PKcs. The XRCC4-like factor (XLF) is a C-NHEJ component that is not required for C-NHEJ of chromosomal signal joins or coding joins because of functional redundancy with ataxia telangiectasia mutated kinase, a protein that also has some functional overlap with DNA-PKcs in this process. Here, we show that XLF has dramatic functional redundancy with DNA-PKcs in the V(D)J SJ joining process, which is nearly abrogated in their combined absence. Moreover, we show that XLF functionally overlaps with DNA-PKcs in normal mouse development, promotion of genomic stability in mouse fibroblasts, and in IgH class switch recombination in mature B cells. Our findings suggest that DNA-PKcs has fundamental roles in C-NHEJ processes beyond end processing that have been masked by functional overlaps with XLF.
经典非同源末端连接（C-NHEJ）是哺乳动物中一种主要的 DNA 双链断裂（DSB）修复途径，它对于通过 V(D)J 重组组装抗原受体可变区基因片段是必需的。重组激活基因内切酶通过在两个 V(D)J 编码基因片段之间和侧翼的重组信号序列（RS）之间产生 DSB 来启动 V(D)J 重组，两个编码末端和两个 RS 末端通过 C-NHEJ 连接形成编码连接和信号连接。在 C-NHEJ 过程中，重组激活基因因子将两个编码末端生成共价封闭的发夹，将 RS 末端生成钝的 5'-磷酸化 DSB。在 C-NHEJ 连接之前，编码末端发夹的打开和加工需要 DNA 依赖性蛋白激酶催化亚基（DNA-PKcs）。然而，不需要加工的 RS 末端的 C-NHEJ 在缺乏 DNA-PKcs 的情况下相对正常发生。XRCC4 样因子（XLF）是 C-NHEJ 的一个组成部分，由于与共济失调毛细血管扩张突变激酶（ataxia telangiectasia mutated kinase，ATM）的功能冗余，它不是染色体信号连接或编码连接的 C-NHEJ 所必需的，ATM 蛋白在这个过程中与 DNA-PKcs 也有一些功能重叠。在这里，我们表明 XLF 在 V(D)J SJ 连接过程中与 DNA-PKcs 具有显著的功能冗余，当两者同时缺失时，这种冗余几乎完全消失。此外，我们还表明，XLF 在正常小鼠发育、促进小鼠成纤维细胞中的基因组稳定性以及成熟 B 细胞中的 IgH 类别转换重组中与 DNA-PKcs 具有功能重叠。我们的发现表明，DNA-PKcs 在 C-NHEJ 过程中具有基本作用，这些作用超出了与 XLF 的功能重叠所掩盖的末端加工。
