Program in Cellular and Molecular Medicine, Howard Hughes Medical Institute, Children's Hospital Boston and Department of Genetics, Immune Disease Institute, Harvard Medical School, Boston, MA 02115, USA.
Proc Natl Acad Sci U S A. 2012 Feb 14;109(7):2473-8. doi: 10.1073/pnas.1121470109. Epub 2012 Jan 30.
Classical nonhomologous DNA end-joining (C-NHEJ), which is a major DNA double-strand break (DSB) repair pathway in mammalian cells, plays a dominant role in joining DSBs during Ig heavy chain (IgH) class switch recombination (CSR) in activated B lymphocytes. However, in B cells deficient for one or more requisite C-NHEJ factors, such as DNA ligase 4 (Lig4) or XRCC4, end-joining during CSR occurs by a distinct alternative end-joining (A-EJ) pathway. A-EJ also has been implicated in joining DSBs found in oncogenic chromosomal translocations. DNA ligase 3 (Lig3) and its cofactor XRCC1 are widely considered to be requisite A-EJ factors, based on biochemical studies or extrachromosomal substrate end-joining studies. However, potential roles for these factors in A-EJ of endogenous chromosomal DSBs have not been tested. Here, we report that Xrcc1 inactivation via conditional gene-targeted deletion in WT or XRCC4-deficient primary B cells does not have an impact on either CSR or IgH/c-myc translocations in activated B lymphocytes. Indeed, homozygous deletion of Xrcc1 does not impair A-EJ of I-SceI-induced DSBs in XRCC4-deficient pro-B-cell lines. Correspondingly, substantial depletion of Lig3 in Lig4-deficient primary B cells or B-cell lines does not impair A-EJ of CSR-mediated DSBs or formation of IgH/c-myc translocations. Our findings firmly demonstrate that XRCC1 is not a requisite factor for A-EJ of chromosomal DSBs and raise the possibility that DNA ligase 1 (Lig1) may contribute more to A-EJ than previously considered.
经典的非同源末端连接（C-NHEJ）是哺乳动物细胞中主要的 DNA 双链断裂（DSB）修复途径，在激活的 B 淋巴细胞中 Ig 重链（IgH）类别转换重组（CSR）过程中，C-NHEJ 在连接 DSB 中起主导作用。然而，在缺乏一种或多种必需的 C-NHEJ 因子（如 DNA 连接酶 4（Lig4）或 XRCC4）的 B 细胞中，CSR 过程中的末端连接是通过一种独特的替代末端连接（A-EJ）途径发生的。A-EJ 也被认为参与了致癌染色体易位中 DSB 的连接。基于生化研究或染色体外底物末端连接研究，DNA 连接酶 3（Lig3）及其协同因子 XRCC1 被广泛认为是必需的 A-EJ 因子。然而，这些因子在 A-EJ 内源性染色体 DSB 中的潜在作用尚未得到测试。在这里，我们报告称，通过条件性基因靶向删除在 WT 或 XRCC4 缺陷型原代 B 细胞中失活 Xrcc1 ，不会影响激活的 B 淋巴细胞中的 CSR 或 IgH/c-myc 易位。事实上，Xrcc1 纯合缺失不会损害 XRCC4 缺陷型前 B 细胞系中 I-SceI 诱导的 DSB 的 A-EJ。相应地，在 Lig4 缺陷型原代 B 细胞或 B 细胞系中大量耗尽 Lig3 不会损害 CSR 介导的 DSB 的 A-EJ 或 IgH/c-myc 易位的形成。我们的研究结果确凿地证明了 XRCC1 不是染色体 DSB 的 A-EJ 的必需因子，并提出了 DNA 连接酶 1（Lig1）可能比以前认为的更有助于 A-EJ 的可能性。
