Ku70 and Ligase IV deficiencies reveal distinct alternative end-joining outcomes in G1-arrested progenitor B cells

Classical nonhomologous end-joining (C-NHEJ) repairs DNA double-stranded breaks (DSBs) throughout interphase but is thought to predominate in G1-phase when homologous recombination is unavailable. Complexes containing the Ku70/80 ("Ku") and XRCC4/Ligase IV (Lig4) core C-NHEJ factors are required, respectively, for sensing and joining DSBs. While such factors are exclusively required for joining RAG1/2-initiated DSBs during V(D)J recombination in G1-phase lymphocyte progenitors, cycling cells deficient for core C-NHEJ factors join chromosomal DSBs by alternative end-joining (A-EJ) pathways. Restriction of V(D)J recombination to C-NHEJ has been attributed to RAG-mediated exclusion of A-EJ; however, it remains unclear whether A-EJ is similarly excluded from more general DSBs in G1. Here, we report that Ku actively and robustly suppresses A-EJ of RAG1/2 and two classes of engineered endonuclease-mediated DSBs in G1-arrested progenitor B cell lines. Thus, while targeted DSBs remain as free broken ends in Lig4-deficient G1-arrested progenitor B cells, deletion of Ku70 in Lig4-deficient cells restores DSB rejoining and translocation to levels observed in Ku70-deficient counterparts. Correspondingly, while V(D)J recombination is abrogated in Ligase4-deficient lines, V(D)J-like joining occurs in Ku70-deficient and Ku70/Lig4 double-deficient lines through a translocation-based A-EJ mechanism. We conclude that in G1, Lig4-deficient progenitor B cells are functionally end-joining deficient due to a near complete Ku-dependent block in A-EJ. Thus, the differential impacts of Ku deficiency versus XRCC4/Ligase IV deficiency on V(D)J recombination, severity of neuronal apoptosis, and embryonic development, including Ku-deficiency rescuing Lig4-deficient embryonic lethality, may be explained by Ku-mediated inhibition of A-EJ in the G1 cell cycle phase. Overall design: We employed JoinT-seq, HTGTS-Rep-Rejoin and LAM-HTGTS to map genome-wide and rejoining repair outcome in G1-arrested murine pro-B cells and to elucidate the roles of Ku70 and Lig4 in repair pathway choice. We performed GRO-seq to study the gene transcription activity in cycling and G1-arrested v-Abl transformed pro-B cells.

经典非同源末端连接（Classical nonhomologous end-joining, C-NHEJ）可在整个细胞间期修复DNA双链断裂（DNA double-stranded breaks, DSBs），但被认为在同源重组无法激活的G1期占据主导地位。包含核心C-NHEJ因子Ku70/80（简称Ku）与XRCC4/连接酶IV（XRCC4/Ligase IV, Lig4）的复合物，分别负责感知并连接DSBs。尽管这类因子是G1期淋巴细胞祖细胞中V(D)J重排（V(D)J recombination）过程中RAG1/2起始的DSB连接所必需的专属因子，但核心C-NHEJ因子缺陷的增殖细胞，可通过替代性末端连接（alternative end-joining, A-EJ）通路完成染色体DSB的连接。V(D)J重排仅能通过C-NHEJ完成这一现象，曾被归因于RAG介导的A-EJ通路排除；然而目前仍不清楚，A-EJ是否同样会被排除在G1期更广泛的DSB修复之外。本研究证实，Ku可在G1期阻滞的祖B细胞系中，强力且特异性地抑制RAG1/2与两类工程核酸酶介导的DSB的A-EJ修复。具体而言，在连接酶IV缺陷的G1期阻滞祖B细胞中，靶向诱导的DSB仍以游离断裂末端形式存在；而在连接酶IV缺陷的细胞中敲除Ku70，可恢复DSB重接与染色体易位的水平，达到与Ku70缺陷细胞相当的程度。相应地，尽管连接酶IV缺陷细胞系中的V(D)J重排完全被消除，但在Ku70缺陷以及Ku70与Lig4双缺陷的细胞系中，可通过基于易位的A-EJ机制发生类V(D)J重接。我们由此得出结论：在G1期，连接酶IV缺陷的祖B细胞存在功能性末端连接缺陷，这是因为A-EJ通路受到近乎完全的Ku依赖性阻滞。因此，Ku缺陷与XRCC4/Ligase IV缺陷对V(D)J重排、神经元凋亡严重程度以及胚胎发育的差异化影响——包括Ku缺陷可挽救Lig4缺陷导致的胚胎致死性——均可通过G1细胞周期时相中Ku介导的A-EJ通路抑制得以解释。实验整体设计：本研究采用JoinT-seq、HTGTS-Rep-Rejoin与LAM-HTGTS技术，对G1期阻滞的小鼠原B细胞开展全基因组定位与重接修复结局分析，以阐明Ku70与Lig4在修复通路选择中的作用。我们还通过GRO-seq技术，研究了增殖及G1期阻滞的v-Abl转化原B细胞中的基因转录活性。