Ku70 suppresses alternative end joining in G1-arrested B cells
Ku70 suppresses alternative end joining in G1-arrested B cells
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DOI：
10.1073/pnas.2103630118
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发表时间：
2021-05-25
影响因子：
11.1
通讯作者：
Frock, Richard L.
中科院分区：
文献类型：
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作者：
Liang, Zhuoyi;Kumar, Vipul;Frock, Richard L.
经典的非同源末端连接（C-NHEJ）在整个间期修复DNA双链断裂（DSB），但在G1期当同源重组不可用时占主导地位。分别需要含有Ku 70/80（“Ku”）和XRCC 4/连接酶IV（Lig 4）核心C-NHEJ因子的复合物用于感测和连接DSB。虽然XRCC 4/Lig 4是在G1期祖淋巴细胞中V（D）J重组期间连接RAG 1/ 2内切核酸酶（“RAG”）引发的DSB所绝对需要的，但缺乏XRCC 4/Lig 4的循环细胞也可以通过替代性末端连接（A-EJ）途径连接染色体DSB。通过XRCC 4/Lig 4介导的连接对V（D）J重组的限制归因于RAG将V（D）J DSB专门引导到C-NHEJ途径中。在这里，我们报告了Ku抑制了Lig 4缺陷型G1停滞祖细胞B细胞系中由RAG 1/2、Cas9：gRNA和锌指核酸内切酶产生的DSB末端的A-EJ。因此，虽然不同的DSB在Lig 4缺陷的G1停滞的祖B细胞中大部分保持为游离的断裂末端，但Ku 70的缺失增加了DSB的重新连接和易位水平，使其达到在Ku 70缺陷的对应物中观察到的水平。相应地，尽管在Lig 4缺陷型G1停滞祖细胞B细胞系中RAG启动的V（D）J DSB连接被废除，但在Ku 70缺陷型和Ku 70/Lig 4双缺陷型细胞系中RAG产生的DSB的连接通过易位样A-EJ机制发生。因此，在G1期阻滞中，Lig 4缺陷的祖B细胞由于Ku依赖性A-EJ阻断而在功能上受到末端连接抑制，这可能与Lig 1的G1期下调有关。最后，我们认为Ku缺陷与Lig 4缺陷对V（D）J重组、神经元凋亡和胚胎发育的不同影响来自Ku介导的在Lig 4缺陷发育中的淋巴细胞和神经元细胞中G1细胞周期阶段A-EJ的抑制。
Classical nonhomologous end joining (C-NHEJ) repairs DNA doublestrand breaks (DSBs) throughout interphase but predominates 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 XRCC4/Lig4 are absolutely required for joining RAG1/ 2 endonuclease ("RAG")-initiated DSBs during V(D)J recombination in G1-phase progenitor lymphocytes, cycling cells deficient for XRCC4/Lig4 also can join chromosomal DSBs by alternative endjoining (A-EJ) pathways. Restriction of V(D)J recombination by XRCC4/Lig4-mediated joining has been attributed to RAG shepherding V(D)J DSBs exclusively into the C-NHEJ pathway. Here, we report that A-EJ of DSB ends generated by RAG1/2, Cas9:gRNA, and Zinc finger endonucleases in Lig4-deficient G1-arrested progenitor B cell lines is suppressed by Ku. Thus, while diverse DSBs remain largely as free broken ends in Lig4-deficient G1-arrested progenitor B cells, deletion of Ku70 increases DSB rejoining and translocation levels to those observed in Ku70-deficient counterparts. Correspondingly, while RAG-initiated V(D)J DSB joining is abrogated in Lig4-deficient G1-arrested progenitor B cell lines, joining of RAGgenerated DSBs in Ku70-deficient and Ku70/Lig4 double-deficient lines occurs through a translocation-like A-EJ mechanism. Thus, in G1-arrested, Lig4-deficient progenitor B cells are functionally endjoining suppressed due to Ku-dependent blockage of A-EJ, potentially in association with G1-phase down-regulation of Lig1. Finally, we suggest that differential impacts of Ku deficiency versus Lig4 deficiency on V(D)J recombination, neuronal apoptosis, and embryonic development results from Ku-mediated inhibition of A-EJ in the G1 cell cycle phase in Lig4-deficient developing lymphocyte and neuronal cells.