Ku70 suppresses alternative end joining in G1-arrested progenitor B cells
Zhuoyi Liang, Vipul Kumar, Marie Le Bouteiller, Jeffrey Zurita, Josefin Kenrick, Sherry Lin, Jiangman Lou, Jianqiao Hu, Adam Yongxin Ye, Cristian Boboilă, Frederick W. Alt, Richard L. Frock
Abstract
Classical nonhomologous end joining (C-NHEJ) repairs DNA double-strand 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 end-joining (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 RAG-generated 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 end-joining 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.