Ku70 suppresses alternative end-joining in G1-arrested progenitor B cells

Liang, Z.; Kumar, V.; Le Bouteiller, M.; Zurita, J.; Kenrick, J.; Lin, S. G.; Lou, J.; Hu, J.; Ye, A. Y.; Boboila, C.; Alt, F. W.; Frock, R. L.

doi:10.1101/2021.02.20.432121

bioRxiv · 10.1101/2021.02.20.432121

Ku70 suppresses alternative end-joining in G1-arrested progenitor B cells

Liang, Z.·Kumar, V.·Le Bouteiller, M.·Zurita, J.·Kenrick, J.·Lin, S. G.·Lou, J.·Hu, J.·Ye, A. Y.·Boboila, C.·Alt, F. W.·Frock, R. L.

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Abstract

Classical nonhomologous end-joining (C-NHEJ) repairs DNA double-stranded 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/Ligase IV are absolutely required for joining RAG1/2-endonucease ("RAG")-initiated DSBs during V(D)J recombination in G1-phase progenitor lymphocytes, cycling cells deficient for XRCC4/Ligase IV also can join chromosomal DSBs by alternative end-joining (A-EJ) pathways. Restriction of V(D)J recombination by XRCC4/Ligase IV-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 Ligase1. 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-defcient developing lymphocyte and neuronal cells. Significance StatementAlternative end-joining (A-EJ) is implicated in oncogenic translocations and mediating DNA double-strand break (DSB) repair in cycling cells when classical nonhomologous endjoining (C-NHEJ) factors of the C-NHEJ Ligase complex are absent. However, V(D)J recombination-associated DSBs that occur in G1 cell cycle-phase progenitor lymphocytes are joined exclusively by the C-NHEJ pathway. Until now, however, the overall mechanisms that join general DSBs in G1-phase progenitor B cells had not been fully elucidated. Here, we report that Ku, a core C-NHEJ double-strand break recognition complex, directs repair of a variety of different targeted DSBs towards C-NHEJ and suppresses A-EJ in G1-phase cells. We suggest this Ku activity explains how Ku-deficiency can rescue the neuronal development and embryonic lethality phenotype of Ligase 4-deficient mice.

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Liang, Z., Kumar, V., Le Bouteiller, M., Zurita, J., Kenrick, J., Lin, S. G., Lou, J., Hu, J., Ye, A. Y., Boboila, C., Alt, F. W., Frock, R. L.. 2021-02-21. Ku70 suppresses alternative end-joining in G1-arrested progenitor B cells. https://doi.org/10.1101/2021.02.20.432121

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