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

View ORCID ProfileZhuoyi Liang, Vipul Kumar, View ORCID ProfileMarie Le Bouteiller, Jeffrey Zurita, View ORCID ProfileJosefin Kenrick, Sherry G. Lin, Jiangman Lou, Jianqiao Hu, Adam Yongxin Ye, Cristian Boboila, View ORCID ProfileFrederick W. Alt, View ORCID ProfileRichard L. Frock
doi: https://doi.org/10.1101/2021.02.20.432121
Zhuoyi Liang
1Howard Hughes Medical Institute, Program in Cellular and Molecular Medicine, Boston Children’s Hospital, Department of Genetics, and Department of Pediatrics, Harvard Medical School, Boston, MA 02115, USA
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  • ORCID record for Zhuoyi Liang
Vipul Kumar
1Howard Hughes Medical Institute, Program in Cellular and Molecular Medicine, Boston Children’s Hospital, Department of Genetics, and Department of Pediatrics, Harvard Medical School, Boston, MA 02115, USA
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Marie Le Bouteiller
2Stanford University School of Medicine, Division of Radiation and Cancer Biology, Department of Radiation Oncology, Stanford, CA 94305, USA
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  • ORCID record for Marie Le Bouteiller
Jeffrey Zurita
1Howard Hughes Medical Institute, Program in Cellular and Molecular Medicine, Boston Children’s Hospital, Department of Genetics, and Department of Pediatrics, Harvard Medical School, Boston, MA 02115, USA
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Josefin Kenrick
2Stanford University School of Medicine, Division of Radiation and Cancer Biology, Department of Radiation Oncology, Stanford, CA 94305, USA
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Sherry G. Lin
1Howard Hughes Medical Institute, Program in Cellular and Molecular Medicine, Boston Children’s Hospital, Department of Genetics, and Department of Pediatrics, Harvard Medical School, Boston, MA 02115, USA
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Jiangman Lou
1Howard Hughes Medical Institute, Program in Cellular and Molecular Medicine, Boston Children’s Hospital, Department of Genetics, and Department of Pediatrics, Harvard Medical School, Boston, MA 02115, USA
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Jianqiao Hu
1Howard Hughes Medical Institute, Program in Cellular and Molecular Medicine, Boston Children’s Hospital, Department of Genetics, and Department of Pediatrics, Harvard Medical School, Boston, MA 02115, USA
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Adam Yongxin Ye
1Howard Hughes Medical Institute, Program in Cellular and Molecular Medicine, Boston Children’s Hospital, Department of Genetics, and Department of Pediatrics, Harvard Medical School, Boston, MA 02115, USA
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Cristian Boboila
1Howard Hughes Medical Institute, Program in Cellular and Molecular Medicine, Boston Children’s Hospital, Department of Genetics, and Department of Pediatrics, Harvard Medical School, Boston, MA 02115, USA
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Frederick W. Alt
1Howard Hughes Medical Institute, Program in Cellular and Molecular Medicine, Boston Children’s Hospital, Department of Genetics, and Department of Pediatrics, Harvard Medical School, Boston, MA 02115, USA
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Richard L. Frock
1Howard Hughes Medical Institute, Program in Cellular and Molecular Medicine, Boston Children’s Hospital, Department of Genetics, and Department of Pediatrics, Harvard Medical School, Boston, MA 02115, USA
2Stanford University School of Medicine, Division of Radiation and Cancer Biology, Department of Radiation Oncology, Stanford, CA 94305, USA
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  • For correspondence: frock@stanford.edu
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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 Statement Alternative 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.

Competing Interest Statement

The authors have declared no competing interest.

Footnotes

  • ↵* Frederick W. Alt and Richard L. Frock, Email: alt{at}enders.tch.harvard.edu, frock{at}stanford.edu

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission.
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Ku70 suppresses alternative end-joining in G1-arrested progenitor B cells
Zhuoyi Liang, Vipul Kumar, Marie Le Bouteiller, Jeffrey Zurita, Josefin Kenrick, Sherry G. Lin, Jiangman Lou, Jianqiao Hu, Adam Yongxin Ye, Cristian Boboila, Frederick W. Alt, Richard L. Frock
bioRxiv 2021.02.20.432121; doi: https://doi.org/10.1101/2021.02.20.432121
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Ku70 suppresses alternative end-joining in G1-arrested progenitor B cells
Zhuoyi Liang, Vipul Kumar, Marie Le Bouteiller, Jeffrey Zurita, Josefin Kenrick, Sherry G. Lin, Jiangman Lou, Jianqiao Hu, Adam Yongxin Ye, Cristian Boboila, Frederick W. Alt, Richard L. Frock
bioRxiv 2021.02.20.432121; doi: https://doi.org/10.1101/2021.02.20.432121

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