Homologous recombination

Non-homologous end joining

Gene

Cell biology

DNA-binding protein

Molecular biology

Biology

Ku80

Ku70

Stem cell

Genetics

Progenitor cell

Chromosomal translocation

Nucleotide excision repair

DNA repair

Transcription factor

DNA mismatch repair

DNA repair protein XRCC4

Cancer Research

Classical non-homologous 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/Ligase4 core C-NHEJ factors are required, respectively, for sensing and joining DSBs. While such factors are exclusively required for joining RAG-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 both RAG- and Cas9-mediated DSBs in G1-arrested progenitor B cell lines. Thus, while Cas9/gRNA DSBs remain as free broken ends in Ligase4-deficient G1-arrested progenitor B cells, deletion of Ku70 in Ligase4-deficient cells restores DSB re-joining 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/Ligase4-double-deficient lines through a translocation-based A-EJ mechanism. We conclude that in G1, Ligase4-deficient progenitor B cells are functionally end-joining deficient due to a complete Ku-dependent block in A-EJ. Thus, the differential impacts of Ku deficiency versus XRCC4/Ligase4 deficiency on V(D)J recombination, severity of neuronal apoptosis, and embryonic development, including ability of Ku-deficiency to rescue Ligase4-deficient embryonic lethality, may be explained by Ku-mediated inhibition of A-EJ in the G1 cell cycle phase. Citation Format: Richard L. Frock, Vipul Kumar, Zhouyi Liang, Jeff Zurita, Zhou Du, Sherry G. Lin, Cristian Boboila, Frederick W. Alt. Ku70 suppresses alternative end-joining in G1-arrested progenitor B cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1745.

Abstract 1745: Ku70 suppresses alternative end-joining in G1-arrested progenitor B cells