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Series GSE162453 Query DataSets for GSE162453
Status Public on May 08, 2021
Title Ku70 and Ligase IV deficiencies reveal distinct alternative end-joining outcomes in G1-arrested progenitor B cells
Organism Mus musculus
Experiment type Other
Summary Classical nonhomologous 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/Ligase IV (Lig4) core C-NHEJ factors are required, respectively, for sensing and joining DSBs. While such factors are exclusively required for joining RAG1/2-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 RAG1/2 and two classes of engineered endonuclease-mediated DSBs in G1-arrested progenitor B cell lines. Thus, while targeted DSBs remain as free broken ends in Lig4-deficient G1-arrested progenitor B cells, deletion of Ku70 in Lig4-deficient cells restores DSB rejoining 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/Lig4 double-deficient lines through a translocation-based A-EJ mechanism. We conclude that in G1, Lig4-deficient progenitor B cells are functionally end-joining deficient due to a near complete Ku-dependent block in A-EJ. Thus, the differential impacts of Ku deficiency versus XRCC4/Ligase IV deficiency on V(D)J recombination, severity of neuronal apoptosis, and embryonic development, including Ku-deficiency rescuing Lig4-deficient embryonic lethality, may be explained by Ku-mediated inhibition of A-EJ in the G1 cell cycle phase.
 
Overall design We employed JoinT-seq, HTGTS-Rep-Rejoin and LAM-HTGTS to map genome-wide and rejoining repair outcome in G1-arrested murine pro-B cells and to elucidate the roles of Ku70 and Lig4 in repair pathway choice.
We performed GRO-seq to study the gene transcription activity in cycling and G1-arrested v-Abl transformed pro-B cells.
 
Contributor(s) Liang Z, Kumar V, Le Bouteiller M, Zurita J, Kenrick J, Lin SG, Lou J, Hu J, Ye AY, Boboila C, Alt FW, Frock RL
Citation(s) 34006647
Submission date Dec 01, 2020
Last update date May 25, 2021
Contact name Marie Le Bouteiller
E-mail(s) marie.lebouteiller@gmail.com
Organization name Stanford University
Department Radiation Oncology
Street address 269 campus drive
City Stanford
State/province CA
ZIP/Postal code 94305
Country USA
 
Platforms (3)
GPL16417 Illumina MiSeq (Mus musculus)
GPL19057 Illumina NextSeq 500 (Mus musculus)
GPL21626 NextSeq 550 (Mus musculus)
Samples (116)
GSM4952240 WT_A_Cas9_Rep1
GSM4952241 WT_A_Cas9_Rep2
GSM4952242 WT_A_Cas9_Rep3
Relations
BioProject PRJNA681890
SRA SRP295405

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Supplementary file Size Download File type/resource
GSE162453_RAW.tar 3.1 Gb (http)(custom) TAR (of BW, TXT)
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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