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Series GSE117628 Query DataSets for GSE117628
Status Public on Jul 26, 2018
Title Kinase-dependent structural role of DNA-PKcs during immunoglobulin class switch recombination
Organism Mus musculus
Experiment type Other
Summary The catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) is a classical nonhomologous end-joining (cNHEJ) factor. Loss of DNA-PKcs diminished mature B cell class switch recombination (CSR) to other isotypes, but not IgG1. Here, we show that expression of the kinase-dead DNA-PKcs (DNA-PKcsKD/KD) severely compromises CSR to IgG1. High-throughput sequencing analyses of CSR junctions reveal frequent accumulation of nonproductive interchromosomal translocations, inversions, and extensive end resection in DNA-PKcsKD/KD, but not DNA-PKcs-/- B cells. Meanwhile, the residual joints from DNA-PKcsKD/KDcells and the efficient Sμ-Sγ1 junctions from DNA-PKcs-/- B cells both display similar preferences for small (2–6 nt) microhomologies (MH). In DNA-PKcs-/- cells, Sμ-Sγ1 joints are more resistant to inversions and extensive resection than Sμ-Se and Sμ-Sμ joints, providing a mechanism for the isotype-specific CSR defects. Together, our findings identify a kinase-dependent role of DNA-PKcs in suppressing MH-mediated end joining and a structural role of DNA-PKcs protein in the orientation of CSR.
Overall design To ascertain how different DNA-PKcs mutations (null vs KD) affect CSR in an isotype dependent manner, we employed the High Throughput Genome Translocation Sequencing (HTGTS) (1) method to analyze CSR junctions in DNA-PKcsKD/KD and DNA-PKcs-/- B cells with preassembly IgH and L chains (HL). We measured switching efficiency, efficiency and size of small MH in DNA-PKcs-/- and DNA-PKcsKD/KD B cells in contrast to cNHEJ-deficient Xrcc4-/- B cells, as well as insertions and deletions than both Sμ-Sμ and Sμ-Sε junctions. Finally our analyses also identified long MH mediated inter-chromosomal translocations in DNA-PKcsKD/KD B cells and a reduced number of G mutations in 5’Sμ in repair deficient B cells.
Since all our experimental mice carry the preassembled IgH on the 129 ackground, we replaced the IgH switch region (from JH4 to the last Cα exon, chr12 114, 494, 415–114, 666, 816) of the C57/BL6-based mm9 with the corresponding region in the AJ851868.3 (NCBI gene accession no. AJ851868.3) 129 IgH sequence (1415966–1592715) to generate the mm9sr (switch region replacement) genome. 1. Hu J, et al. (2016) Detecting DNA double-stranded breaks in mammalian genomes by linear amplification-mediated high-throughput genome-wide translocation sequencing. Nat Protoc 11:853–871.
Contributor(s) Crowe JL, Shao Z, Wang XS, Zha S
Citation(s) 30072430
Submission date Jul 25, 2018
Last update date Jan 27, 2019
Contact name Zhengping Shao
Phone 212-851-4785
Organization name Columbia University Medical Center
Department ICG
Lab Shan Zha Lab
Street address 1130 St. Nicholas Ave
City New Yprk
State/province New York
ZIP/Postal code 10033
Country USA
Platforms (1)
GPL16417 Illumina MiSeq (Mus musculus)
Samples (16)
GSM3305295 wild type-1
GSM3305296 wild type-2
GSM3305297 wild type-3
BioProject PRJNA482753
SRA SRP155142

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Supplementary file Size Download File type/resource
GSE117628_DNA_PKcs_KD_KD_pool_results.xlsx.gz 638.4 Kb (ftp)(http) XLSX
GSE117628_DNA_PKcs_KO_pool_results.xlsx.gz 3.5 Mb (ftp)(http) XLSX
GSE117628_P53_KO_pool_results.xlsx.gz 2.7 Mb (ftp)(http) XLSX
GSE117628_RAW.tar 10.5 Mb (http)(custom) TAR (of XLSX)
GSE117628_Xrcc4_KO_pool_results.xlsx.gz 3.8 Mb (ftp)(http) XLSX
GSE117628_wild_type_pool_results.xlsx.gz 4.9 Mb (ftp)(http) XLSX
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Raw data are available in SRA
Processed data provided as supplementary file
Processed data are available on Series record

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