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| Status |
Public on Apr 17, 2019 |
| Title |
Phosphorylation at the S2056 1 cluster of DNA-PKcs is dispensable for lymphocyte development and class switch recombination in mice. |
| Organism |
Mus musculus |
| Experiment type |
Other
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| Summary |
The classical non-homologous end-joining (cNHEJ) pathway is a major DNA double-strand break repair pathway in mammalian cells and is required for lymphocyte development and maturation. The DNA-dependent protein kinase (DNA-PK) is a cNHEJ factor that encompasses the Ku70-Ku80 (KU) heterodimer and the large catalytic subunit (DNA-PKcs). In mouse models, loss of DNA-PKcs (DNA-PKcs-/-) abrogates end-processing (e.g., hairpin-opening), but not end-ligation, while expression of the kinase-dead DNA-PKcs protein (DNA-PKcsKD/KD) abrogates end-ligation, suggesting a kinases-dependent structural function of DNA-PKcs during cNHEJ. Lymphocyte development is abolished in DNA-PKcs-/- and DNA-PKcsKD/KD mice due to the requirement for both hairpin-opening and end-ligation during V(D)J recombination. DNA-PKcs itself is the best-characterized substrate of DNA-PK. The S2056-cluster is the best characterized auto-phosphorylation site on human DNA-PKcs. Here we show that radiation can induce phosphorylation of murine DNA-PKcs at the corresponding S2053 and generated knockin mouse models with alanine- (DNA-PKcsPQR) or phospho-mimetic aspartate (DNA-PKcsSD) substitutions at the S2053 cluster. Despite moderate radiation sensitivity in the DNA-PKcsPQR/PQR fibroblasts and lymphocytes, both DNA-PKcsPQR/PQR and DNA-PKcsSD/SD mice retain normal kinase activity, and undergo efficient V(D)J recombination and class switch recombination, indicating that phosphorylation at the S2053-cluster of mouse DNA-PKcs (corresponding to S2056 of human DNA-PKcs), although important for radiation resistance, is dispensable for the end-ligation and hairpin-opening function of DNA-PK essential for lymphocyte development.
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| Overall design |
HTGTS analyzes thousands of CSR junctions between the 5’ of Sμ and its joining partner genome wide, including both internal deletion (Sμ-Sμ) and potentially productive switching (Sμ-Sγ1). We also used HTGTS to uncover a prominent shift to MH, extensive erosion within switch region, and relative increase of inversion in DNA-PKcs-/- B cells with only moderate CSR defects. So we performed HTGTS analyses of CSR junctions from DNA-PKcsPQR/PQR B cells along with control DNA-PKcs-/- B cells. While the results confirmed S region erosion and increased MH usage in DNA-PKcs-/- B cells, no alteration in MH usage and Sµ junction distribution is noted in DNA-PKcsPQR/PQR B cells.
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| Contributor(s) |
Wang XS, Crowe JL, Shao Z, Lee BJ, Zha S |
| Citation missing |
Has this study been published? Please login to update or notify GEO. |
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| Submission date |
Apr 16, 2019 |
| Last update date |
Apr 20, 2019 |
| Contact name |
Zhengping Shao |
| E-mail(s) |
zs2275@cumc.columbia.edu
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| Phone |
212-851-4785
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| Organization name |
Columbia University Medical Center
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| Department |
ICG
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| Lab |
Shan Zha Lab
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| Street address |
1130 St. Nicholas Ave
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| City |
New Yprk |
| State/province |
New York |
| ZIP/Postal code |
10033 |
| Country |
USA |
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| Platforms (1) |
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| Samples (3) |
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| Relations |
| BioProject |
PRJNA533107 |
| SRA |
SRP192737 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE129895_DNA_PKcs_KO_pool_results.xlsx |
4.2 Mb |
(ftp)(http) |
XLSX |
| GSE129895_DNA_PKcs_PQR_pool_results.xlsx |
1.5 Mb |
(ftp)(http) |
XLSX |
| GSE129895_RAW.tar |
1.3 Mb |
(http)(custom) |
TAR (of XLSX) |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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