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In vitro Naïve B-cell Class Switching Stimulation with ATM-inhibitor and DNA-PKcs-inhibitor

ABSTRACT: In vitro Naïve B-cell Class Switching Stimulation with ATM-inhibitor and DNA-PKcs-inhibitor

PROVIDER: PRJNA935746 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR23498159_1.fastq.gz	Fastqsanger.gz
	SRR23498159_2.fastq.gz	Fastqsanger.gz
	SRR23498160_1.fastq.gz	Fastqsanger.gz
	SRR23498160_2.fastq.gz	Fastqsanger.gz
	SRR23498161_1.fastq.gz	Fastqsanger.gz

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In vitro Naïve B-cell Class Switching Stimulation with ATM-inhibitor and DNA-PKcs-inhibitor

Project description:Analyze Class switch recombination of naïve B-cells in vitro mimicking Ataxia Telangiectasia using a small molecule inhibitor (KU-55933). We measure switched IgG and IgG memory B-cells, and analyzed transcriptomics (this data).

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Kinase-dependent structural role of DNA-PKcs during immunoglobulin class switch recombination

Project description: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.

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DNA-PKcs phosphorylation at the T2609 cluster alters the repair pathway choice during immunoglobulin class switch recombination

Project description:The DNA-dependent protein kinase (DNA-PK), which is composed of the KU heterodimer and the large catalytic subunit (DNA-PKcs), is a classical non-homologous end-joining (cNHEJ) factor. Naïve B cells undergo class switch recombination (CSR) to generate antibodies with different isotypes by joining two DNA double-strand breaks at different switching regions via the cNHEJ pathway. DNA-PK and the cNHEJ pathway play important roles in the DNA repair phase of CSR. To initiate cNHEJ, KU binds to DNA ends and recruits and activates DNA-PK. Activated DNA-PK phosphorylates DNA-PKcs at the S2056 and T2609 clusters. Loss of T2609 cluster phosphorylation increases radiation sensitivity but whether T2609 phosphorylation has a role in physiological DNA repair remains elusive. Using the DNA-PKcs5A mouse model carrying alanine substitutions at the T2609 cluster, here we show that loss of T2609 phosphorylation of DNA-PKcs does not affect the CSR efficiency. Yet, the CSR junctions recovered from DNA-PKcs5A/5A B cells reveal increased chromosomal translocations, extensive use of distal switch regions (consistent with end-resection), and preferential usage of micro-homology – all signs of the alternative end-joining pathway. Thus, these results uncover a role of DNA-PKcs T2609 phosphorylation in promoting cNHEJ repair pathway choice during CSR.

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Project description:In depth analysis of double strand break signaling was performed using mouse Pre-B cells and Human HCT116 cells. Our analysis included the induction of double strand breaks with ionizing radiation with the addition of ATM and DNA-PKcs inhibitors, alone and in combination.

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Phosphorylation at the S2056 1 cluster of DNA-PKcs is dispensable for lymphocyte development and class switch recombination in mice.

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Project description:The non-homologous end-joining (NHEJ) pathway is a major DNA double-strand break repair pathway in mammals and is essential for lymphocyte development. Ku70 and Ku80 heterodimer (KU) initiates NHEJ, thereby recruiting and activating the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs). While DNA-PKcs deletion only moderately impairs end-ligation, the expression of Kinase-dead DNA-PKcs completely abrogates NHEJ. Active DNA-PK phosphorylates DNA-PKcs at two clusters – PQR around S2056 (S2053 in mouse) and ABCDE around T2609. Alanine substitution at the S2056 cluster moderately compromises end-ligation on plasmid-based assays. But mice carrying alanine substitution at all 5 serine residues within the S2056 cluster (DNA-PKcsPQR/PQR) display no defect in lymphocyte development, leaving the physiological significance of S2056 cluster phosphorylation elusive. XLF is a non-essential NHEJ factor. Xlf-/- mice have substantial peripheral lymphocytes that are completely abolished by the loss of DNA-PKcs, the related ATM kinases, other chromatin associated DNA damage response factors (e.g., 53BP1, MDC1, H2AX, MRI, etc.) or RAG2-C-terminal regions, suggesting functional redundancy. While ATM inhibition does not further compromise end-ligation, here we show that in XLF-deficient background, DNA-PKcs S2056 cluster phosphorylation is critical for normal lymphocyte development. Chromosomal V(D)J recombination from DNA-PKcsPQR/PQRXlf-/- B cells is efficient but often has large deletions that jeopardize lymphocyte development. Class switch recombination junctions from DNA-PKcsPQR/PQRXlf-/- mice are less efficient and the residual junctions display decreased fidelity and increased deletion. These findings establish a role for DNA-PKcs S2056 cluster phosphorylation in physiological chromosomal NHEJ, implying that S2056 cluster phosphorylation contributes to the synergy between XLF and DNA-PKcs in end-ligation.

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Project description:Kinase-dependent structural role of DNA-PKcs during immunoglobulin class switch recombination

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