Related Experiment Video
Updated: Mar 30, 2026
Induction and Assessment of Class Switch Recombination in Purified Murine B Cells
Published on: August 13, 2010
DNA-PKcs Is Involved in Ig Class Switch Recombination in Human B Cells
Andrea Björkman1, Likun Du1, Kerstin Felgentreff2
1Department of Laboratory Medicine, Karolinska Institutet, 141 86 Stockholm, Sweden;
Insights
DNA-dependent protein kinase, catalytic subunit (DNA-PKcs) deficiency impairs DNA repair pathways essential for B cell development. Studies in human and mouse models reveal a shift towards alternative end-joining during class switch recombination when DNA-PKcs is defective.
Area of Science:
- Immunology
- Molecular Biology
- Genetics
Background:
- Nonhomologous end-joining (NHEJ) is a critical DNA repair pathway in mammals.
- NHEJ is essential for V(D)J recombination and class switch recombination (CSR) in B cells.
- DNA-dependent protein kinase, catalytic subunit (DNA-PKcs) is a key component of NHEJ, but its role in CSR is debated.
Purpose of the Study:
- To investigate the role of DNA-PKcs in class switch recombination (CSR) during B cell development.
- To analyze recombination junctions in DNA-PKcs-deficient cells from human patients and mouse models.
- To determine if DNA-PKcs kinase activity is essential for its function in CSR and NHEJ.
Main Methods:
- Analysis of recombination junction patterns in B cells from two DNA-PKcs-deficient human patients.
- Comparison with recombination data from DNA-PKcs-deficient mouse B cells.
- Assessment of DNA-PKcs mutations affecting kinase activity and Artemis activation.
Main Results:
- A shift towards microhomology-based alternative end-joining (AEJ) was observed in CSR junctions from both human and mouse DNA-PKcs-deficient B cells.
- This suggests that classical NHEJ is impaired in CSR when DNA-PKcs is defective.
- Cells from a patient with diminished DNA-PKcs expression and activity showed more severe alterations, indicating potential Artemis-independent functions.
Conclusions:
- DNA-PKcs is crucial for efficient classical NHEJ during class switch recombination (CSR).
- Deficiency in DNA-PKcs leads to increased reliance on alternative end-joining (AEJ) pathways.
- DNA-PKcs kinase activity may have additional, Artemis-independent roles in CSR and NHEJ processes.
Abstract:
Nonhomologous end-joining (NHEJ) is one of the major DNA double-strand break repair pathways in mammalian cells and is required for both V(D)J recombination and class switch recombination (CSR), two Ig gene-diversification processes occurring during B cell development. DNA-dependent protein kinase, catalytic subunit (DNA-PKcs) is a component of the classical NHEJ machinery and has a critical function during V(D)J recombination. However, its role in CSR has been controversial. In this study, we examined the pattern of recombination junctions from in vivo-switched B cells from two DNA-PKcs-deficient patients. One of them harbored mutations that did not affect DNA-PKcs kinase activity but caused impaired Artemis activation; the second patient had mutations resulting in diminished DNA-PKcs protein expression and kinase activity. These results were compared with those from DNA-PKcs-deficient mouse B cells. A shift toward the microhomology-based alternative end-joining at the recombination junctions was observed in both human and mouse B cells, suggesting that the classical NHEJ pathway is impaired during CSR when DNA-PKcs is defective. Furthermore, cells from the second patient showed additional or more severe alterations in CSR and/or NHEJ, which may suggest that DNA-PKcs and/or its kinase activity have additional, Artemis-independent functions during these processes.
Related Concept Videos
01:28Diversity of Antigen Receptors
Before encountering any antigen, lymphocytes express these receptors. On B cells, the antigen receptor is a membrane-bound antibody molecule called BCR; on T cells, it is a T cell receptor or TCR. B and T cell receptors are composed of two...
02:53Conservative Site-specific Recombination and Phase Variation
The recognition sites for Cre recombinase called LoxP...
01:24B Cell Activation and Differentiation
When naive B cells encounter a specific antigen that can bind to the B cell receptor (BCR) on their surface, they undergo sensitization to respond to the antigen's presence. Sensitization begins with...
02:32Exon Recombination
Exon shuffling follows “splice frame rules.” Each exon...
02:31Homologous Recombination
02:31Homologous Recombination