scholarly journals HMCES Functions in the Alternative End-Joining Pathway of the DNA DSB Repair during Class Switch Recombination in B Cells

2020 ◽  
Vol 77 (5) ◽  
pp. 1154 ◽  
Author(s):  
Vipul Shukla ◽  
Levon Halabelian ◽  
Sanjana Balagere ◽  
Daniela Samaniego-Castruita ◽  
Douglas E. Feldman ◽  
...  
Keyword(s):  
B Cells ◽  
Class Switch Recombination ◽  
End Joining ◽  
Dsb Repair ◽  
Class Switch ◽  
Alternative End Joining

scholarly journals Alternative end-joining catalyzes class switch recombination in the absence of both Ku70 and DNA ligase 4

2010 ◽  
Vol 207 (2) ◽  
pp. 417-427 ◽  
Author(s):  
Cristian Boboila ◽  
Catherine Yan ◽  
Duane R. Wesemann ◽  
Mila Jankovic ◽  
Jing H. Wang ◽  
...  
Keyword(s):  
B Cells ◽  
Class Switch Recombination ◽  
Strand Break ◽  
Nonhomologous End Joining ◽  
Dna Ligase ◽  
End Joining ◽  
Dsb Repair ◽  
Class Switch ◽  
Dna Double Strand Break ◽  
Alternative End Joining

The classical nonhomologous end-joining (C-NHEJ) DNA double-strand break (DSB) repair pathway employs the Ku70/80 complex (Ku) for DSB recognition and the XRCC4/DNA ligase 4 (Lig4) complex for ligation. During IgH class switch recombination (CSR) in B lymphocytes, switch (S) region DSBs are joined by C-NHEJ to form junctions either with short microhomologies (MHs; “MH-mediated” joins) or no homologies (“direct” joins). In the absence of XRCC4 or Lig4, substantial CSR occurs via “alternative” end-joining (A-EJ) that generates largely MH-mediated joins. Because upstream C-NHEJ components remain in XRCC4- or Lig4-deficient B cells, residual CSR might be catalyzed by C-NHEJ using a different ligase. To address this, we have assayed for CSR in B cells deficient for Ku70, Ku80, or both Ku70 and Lig4. Ku70- or Ku80-deficient B cells have reduced, but still substantial, CSR. Strikingly, B cells deficient for both Ku plus Lig4 undergo CSR similarly to Ku-deficient B cells, firmly demonstrating that an A-EJ pathway distinct from C-NHEJ can catalyze CSR end-joining. Ku-deficient or Ku- plus Lig4-deficient B cells are also biased toward MH-mediated CSR joins; but, in contrast to XRCC4- or Lig4-deficient B cells, generate substantial numbers of direct CSR joins. Our findings suggest that more than one form of A-EJ can function in CSR.

scholarly journals HMCES Functions in the Alternative End-Joining Pathway of the DNA DSB Repair during Class Switch Recombination in B Cells

2020 ◽  
Vol 77 (2) ◽  
pp. 384-394.e4 ◽  
Author(s):  
Vipul Shukla ◽  
Levon Halabelian ◽  
Sanjana Balagere ◽  
Daniela Samaniego-Castruita ◽  
Douglas E. Feldman ◽  
...  
Keyword(s):  
B Cells ◽  
Class Switch Recombination ◽  
End Joining ◽  
Dsb Repair ◽  
Class Switch ◽  
Alternative End Joining

scholarly journals CtIP-mediated DNA resection is dispensable for IgH class switch recombination by alternative end-joining

2020 ◽  
Vol 117 (41) ◽  
pp. 25700-25711
Author(s):  
Xiaobin S. Wang ◽  
Junfei Zhao ◽  
Foon Wu-Baer ◽  
Zhengping Shao ◽  
Brian J. Lee ◽  
...  
Keyword(s):  
B Cells ◽  
High Throughput Sequencing ◽  
Class Switch Recombination ◽  
End Joining ◽  
Atm Kinase ◽  
Class Switch ◽  
Switch Regions ◽  
Alternative End Joining ◽  
Dna End Resection ◽  
End Resection

To generate antibodies with different effector functions, B cells undergo Immunoglobulin Heavy Chain (IgH) class switch recombination (CSR). The ligation step of CSR is usually mediated by the classical nonhomologous end-joining (cNHEJ) pathway. In cNHEJ-deficient cells, a remarkable ∼25% of CSR can be achieved by the alternative end-joining (Alt-EJ) pathway that preferentially uses microhomology (MH) at the junctions. While A-EJ-mediated repair of endonuclease-generated breaks requires DNA end resection, we show that CtIP-mediated DNA end resection is dispensable for A-EJ-mediated CSR using cNHEJ-deficient B cells. High-throughput sequencing analyses revealed that loss of ATM/ATR phosphorylation of CtIP at T855 or ATM kinase inhibition suppresses resection without altering the MH pattern of the A-EJ-mediated switch junctions. Moreover, we found that ATM kinase promotes Alt-EJ-mediated CSR by suppressing interchromosomal translocations independent of end resection. Finally, temporal analyses reveal that MHs are enriched in early internal deletions even in cNHEJ-proficient B cells. Thus, we propose that repetitive IgH switch regions represent favored substrates for MH-mediated end-joining contributing to the robustness and resection independence of A-EJ-mediated CSR.

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

2018 ◽  
Vol 115 (34) ◽  
pp. 8615-8620 ◽  
Author(s):  
Jennifer L. Crowe ◽  
Zhengping Shao ◽  
Xiaobin S. Wang ◽  
Pei-Chi Wei ◽  
Wenxia Jiang ◽  
...  
Keyword(s):  
B Cells ◽  
High Throughput Sequencing ◽  
Class Switch Recombination ◽  
End Joining ◽  
Extensive Resection ◽  
Structural Role ◽  
Class Switch ◽  
Cell Class ◽  
Dependent Protein

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/KD cells 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-Sε 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.

scholarly journals Alternative end-joining catalyzes class switch recombination in the absence of both Ku70 and DNA ligase 4

2013 ◽  
Vol 210 (3) ◽  
pp. 641-641 ◽  
Author(s):  
Cristian Boboila ◽  
Catherine Yan ◽  
Duane R. Wesemann ◽  
Mila Jankovic ◽  
Jing H. Wang ◽  
...  
Keyword(s):  
Class Switch Recombination ◽  
Dna Ligase ◽  
End Joining ◽  
Class Switch ◽  
Alternative End Joining

scholarly journals Alternative end-joining catalyzes class switch recombination in the absence of both Ku70 and DNA ligase 4

2010 ◽  
Vol 188 (4) ◽  
pp. i7-i7
Author(s):  
Cristian Boboila ◽  
Catherine Yan ◽  
Duane R. Wesemann ◽  
Mila Jankovic ◽  
Jing H. Wang ◽  
...  
Keyword(s):  
Class Switch Recombination ◽  
Dna Ligase ◽  
End Joining ◽  
Class Switch ◽  
Alternative End Joining

scholarly journals Multiple DSB Resection Activities Redundantly Promote Alternative End Joining-Mediated Class Switch Recombination

2021 ◽  
Vol 9 ◽  
Author(s):  
Xikui Sun ◽  
Jingning Bai ◽  
Jiejie Xu ◽  
Xiaoli Xi ◽  
Mingyu Gu ◽  
...  
Keyword(s):  
High Throughput Sequencing ◽  
Class Switch Recombination ◽  
Class Switching ◽  
End Joining ◽  
Independent Manner ◽  
Class Switch ◽  
Alternative End Joining ◽  
A Cell ◽  
Non Homologous End Joining ◽  
Antibody Class

Alternative end joining (A-EJ) catalyzes substantial level of antibody class switch recombination (CSR) in B cells deficient for classical non-homologous end joining, featuring increased switch (S) region DSB resection and junctional microhomology (MH). While resection has been suggested to initiate A-EJ in model DSB repair systems using engineered endonucleases, the contribution of resection factors to A-EJ-mediated CSR remains unclear. In this study, we systematically dissected the requirement for individual DSB resection factors in A-EJ-mediated class switching with a cell-based assay system and high-throughput sequencing. We show that while CtIP and Mre11 both are mildly required for CSR in WT cells, they play more critical roles in mediating A-EJ CSR, which depend on the exonuclease activity of Mre11. While DNA2 and the helicase/HRDC domain of BLM are required for A-EJ by mediating long S region DSB resection, in contrast, Exo1’s resection-related function does not play any obvious roles for class switching in either c-NHEJ or A-EJ cells, or mediated in an AID-independent manner by joining of Cas9 breaks. Furthermore, ATM and its kinase activity functions at least in part independent of CtIP/Mre11 to mediate A-EJ switching in Lig4-deficient cells. In stark contrast to Lig4 deficiency, 53BP1-deficient cells do not depend on ATM/Mre11/CtIP for residual joining. We discuss the roles for each resection factor in A-EJ-mediated CSR and suggest that the extent of requirements for resection is context dependent.

scholarly journals BRCT-domain protein BRIT1 influences class switch recombination

2017 ◽  
Vol 114 (31) ◽  
pp. 8354-8359 ◽  
Author(s):  
Wei-Feng Yen ◽  
Ashutosh Chaudhry ◽  
Bharat Vaidyanathan ◽  
William T. Yewdell ◽  
Joseph N. Pucella ◽  
...  
Keyword(s):  
B Cells ◽  
Ex Vivo ◽  
Cytidine Deaminase ◽  
Class Switch Recombination ◽  
B Cell Lymphoma ◽  
Brct Domain ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Class Switch ◽  
Domain Protein

DNA double-strand breaks (DSBs) serve as obligatory intermediates for Ig heavy chain (Igh) class switch recombination (CSR). The mechanisms by which DSBs are resolved to promote long-range DNA end-joining while suppressing genomic instability inherently associated with DSBs are yet to be fully elucidated. Here, we use a targeted short-hairpin RNA screen in a B-cell lymphoma line to identify the BRCT-domain protein BRIT1 as an effector of CSR. We show that conditional genetic deletion of BRIT1 in mice leads to a marked increase in unrepaired Igh breaks and a significant reduction in CSR in ex vivo activated splenic B cells. We find that the C-terminal tandem BRCT domains of BRIT1 facilitate its interaction with phosphorylated H2AX and that BRIT1 is recruited to the Igh locus in an activation-induced cytidine deaminase (AID) and H2AX-dependent fashion. Finally, we demonstrate that depletion of another BRCT-domain protein, MDC1, in BRIT1-deleted B cells increases the severity of CSR defect over what is observed upon loss of either protein alone. Our results identify BRIT1 as a factor in CSR and demonstrate that multiple BRCT-domain proteins contribute to optimal resolution of AID-induced DSBs.

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