Functional redundancy between the XLF and DNA-PKcs DNA repair factors in V(D)J recombination and nonhomologous DNA end joining

Oksenych, Valentyn; Kumar, Vipul; Liu, Xiangyu; Guo, Chunguang; Schwer, Bjoern; Zha, Shan; Alt, Frederick W.

doi:10.1073/pnas.1222573110

“…Thus, although ATM deficiency only mildly impacts V(D)J recombination, this process is abrogated in developing pro-B cells dually deficient for XLF and ATM or downstream DSB response factors (16)(17)(18). XLF also is functionally redundant with DNA-PKcs in V(D)J recombination signal end joining (19). Potential processes in which XLF and DSBR factors may be functionally redundant are not well-characterized but may include tethering ends or facilitating their joining (6,16).…”

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confidence: 99%

PAXX and XLF DNA repair factors are functionally redundant in joining DNA breaks in a G1-arrested progenitor B-cell line

Kumar

¹

,

Fw

²

,

Frock

³

2016

Proc. Natl. Acad. Sci. U.S.A.

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Classical nonhomologous end joining (C-NHEJ) is a major mammalian DNA double-strand break (DSB) repair pathway. Core C-NHEJ factors, such as XRCC4, are required for joining DSB intermediates of the G1 phase-specific V(D)J recombination reaction in progenitor lymphocytes. Core factors also contribute to joining DSBs in cycling mature B-lineage cells, including DSBs generated during antibody class switch recombination (CSR) and DSBs generated by ionizing radiation. The XRCC4-like-factor (XLF) C-NHEJ protein is dispensable for V(D)J recombination in normal cells, but because of functional redundancy, it is absolutely required for this process in cells deficient for the ataxia telangiectasia-mutated (ATM) DSB response factor. The recently identified paralogue of XRCC4 and XLF (PAXX) factor has homology to these two proteins and variably contributes to ionizing radiation-induced DSB repair in human and chicken cells. We now report that PAXX is dispensable for joining V(D)J recombination DSBs in G1-arrested mouse pro-B-cell lines, dispensable for joining CSR-associated DSBs in a cycling mouse B-cell line, and dispensable for normal ionizing radiation resistance in both G1-arrested and cycling pro-B lines. However, we find that combined deficiency for PAXX and XLF in G1-arrested pro-B lines abrogates DSB joining during V(D)J recombination and sensitizes the cells to ionizing radiation exposure. Thus, PAXX provides core C-NHEJ factor-associated functions in the absence of XLF and vice versa in G1-arrested pro-B-cell lines. Finally, we also find that PAXX deficiency has no impact on V(D)J recombination DSB joining in ATM-deficient pro-B lines. We discuss implications of these findings with respect to potential PAXX and XLF functions in C-NHEJ.T o repair DNA double-strand breaks (DSBs), mammalian cells use two major DSB repair pathways: homologous recombination (HR) and classical nonhomologous end joining (C-NHEJ). HR requires a long homologous template for repair and is active in S/G2 cell cycle phase (1). In contrast, C-NHEJ, which directly ligates DSBs with short (1-4 bp) or no homologies, functions throughout interphase, including during the G1 cell cycle phase (2-4). C-NHEJ repairs diverse DSBs, including those arising ectopically by ionizing radiation as well as DSBs generated as intermediates during V(D)J recombination in developing progenitor (pro)-B and -T lymphocytes and Ig heavy chain (IgH) class switch recombination (CSR) in activated mature B lymphocytes (5). The C-NHEJ pathway has a number of characterized members, including "core" C-NHEJ factors that were so-named in part based on their requirement for joining known types of ends via C-NHEJ and their evolutionary conservation (6). Such core factors include the Ku70 and Ku80 proteins, which form the "Ku" end recognition complex, and the XRCC4 and DNA Ligase4 proteins, which form the C-NHEJ ligation complex (5, 6).Exons that encode Ig and T-cell receptor (TCR) variable region exons are assembled from germline V, D, and J gene segments. This V(D)J recombinatio...

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“…DNA-PKcs has redundant functions with ATM and Cernunnos during DNA repair (49)(50)(51)(52). Double deficiency in DNA-PKcs and ATM or Cernunnos results in more severe defects in both CSR (17,51) and V(D)J recombination (51) than does single deficiency in DNA-PKcs, ATM, or Cernunnos in mice.…”

Section: Discussionmentioning

confidence: 99%

“…Double deficiency in DNA-PKcs and ATM or Cernunnos results in more severe defects in both CSR (17,51) and V(D)J recombination (51) than does single deficiency in DNA-PKcs, ATM, or Cernunnos in mice. The convergent roles could be due to shared phosphorylating substrates (51,52) between the proteins or caused by other overlapping functions, such as in end-protection (51).…”

Section: Discussionmentioning

confidence: 99%

“…Double deficiency in DNA-PKcs and ATM or Cernunnos results in more severe defects in both CSR (17,51) and V(D)J recombination (51) than does single deficiency in DNA-PKcs, ATM, or Cernunnos in mice. The convergent roles could be due to shared phosphorylating substrates (51,52) between the proteins or caused by other overlapping functions, such as in end-protection (51). However, the redundancy, at least between DNA-PKcs and Cernunnos, does not seem to be identical in human and mouse cells, because Cernunnos-deficient patients have a much more severe switching defect and immunodeficiency than do the corresponding knockout mice (37,53).…”

Section: Discussionmentioning

confidence: 99%

“…However, the redundancy, at least between DNA-PKcs and Cernunnos, does not seem to be identical in human and mouse cells, because Cernunnos-deficient patients have a much more severe switching defect and immunodeficiency than do the corresponding knockout mice (37,53). Furthermore, developmental problems were not observed in DNA-PKcs-deficient or Cernunnos-deficient mice, but they were present in mice deficient in both proteins (51). In contrast, both DNA-PKcs-deficient (23) and Cernunnosdeficient (54) humans present with growth delay, as well as neurologic abnormalities.…”

Section: Discussionmentioning

confidence: 99%

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DNA-PKcs Is Involved in Ig Class Switch Recombination in Human B Cells

Björkman

¹

,

Du

²

,

Felgentreff

³

et al. 2015

The Journal of Immunology

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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.

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Robust DNA repair in PAXX‐deficient mammalian cells

Dewan

¹

,

Xing

²

,

Lundbæk

³

et al. 2018

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To ensure genome stability, mammalian cells employ several DNA repair pathways. Nonhomologous DNA end joining ( NHEJ ) is the DNA repair process that fixes double‐strand breaks throughout the cell cycle. NHEJ is involved in the development of B and T lymphocytes through its function in V(D)J recombination and class switch recombination ( CSR ). NHEJ consists of several core and accessory factors, including Ku70, Ku80, XRCC 4, DNA ligase 4, DNA ‐ PK cs, Artemis, and XLF . Paralog of XRCC 4 and XLF ( PAXX ) is the recently described accessory NHEJ factor that structurally resembles XRCC 4 and XLF and interacts with Ku70/Ku80. To determine the physiological role of PAXX in mammalian cells, we purchased and characterized a set of custom‐generated and commercially available NHEJ ‐deficient human haploid HAP 1 cells, PAXX Δ , XRCC 4 Δ , and XLF Δ . In our studies, HAP 1 PAXX Δ cells demonstrated modest sensitivity to DNA damage, which was comparable to wild‐type controls. By contrast, XRCC 4 Δ and XLF Δ HAP 1 cells possessed significant DNA repair defects measured as sensitivity to double‐strand break inducing agents and chromosomal breaks. To investigate the role of PAXX in CSR , we generated and characterized Paxx −/− and Aid −/− murine lymphoid CH 12F3 cells. CSR to IgA was nearly at wild‐type levels in the Paxx −/− cells and completely ablated in the absence of activation‐induced cytidine deaminase ( AID ). In addition, Paxx −/− CH 12F3 cells were hypersensitive to zeocin when compared to wild‐type controls. We concluded that Paxx ‐deficient mammalian cells maintain robust NHEJ and CSR .

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Functional redundancy between the XLF and DNA-PKcs DNA repair factors in V(D)J recombination and nonhomologous DNA end joining

Cited by 84 publications

References 46 publications

PAXX and XLF DNA repair factors are functionally redundant in joining DNA breaks in a G1-arrested progenitor B-cell line

PAXX and XLF DNA repair factors are functionally redundant in joining DNA breaks in a G1-arrested progenitor B-cell line

DNA-PKcs Is Involved in Ig Class Switch Recombination in Human B Cells

Robust DNA repair in PAXX‐deficient mammalian cells

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