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. 2004 May 24;165(4):459-64.
doi: 10.1083/jcb.200403021.

53BP1 is required for class switch recombination

Irene M Ward  1 , Bernardo Reina-San-Martin, Alexandru Olaru, Kay Minn, Koji Tamada, Julie S Lau, Marilia Cascalho, Lieping Chen, Andre Nussenzweig, Ferenc Livak, Michel C Nussenzweig, Junjie Chen
Affiliations

53BP1 is required for class switch recombination

Irene M Ward et al. J Cell Biol. .

Abstract

53BP1 participates early in the DNA damage response and is involved in cell cycle checkpoint control. Moreover, the phenotype of mice and cells deficient in 53BP1 suggests a defect in DNA repair (Ward et al., 2003b). Therefore, we asked whether or not 53BP1 would be required for the efficient repair of DNA double strand breaks. Our data indicate that homologous recombination by gene conversion does not depend on 53BP1. Moreover, 53BP1-deficient mice support normal V(D)J recombination, indicating that 53BP1 is not required for "classic" nonhomologous end joining. However, class switch recombination is severely impaired in the absence of 53BP1, suggesting that 53BP1 facilitates DNA end joining in a way that is not required or redundant for the efficient closing of RAG-induced strand breaks. These findings are similar to those observed in mice or cells deficient in the tumor suppressors ATM and H2AX, further suggesting that the functions of ATM, H2AX, and 53BP1 are closely linked.

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Figures

Figure 1.
Figure 1.
53BP1 and DNA damage repair. (A) Increased apoptosis in thymocytes derived from irradiated 53BP1−/− mice. Thymocytes were isolated from wild-type and 53BP1-deficient mice 8 h after irradiation with 5 Gy of IR and stained with annexin–FITC and PI. (B) 53BP1−/− embryonic cells show normal levels of DNA DSB rejoining. Wild-type and 53BP1-deficient cells were irradiated with different doses of IR and either allowed to recover (R) for 15 min or 24 h, as indicated, or immediately processed for PFGE analysis.
Figure 2.
Figure 2.
53BP1 is not required for homology-directed repair. Wild-type or 53BP1-deficient embryonic cells were cotransfected with an I-SceI repair substrate (DR-GFP) composed of two differentially mutated GFP and an I-SceI expression plasmid (pCBASce). Reconstitution of the GFP reporter gene by HR was assessed 46 h later by flow cytometry. As a control, cells were transfected with either DR-GFP alone or with a functional GFP expression plasmid (pIRES GFPpuro).
Figure 3.
Figure 3.
53BP1 is not required for NHEJ. Wild-type or 53BP1-deficient embryonic cells as well as DNA-PKcs–deficient and –reconstituted cells were transfected with plasmid pDVG94 linearized in such a way that joining on a particular microhomology creates a novel BstXI restriction site. 48 h later, the plasmid was recovered from the cells and the joining region was amplified by PCR. An aliquot of the PCR reaction was digested with BstXI, and uncut (180 bp) or BstXI-cut (120 bp) fragments were separated by gel electrophoresis.
Figure 4.
Figure 4.
Normal TCR gene rearrangements in the absence of 53BP1. Serial dilutions of thymus DNA from 1-mo-old 53BP1-deficient mice and their wild-type littermates were PCR-amplified with primers specific for various TCRα, β, γ, or δ rearrangements or TCRα signal joints. The PCR products were Southern blotted and hybridized with the different gene-specific reverse probes as described in Materials and methods. PCR of the nonrearranging gene RAG-2 served as control for DNA quantity and integrity.
Figure 5.
Figure 5.
CSR is dependent on 53BP1. Cell division (A) and surface IgG1 expression (B) as measured by flow cytometry on live CFSE-labeled wild type (WT) and 53BP1−/− B cells stimulated with LPS plus IL-4 for 4 d. Results are representative of four experiments. Real-time RT-PCR for μ and γ1 sterile transcripts (C) and γ1 circle transcript (D) in wild-type (solid bars) and 53BP1−/− (open bars) B cells stimulated with LPS and IL-4 for 3 d. Results from four independent cultures are expressed as fold induction relative to wild-type B cells.
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