Robust chromosomal DNA repair via alternative end-joining in the absence of X-ray repair cross-complementing protein 1 (XRCC1) - PubMed (original) (raw)

. 2012 Feb 14;109(7):2473-8.

doi: 10.1073/pnas.1121470109. Epub 2012 Jan 30.

Valentyn Oksenych, Monica Gostissa, Jing H Wang, Shan Zha, Yu Zhang, Hua Chai, Cheng-Sheng Lee, Mila Jankovic, Liz-Marie Albertorio Saez, Michel C Nussenzweig, Peter J McKinnon, Frederick W Alt, Bjoern Schwer

Affiliations

Robust chromosomal DNA repair via alternative end-joining in the absence of X-ray repair cross-complementing protein 1 (XRCC1)

Cristian Boboila et al. Proc Natl Acad Sci U S A. 2012.

Erratum in

Abstract

Classical nonhomologous DNA end-joining (C-NHEJ), which is a major DNA double-strand break (DSB) repair pathway in mammalian cells, plays a dominant role in joining DSBs during Ig heavy chain (IgH) class switch recombination (CSR) in activated B lymphocytes. However, in B cells deficient for one or more requisite C-NHEJ factors, such as DNA ligase 4 (Lig4) or XRCC4, end-joining during CSR occurs by a distinct alternative end-joining (A-EJ) pathway. A-EJ also has been implicated in joining DSBs found in oncogenic chromosomal translocations. DNA ligase 3 (Lig3) and its cofactor XRCC1 are widely considered to be requisite A-EJ factors, based on biochemical studies or extrachromosomal substrate end-joining studies. However, potential roles for these factors in A-EJ of endogenous chromosomal DSBs have not been tested. Here, we report that Xrcc1 inactivation via conditional gene-targeted deletion in WT or XRCC4-deficient primary B cells does not have an impact on either CSR or IgH/c-myc translocations in activated B lymphocytes. Indeed, homozygous deletion of Xrcc1 does not impair A-EJ of I-SceI-induced DSBs in XRCC4-deficient pro-B-cell lines. Correspondingly, substantial depletion of Lig3 in Lig4-deficient primary B cells or B-cell lines does not impair A-EJ of CSR-mediated DSBs or formation of IgH/c-myc translocations. Our findings firmly demonstrate that XRCC1 is not a requisite factor for A-EJ of chromosomal DSBs and raise the possibility that DNA ligase 1 (Lig1) may contribute more to A-EJ than previously considered.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

XRCC1 is dispensable for normal and A-EJ–mediated CSR to IgG1. (A) Splenic B cells from Ctrl, CX1, or CX1X4 mice were stained with IgM and B220 antibodies and analyzed by flow cytometry. (B) Deletion levels in CX1 and CX1X4 splenic B cells. Extracts from purified activated B cells or thymocytes from CX1 (n = 2), CX1X4 (n = 2), or Ctrl mice were immunoblotted with the indicated antibodies. (C) Elevated genotoxic stress sensitivity of mature CX1 and CX1X4 B cells activated with αCD40/IL-4 for 3 d. B cells from at least three mice per genotype were used for each MMS concentration. (D) Surface IgG1 expression of B cells stimulated with αCD40/IL-4 for 4 d. A total of 13 Ctrl, 15 CX1, 8 CX4, and 7 CX1X4 mice were analyzed in independent experiments. _AID_−/− (AID KO) mice were included as negative Ctrls. The results of IgG1 time course experiments are presented in

Fig. S1_D_

. Data are means ± SEM.

Fig. 2.

Fig. 2.

XRCC1 deficiency does not affect switch region junctions, IgH genomic instability, or IgH/c-myc translocations. (A) Levels of MH-mediated Sμ-Sγ1 joins (Left) and Sμ-Sε joins (Right) in Ctrl, CX1, CX4, and CX1X4 activated B cells. S region junction data are shown in detail in

Table S1

. (B) CX1 B cells do not display altered levels of IgH locus breaks. The percentage of unrepaired IgH locus breaks in activated Ctrl, CX1, and CX4 B cells is shown. Data are means ± SEM. A summary of all FISH data is included in

Table S2

. (C) XRCC1 is dispensable for IgH/c-myc translocations in CX1 and CX1X4 B cells. IgH/c-myc translocations were amplified from B cells cultured with αCD40/IL-4 for 4 d. Data are means ± SEM. One-way ANOVA analysis with a Tukey posttest was used for calculation of statistical significance. n.s., not significant; *P < 0.05; **P < 0.001; ***P < 0.0001.

Fig. 3.

Fig. 3.

_Xrcc4_−/−_Xrcc1_−/−-transformed pro-B lines do not display XRCC1-dependent end-joining defects. (A) Western blot analysis revealed complete absence of XRCC1 and XRCC4 proteins in subclones from two independent v-Abl–transformed pro-B lines. Lig3 levels are dramatically reduced in XRCC1 null cells. (B) (Upper) Illustration of I-SceI chromosomal reporter. (Lower) Quantification of I-SceI chromosomal joining for X4c/cX1c/c (n = 7), X4−/− (n = 4), and X4−/−X1−/− (n = 7) v-Abl clonal lines with single-copy I-SceI chromosomal substrates integrated in the same location (clone 26). Data are means ± SEM. One-way ANOVA analysis with a Tukey posttest was used for calculation of statistical significance. n.s., not significant; ***P < 0.0001.

Tables S3

and

S4

list details on I-SceI joining experiments. (C) Xrcc1 deletion does not affect the nature of I-SceI junctions in XRCC4 null cells. I-SceI junctions were amplified from v-Abl cells of the indicated genotypes. Data are means ± SEM.

Fig. 4.

Fig. 4.

shRNA-mediated depletion of Lig3 in B-cell lines does not affect CSR or frequency of IgH/c-myc translocations. (A) Lig3 protein levels in CH12F3 cells of the indicated genotypes expressing either Ctrl or Lig3 shRNA. Blots were stripped and probed for tubulin as a loading Ctrl. Unstim., unstimulated. (B) Representative example of flow cytometry results for IgA expression of WT or _Lig4_−/− CH12F3 cells stably expressing either Ctrl or Lig3 shRNA on day 3 of stimulation with IL-4/αCD40/TGF-β. Cultures propagated without cytokines and analyzed in parallel contained <1% IgA+ cells. (C) Summary of CSR experiments in CH12F3 B-cell lines. Means ± SEM from seven (WT shCtrl and WT shLig3) or six (_Lig4_−/− shCtrl and _Lig4_−/− shLig3) independent experiments are shown. (D) Lig3 depletion does not affect the frequency of IgH/c-myc translocations. Translocations were analyzed in CH12F3 cells stimulated for 3 d with IL-4/αCD40/TGF-β. Results from three independent knockdown experiments using WT shCtrl, WT shLig3, _Lig4_−/− shCtrl, or _Lig4_−/− shLig3 CH12F3 cells are shown. One-way ANOVA analysis with a Tukey posttest was used to assess statistical significance. Data are means ± SEM. n.s., not significant; **P < 0.01.

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