The C-terminal domain of Cernunnos/XLF is dispensable for DNA repair in vivo

Abstract

The core nonhomologous end-joining DNA repair pathway is composed of seven factors: Ku70, Ku80, DNA-PKcs, Artemis, XRCC4 (X4), DNA ligase IV (L4), and Cernunnos/XLF (Cernunnos). Although Cernunnos and X4 are structurally related and participate in the same complex together with L4, they have distinct functions during DNA repair. L4 relies on X4 but not on Cernunnos for its stability, and L4 is required for optimal interaction of Cernunnos with X4. We demonstrate here, using in vitro-generated Cernunnos mutants and a series of functional assays in vivo, that the C-terminal region of Cernunnos is dispensable for its activity during DNA repair.

FIG. 1.

Evidence for distinct functions of Cernunnos and X4. (A) Mean results of three V(D)J recombination assays on chromosomal substrates integrated into Cernunnos-deficient fibroblasts (Cer-RSS) and WT OTel fibroblasts (OTel-RSS). Relative V(D)J recombination is calculated based on the recombination frequency obtained by transfection with RAG1 and RAG2 for OTel-RSS and with RAG1, RAG2, and Cernunnos for Cer-RSS cells. ns, nonsignificant difference (P > 0.005); *, statistically significant difference (P < 0.005); ***, highly statistically significant difference (P < 0.001). (Inset) Expression of V5-tagged Cernunnos and X4 constructs in 293T cells. (B) V(D)J recombination assay in X4KO and WT MEFs using the pBlueRec system. Recombination of the substrate was analyzed by PCR. (C, D, and E) Survival of Cernunnos-deficient cells (C) and X4KO (D) and WT (E) MEFs transduced with the pMND retrovirus containing Cernunnos or X4 14 days after bleomycin treatment.

FIG. 2.

The Cernunnos-X4-L4 complex. (A) Whole-cell lysates (WCL) from X4KO MEFs, either left untransfected or transfected with a V5-tagged Cernunnos or X4 expression construct, and from L4KO and WT MEFs were analyzed by WB with the indicated antibodies. (B) WCL from WT fibroblasts and Cernunnos-deficient cells transfected with a V5-tagged Cernunnos or X4 expression construct as for panel A. (C) WCL from N114P2 (L4KO) or Nalm6 (parental) cells, transduced (+) or not (−) with pMND-Cernunnos-Myc-IRES-GFP, were immunoprecipitated with irrelevant antibodies (IgG) or anti-Myc antibodies and then blotted and revealed as indicated. *, endogenous X4; **, V5-tagged X4; ***, IgG heavy chain.

FIG. 3.

Analysis of Cernunnos domains. (A) Schematic representation of WT Cernunnos and its mutants. (B) Mean results of three V(D)J recombination assays on chromosomal substrates integrated into Cernunnos-deficient cells (Cer-RSS), calculated as described in the legend to Fig. 1A. (C) Survival of Cernunnos-deficient cells, transduced with the pMND vector containing WT or mutant (1-230, 1-178, 1-167, or 1-141) Cernunnos or WT X4, 14 days after bleomycin treatment. Results are expressed as in Fig. 1C. (D) Percentage of IRIF-positive cells. Forty cells, transduced (GFP⁺) or not (GFP⁻) and either left untreated or subjected to IR, were scored for DAPI, GFP, and γH2AX foci 2 h and 24 h after IR (2 Gy). A cell was considered IRIF positive if it had more than 10 IRIFs. (E) Whole-cell lysates (WCL) from 293T cells transfected with V5-tagged WT or mutant (1-230, 1-178, or 1-167) Cernunnos were immunoprecipitated with irrelevant (IgG) and anti-V5 rabbit antibodies. (F) 293T cells were cotransfected with Myc-tagged WT Cernunnos and V5-tagged WT or mutant (1-230, 1-178, or 1-167) Cernunnos. For panels E and F, the immunoprecipitates were analyzed by WB as indicated.

FIG. 4.

Analysis of chimeras between Cernunnos and X4. (A) Summary of chimeras resulting from swapping of domains between WT Cernunnos and X4. (B) Mean results of three V(D)J recombination assays on chromosomal substrates integrated into Cernunnos-deficient cells (Cer-RSS). Results are expressed as described in the legend to Fig. 1A. (C) Survival of Cernunnos-deficient cells, transduced with the pMND-Myc-IRES-GFP vector containing WT Cernunnos, C1X2, X1C2, or WT X4, 14 days after bleomycin treatment. Results are expressed as in Fig. 1C. (D) Survival of Cernunnos-deficient cells transduced with an empty MND vector or with the vector carrying WT Cernunnos or C1X2, without any treatment. Cells were monitored for 21 days. J1, day 1; J21, day 21. Asterisks indicate statistical significance (P < 0.05). (E) 293T cells were transfected with V5-tagged WT Cernunnos, C1X2, X1C2, or WT X4. Whole-cell lysates (WCL) were immunoprecipitated with irrelevant (IgG) and anti-V5 rabbit antibodies. (F) 293T cells were cotransfected with Myc-tagged WT Cernunnos and V5-tagged WT Cernunnos, C1X2, or X1C2. For panels E and F, WCL were treated as for Fig. 3E.