Delineation of the Xrcc4-interacting region in the globular head domain of cernunnos/XLF

Abstract

In mammals, the majority of DNA double-strand breaks are processed by the nonhomologous end-joining (NHEJ) pathway, composed of seven factors: Ku70, Ku80, DNA-PKcs, Artemis, Xrcc4 (X4), DNA-ligase IV (L4), and Cernunnos/XLF. Cernunnos is part of the ligation complex, constituted by X4 and L4. To improve our knowledge on the structure and function of Cernunnos, we performed a systematic mutagenesis study on positions selected from an analysis of the recent three-dimensional structures of this factor. Ten of 27 screened mutants were nonfunctional in several DNA repair assays. Outside amino acids critical for the expression and stability of Cernunnos, we identified three amino acids (Arg(64), Leu(65), and Leu(115)) essential for the interaction with X4 and the proper function of Cernunnos. Docking the crystal structures of the two factors further validated this probable interaction surface of Cernunnos with X4.

FIGURE 1.

Detailed views of the experimental three-dimensional structure of human Cernunnos (Protein Data Bank 2R9A (6)). This figure shows the positions of the amino acids of four groups, as selected for this point mutational analysis. Group 1 corresponds to two sets of conserved amino acids, which were predicted to cluster at the surface of the globular head domain (with Leu⁶¹, Phe¹¹⁷, and Trp¹¹⁹ (green) actually buried in the experimental structure). Group 2 corresponds to a set of amino acids predicted to be included in the interface between the head and stalk domains. Group 3 includes conserved amino acids in the stalk domain (L4 binding region of X4), whereas group 4 is formed by amino acids, which are associated with naturally occurring mutations.

FIGURE 2.

Point mutants of Cernunnos in V(D)J recombination. Mean results of three V(D)J recombination assays on chromosomal substrates, stably integrated in the genome of Cernunnos-deficient fibroblasts (Cer-RSS) are shown. Relative V(D)J recombination is calculated based on the recombination frequency obtained with transfection of Rag1, Rag2, and WT Cernunnos. ns, nonsignificant difference (p > 0.005); **, statistically significant (p < 0.005); ***, highly statistically significant (p < 0.001).

FIGURE 3.

Protein expression and localization of the selected point mutants of Cernunnos. A, 100 μg of total proteins obtained from 293T cells, transfected with V5-tagged, WT or mutant Cernunnos expression constructs or not, were analyzed by WB with the indicated antibodies. B, immunofluorescence experiments were performed on glass slide-immobilized Cernunnos-defective cells, which have been transduced with the pMND-myc-ires-GFP retrovirus containing Cernunnos (WT or the indicated mutant). Nuclei were stained with DAPI. The secondary anti-mouse Alexa Fluor 546 antibodies were exposed myc-tagged proteins.

FIGURE 4.

DNA repair efficiency of Cernunnos mutants. A and D, survival of Cernunnos-deficient cells, transduced with the pMND vector containing WT or the indicated Cernunnos mutants, 14 days after bleomycin treatment is shown. B and E, survival of empty MND vector, WT or the indicated mutant Cernunnos, complemented Cernunnos-deficient cells, without any treatment. Cells were followed for 57 days. C and F, percentage of IRIF-positive cells is shown. 40 transduced cells were scored for DAPI and γH2AX foci and IR-untreated, 2 and 24 h after IR (2 Gray). A cell was considered IRIF-positive if it had >10 IRIFs.

FIGURE 5.

Interactions between mutants of Cernunnos and the X4-L4 complex. A, 293T cells were co-transfected with myc-tagged WT Cernunnos and the V5-tagged Cernunnos (WT or the indicated mutant). The obtained whole cell lysates (WCL) were immunoprecipitated (IP) with rabbit irrelevant (IgG) and anti-V5 antibodies and were analyzed by WB with the indicated antibodies. B, 293T cells were transfected with V5-tagged WT or R57G Cernunnos. Whole cell lysates were immunoprecipitated with rabbit irrelevant (IgG) and anti-V5 antibodies and revealed as indicated. C, 293T cells were transfected with V5-tagged WT or mutant Cernunnos. Whole cell lysates were immunoprecipitated and treated as Fig. 4C.

FIGURE 6.

Structuring and interacting residues of human Cernunnos. Ribbon representation of the three-dimensional structure of human Cernunnos (Protein Data Bank code 2R9A (6)) highlights residues analyzed in this study and playing important role for the Cernunnos structure (“structuring” residues, left) and function (“interacting” residues, right). At the left are two boxes, with emphasis on the neighborhood of residues participating in the hydrophobic core of the head domain (gray box: Phe¹¹⁷, Trp¹¹⁹, Cys¹²³) or in the stabilization of the head domain structure (gray box: Arg⁵⁷; red box: Trp¹³, Lys²⁶).

FIGURE 7.

Physical interactions between WT or mutant Cernunnos/XLF and Xrcc4 proteins measured by calorimetry. All experiments were performed at 10 °C. Upper panels show the binding isotherms. Lower panels show integrated heats, after subtraction of heats from control experiments. The black lines represent least-square fits of data. A, ITC binding isotherm obtained when WT Cernunnos was injected into the X4-containing cell. B, ITC binding isotherm obtained for Cernunnos L115D injected into the X4-containing cell. C, interaction between Cernunnos R64E and X4. D, interaction between Cernunnos L65D and X4.

FIGURE 8.

Docking of Cernunnos and X4 crystal structures. Both dimeric proteins are represented as schematics with Cernunnos monomers in green and yellow and X4 monomers in blue and light blue. The model was extracted from a set of structural models satisfying the constraints from conservation. Interaction is mediated by the distal end of the head of Cernunnos and X4 and is remote from L4 (magenta) binding site that is located on the X4 coiled-coil. Positions of the variants analyzed in this study are represented in stick in gray for VDJ neutral position, in orange for unstable variant, and in magenta for interacting residues. The inset presents a focus of the interface proposed with the residues identified in this study as essential for the interaction.