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. 2017 May 13:6:e22900.
doi: 10.7554/eLife.22900.

Mutational phospho-mimicry reveals a regulatory role for the XRCC4 and XLF C-terminal tails in modulating DNA bridging during classical non-homologous end joining

Davide Normanno  1 , Aurélie Négrel  1 , Abinadabe J de Melo  1 , Stéphane Betzi  1 , Katheryn Meek  2   3 , Mauro Modesti  1
Affiliations

Mutational phospho-mimicry reveals a regulatory role for the XRCC4 and XLF C-terminal tails in modulating DNA bridging during classical non-homologous end joining

Davide Normanno et al. Elife. .

Abstract

XRCC4 and DNA Ligase 4 (LIG4) form a tight complex that provides DNA ligase activity for classical non-homologous end joining (the predominant DNA double-strand break repair pathway in higher eukaryotes) and is stimulated by XLF. Independently of LIG4, XLF also associates with XRCC4 to form filaments that bridge DNA. These XRCC4/XLF complexes rapidly load and connect broken DNA, thereby stimulating intermolecular ligation. XRCC4 and XLF both include disordered C-terminal tails that are functionally dispensable in isolation but are phosphorylated in response to DNA damage by DNA-PK and/or ATM. Here we concomitantly modify the tails of XRCC4 and XLF by substituting fourteen previously identified phosphorylation sites with either alanine or aspartate residues. These phospho-blocking and -mimicking mutations impact both the stability and DNA bridging capacity of XRCC4/XLF complexes, but without affecting their ability to stimulate LIG4 activity. Implicit in this finding is that phosphorylation may regulate DNA bridging by XRCC4/XLF filaments.

Keywords: NHEJ; XLF; XRCC4; biochemistry; chromosomes; genes; human.

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Conflict of interest statement

The authors declare that no competing interests exist.

Figures

Figure 1.
Figure 1.. Structural organization of XRCC4 and XLF homodimers.
(A) Structure of XRCC4 homodimer based on PDB 3II6, including residues 1–203 of XRCC4 and a schematic representation of the C-terminal disordered tails. In the bottom panel, also residues 654–911 of LIG4 are shown (green). (B) Structure of XLF homodimer based on PDB 2R9A, including residues 1–224 and a schematic representation of the C-terminal tails. Purple stars indicate the residues modified for phospho-mimicking or -blocking, and all were mutated, respectively, to Asp or Ala in this study. Residues are numbered using as reference Uniprot Q13426 isoform 1 and Q9H9Q4 isoform 1 for XRCC4 and XFF, respectively. DOI: http://dx.doi.org/10.7554/eLife.22900.003
Figure 1—figure supplement 1.
Figure 1—figure supplement 1.. Coomassie stained reducing SDS-PAGE analysis of recombinant XRCC4 and XLF variants produced in bacteria (1 μg/lane).
DOI: http://dx.doi.org/10.7554/eLife.22900.004
Figure 2.
Figure 2.. Wild-type XRCC4 and XLF form super-complexes in vitro that bridge DNA favoring molecular collision rates that enhance DNA ligation.
(A) EMSA using a 2.7 kb DNA fragment with protruding ends and the indicated proteins resolved by agarose gel electrophoresis and DNA detected by ethidium bromide staining. (B) EMSA using a blunt-ended 2.7 kb DNA fragment and the indicated proteins resolved by agarose gel electrophoresis and DNA detected by ethidium bromide staining. (C) (D) (E) T4 DNA ligase assay using a 2.7 kb DNA fragment with cohesive (left) or blunt ends (right) and the indicated proteins. Ligation products were deproteinized and resolved by agarose gel electrophoresis followed by detection by ethidium bromide staining. NC = nicked circle, CCC = covalently closed circle. (F) Schematic of the DNA pull-down/bridging assay. A one-end biotinylated 1 kb DNA fragment is first attached to streptavidin-coated magnetic beads and then incubated with a blunt-ended 2.7 kb DNA fragment in the presence of the proteins of interest. (G) Bridging assays for the indicated proteins resolved by agarose gel electrophoresis after deproteinization followed by DNA detection by ethidium bromide staining. DOI: http://dx.doi.org/10.7554/eLife.22900.005
Figure 2—figure supplement 1.
Figure 2—figure supplement 1.. T4 DNA ligase assay using a 2.7 kb DNA fragment with cohesive (left) or blunt ends (right) at three different protein equimolar concentrations.
(A) 2 μM each, (B) 1 μM each, and (C) 0.5 μM each. Ligation products were deproteinized and resolved by agarose gel electrophoresis followed by DNA detection by ethidium bromide staining. NC = nicked circle, CCC = covalently closed circle. DOI: http://dx.doi.org/10.7554/eLife.22900.006
Figure 3.
Figure 3.. Formation of DNA/XRCC4/XLF super-complexes is not disrupted by phosphorylation site mutation.
(A) EMSAs showing DNA binding of XRCC4 variants in isolation (top panel) and cooperatively with XLF-WT (bottom panel) resolved by agarose gel electrophoresis and detected by ethidium bromide staining. (B) EMSAs showing DNA binding of XLF variants in isolation (top panel) and cooperatively with XRCC4-WT (bottom panel) resolved by agarose gel electrophoresis and detected by ethidium bromide staining. DOI: http://dx.doi.org/10.7554/eLife.22900.007
Figure 4.
Figure 4.. Phosphorylation site mutations do not alter XRCC4-XLF direct interaction.
XRCC4 and XLF were cross-linked in isolation or in equimolar combination with BS3. Cross-linked species were resolved by reducing and denaturing SDS-PAGE and detected by Coomassie staining. (A) XRCC4 and XLF WT-WT combination (left panel) and XRCC4-WT combined with XLF-L115A, that has a point mutation that diminished affinity for XRCC4 (right panel). (B) XRCC4-WT (left panel), XRCC4-Ala (middle panel) and XRCC4-Asp (right panel) respectively cross-linked to all the XLF variants. M = protein molecular weight size ladder. DOI: http://dx.doi.org/10.7554/eLife.22900.008
Figure 5.
Figure 5.. Thermodynamic analysis of wild-type and phospho-mimetic XRCC4-XLF interaction.
Isothermal titration micro-calorimetry data obtained at 37°C injecting (A) XLF-WT (154 μM) into XRCC4-WT (12 μM), (B) XLF-Ala (250 μM) into XRCC4-Ala (19 μM), (C) XLF-Asp (250 μM) into XRCC4-Asp (19 μM). Top panels display the thermogram of the heat exchanged as a function of time after baseline (red lines) subtraction. Bottom panels show the result of the (numerical) integration of the peaks as a function of the molar ratio between the titrant (XLF) and the analyte (XRCC4); black lines are non-linear least squares fits of the data. Data are representative results out of two experimental replicas each composed of a technical duplicate (see Table 1 for details). Source data are included in Table 1—source data 1. (D) Equilibrium constant (Kd) (top) and thermodynamic parameters (free energy ΔG0, black bars; enthalpy ΔH0, dark grey bars; and entropy –TΔS0, light grey bars) (bottom) obtained from sigmoidal fit (black lines in bottom panels of (A, B, C) of ITC curves. Bars show mean values and error bars represent the standard error of the mean (SEM) of the four experimental runs performed per each condition. Thermodynamic parameter values are reported in Table 1. DOI: http://dx.doi.org/10.7554/eLife.22900.009
Figure 5—figure supplement 1.
Figure 5—figure supplement 1.. Isothermal titration micro-calorimetry thermograms.
Top panels display the heat exchanged as a function of time, after baseline (red lines) subtraction. Bottom panels show the result of the (numerical) integration of the peaks as a function of the molar ratio between the titrant (XLF) and the analyte (XRCC4). Thermograms have been obtained (A) at 10°C injecting XLF-WT (168 μM) into XRCC4-WT (17 μM) and at 25°C injecting (B) XLF-WT (330 μM) into XRCC4-WT (25 μM), (C) XLF-Ala (330 μM) into XRCC4-Ala (25 μM), and (D) XLF-Asp (330 μM) into XRCC4-Asp (25 μM). Control isothermal titration micro-calorimetry thermograms obtained at 37°C injecting (E) buffer into XRCC4-WT (12 μM) and (F) XLF-WT (154 μM) into buffer (the first large peak might represent XLF homodimer dissociation heat due to XLF dilution). Source data are included in Table 1—source data 1 (panels A–D) and reported in Figure 5—figure supplement 1—source data 1 (panels E and F). DOI: http://dx.doi.org/10.7554/eLife.22900.010
Figure 6.
Figure 6.. Blocking XRCC4 and XLF phosphorylation sites enhances DNA bridging; phospho-mimicking mutations abate DNA bridging.
All combinations of XRCC4 variants with XLF variants tested in ability to stimulate T4 DNA ligase cohesive end ligation (A) or blunt end ligation (B). Ligation products were deproteinized and resolved by agarose gel electrophoresis followed by detection by ethidium bromide staining. DOI: http://dx.doi.org/10.7554/eLife.22900.014
Figure 7.
Figure 7.. Blocking XRCC4 and XLF phosphorylation sites enhances DNA bridging; phospho-mimicking mutations abate DNA bridging.
DNA bridging assays using a short blunt ended 0.5 kb DNA (A) or a long blunt ended 2.7 kb DNA (B) and the indicated proteins. Samples were deproteinized and resolved by agarose gel electrophoresis followed by detection by ethidium bromide staining. DOI: http://dx.doi.org/10.7554/eLife.22900.015
Figure 8.
Figure 8.. Phospho-mimicking XRCC4 and XLF mutants associate with DNA, but dissociate rapidly.
(A) Scheme of the SPR assay with 400 bp blunt end DNA anchored to the streptavidin coated chip via biotin-streptavidin linkage. (B) From left to right, sensorgrams of XRCC4-WT, XLF-WT, and equi-molar mix of XRCC4-WT and XLF-WT at the indicated concentrations. Source data are provided in Figure 8—source data 1, 2 and 3, respectively. (C) Sensorgrams of all XRCC4 and XLF variant combinations each at 1 μM concentration. Source data are provided in Figure 8—source data 4. (D) Equilibrium affinity of the different combinations of XRCC4 and XLF variants. Source data are provided in Figure 8—source data 5. (E) Dissociation rates for the different combinations of XRCC4 and XLF variants. koff_fast (dark grey symbols) and koff_slow (light grey symbols) correspond, respectively, to the initial (fast) and late (slow) phase in the biphasic dissociation curves. Error bars fall within symbol dimension. Source data are provided in Figure 8—source data 5. DOI: http://dx.doi.org/10.7554/eLife.22900.016
Figure 8—figure supplement 1.
Figure 8—figure supplement 1.. Sensorgrams of XRCC4 and XLF variants and truncations.
(A) Sensorgrams of XRCC4 variants (left) or XLF variants (right) at 1 μM concentration in isolation. Source data are provided in Figure 8—figure supplement 1—source data 1. (B) Sensorgrams comparison between XRCC4 and XLF WT-WT (black, left scale) and Asp-Asp (red, right scale) at equimolar concentration (1 μM each) showing the faster dissociation of the Asp-Asp combination and the deviation in both cases from a single exponential decay (dashed lines) and the biphasic behavior (solid lines). Source data are provided in Figure 8—figure supplement 1—source data 2. (C) Sensorgrams of XRCC4-WT and truncation XRCC4(1–157) lacking the C-terminal tail at 1 μM concentration in isolation (left); and sensorgrams of XLF-WT and truncation XLF(1-224) lacking the C-terminal tails at 1 μM concentrations in isolation (right). Source data are provided in Figure 8—figure supplement 1—source data 3. (D) Sensorgrams of equimolar combinations (1 μM each) of the WT and truncated XRCC4 and XLF proteins as indicated. Source data are provided in Figure 8—figure supplement 1—source data 3. DOI: http://dx.doi.org/10.7554/eLife.22900.022
Figure 9.
Figure 9.. XRCC4 and XLF phospho-mimetics do not bridge DNA ends but are completely sufficient in stimulating LIG4-XRCC4 activity.
Stimulation of LIG4/XRCC4-WT, LIG4/XRCC4-WT-eGFP, LIG4/XRCC4-Ala-eGFP or LIG4/XRCC4-Asp-eGFP cohesive end ligation by XLF-WT (A), XLF-Ala (B) or XLF-Asp (C). Ligation products were deproteinized and resolved by agarose gel electrophoresis followed by detection by ethidium bromide staining. DOI: http://dx.doi.org/10.7554/eLife.22900.026
Figure 9—figure supplement 1.
Figure 9—figure supplement 1.. Coomassie stained reducing SDS-PAGE analysis of recombinant LIG4/XRCC4 variants purified after overexpression in bacteria (1 μg/lane).
DOI: http://dx.doi.org/10.7554/eLife.22900.027
Figure 10.
Figure 10.. XRCC4 and XLF phosphorylation site mutation does not affect end joining of episomal substrates.
Fluorescent substrates (depicted in top panels) were utilized to detect V(D)J coding and signal joints in XRCC4/XLF-deficient 293 T cells transiently expressing full-length RAG1, RAG2, and WT, Ala, or Asp mutants of XRCC4 and XLF (A), or joining of I-Sce1 induced DSBs (B). Bottom panels show percent recombination of episomal fluorescent substrates. Error bars indicate SEM from five independent experiments. DOI: http://dx.doi.org/10.7554/eLife.22900.028
Figure 11.
Figure 11.. XRCC4 and XLF complexes do not promote a-NHEJ.
The fluorescent substrate (depicted in the top panel) was utilized to detect joining of I-Sce1 induced DSBs. Bottom panel shows percent recombination of episomal fluorescent I-Sce1 end joining substrate in XRCC4/XLF-deficient 293 T cells transiently expressing I-Sce1, XRCC4 alone, WT XRCC4 and XLF, or an XRCC4 mutant that is defective in LIG4 interaction and WT XLF as indicated. Error bars indicate SEM from three independent experiments. DOI: http://dx.doi.org/10.7554/eLife.22900.029
Figure 12.
Figure 12.. XRCC4 and XLF phosphorylation site mutants do not fully reverse sensitivity to radio-mimetic drugs.
(A) Immunoblots of stable expression of WT, Ala, or Asp mutants of XRCC4 and XLF in 293 T cells that lack both XRCC4 and XLF. * non-specific species. (B) 293 T cells expressing WT, Ala, Asp, or no XRCC4 or XLF were exposed to Zeocin (500 μg/ml for 1 hr) or not and analyzed after 1 hr or 24 hr by immunoblotting for XRCC4, β-actin, or γ-H2AX as indicated. Zeocin (C) and Neocarzinostatin (D) sensitivity of 293T XRCC4/XLF double deficient cell strains complemented by stable expression of equivalent levels of WT, Ala, or Asp mutants of XRCC4 and XLF. Error bars indicate SEM from at least three independent experiments. DOI: http://dx.doi.org/10.7554/eLife.22900.030
Figure 12—figure supplement 1.
Figure 12—figure supplement 1.. XRCC4 and XLF phosphorylation site mutants do not fully reverse sensitivity to radio-mimetic drugs.
(Left) Immunoblot showing comparable to endogenous, stable expression of WT, Ala, or Asp mutants of XRCC4 and XLF in previously described XRCC4 deficient HCT116 cells that were deleted for XLF. Parental cell strains, as well as XLF deficient cells that were generated by AAV targeting are also depicted. (Right) Neocarzinostatin sensitivity of the indicated cell strains stably expressing WT, Ala, or Asp mutants of XRCC4 and XLF. Error bars indicate SEM from at least three independent experiments. DOI: http://dx.doi.org/10.7554/eLife.22900.031
Figure 12—figure supplement 2.
Figure 12—figure supplement 2.. XRCC4 and XLF phosphorylation site mutants do not fully reverse sensitivity to radio-mimetic drugs.
(Left) Immunoblot showing comparable to endogenous, stable expression of WT, Ala, or Asp mutants of XRCC4 and XLF in XRCC4-deficient hamster XR-1 cells deleted for XLF. (Right) Neocarzinostatin sensitivity of the indicated cell strains stably expressing WT, Ala, or Asp mutants of XRCC4 and XLF. Error bars indicate SEM from at least three independent experiments. DOI: http://dx.doi.org/10.7554/eLife.22900.032
Figure 13.
Figure 13.. Phospho-mimicking XRCC4 and XLF alters repair of chromosomal DNA DSBs.
(A) Diagram of region on chromosome nine targeted by three different gRNAs and position of primers utilized to detect chromosomal deletions induced by Cas9 and indicated gRNAs. (B) Ethidium bromide staining of PCR amplifications of DNA isolated from cells expressing WT, Ala, Asp, or no XRCC4 and XLF and transfected with the different combinations of gRNAs as indicated. A no gRNA control DNA of parental 293 T cells is also included to help define products induced by Cas9 induced deletions. (C) Summary of sequenced joints utilizing gRNAs 1 and 3 that result in a 4 kb chromosomal deletion. (D) Graphs represent the percentage of joints deleting increasing numbers of nucleotides from predicted double strand break site. DOI: http://dx.doi.org/10.7554/eLife.22900.033
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DOI: http://dx.doi.org/10.7554/eLife.22900.034
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