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Sliding sleeves of XRCC4–XLF bridge DNA and connect fragments of broken DNA

Nature volume 535, pages 566–569 (2016)Cite this article

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A Corrigendum to this article was published on 22 March 2017

Abstract

Non-homologous end joining (NHEJ) is the primary pathway for repairing DNA double-strand breaks (DSBs) in mammalian cells1. Such breaks are formed, for example, during gene-segment rearrangements in the adaptive immune system or by cancer therapeutic agents. Although the core components of the NHEJ machinery are known, it has remained difficult to assess the specific roles of these components and the dynamics of bringing and holding the fragments of broken DNA together. The structurally similar XRCC4 and XLF proteins are proposed to assemble as highly dynamic filaments at (or near) DSBs2. Here we show, using dual- and quadruple-trap optical tweezers combined with fluorescence microscopy, how human XRCC4, XLF and XRCC4–XLF complexes interact with DNA in real time. We find that XLF stimulates the binding of XRCC4 to DNA, forming heteromeric complexes that diffuse swiftly along the DNA. Moreover, we find that XRCC4–XLF complexes robustly bridge two independent DNA molecules and that these bridges are able to slide along the DNA. These observations suggest that XRCC4–XLF complexes form mobile sleeve-like structures around DNA that can reconnect the broken ends very rapidly and hold them together. Understanding the dynamics and regulation of this mechanism will lead to clarification of how NHEJ proteins are involved in generating chromosomal translocations3,4.

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Figure 1: Interaction of XRCC4 and XLF with DNA.
Figure 2: DNA bridging by XRCC4 and XLF.
Figure 3: Mobility of XRCC4–XLF bridges.
Figure 4: XRCC4–XLF complexes keep DNA fragments together.

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Acknowledgements

We thank P. Fourquet and S. Audebert from the CRCM Proteomics facility for help with MS analysis and G. King and A. S. Biebricher for help with the optical tweezers instruments. This work was supported by VICI grants (G.J.L.W. and E.J.G.P.), a VENI grant (I.H.) and a TopTalent grant (A.C.) from the Nederlandse Organisatie voor Wetenschappelijk Onderzoek, a European Research Council starting grant (G.J.L.W.), the French National Cancer Institute (grant PLBIO13-103) (M.M.), the ARC Foundation for Cancer Research (M.M.), the A*MIDEX project (no. ANR-11-IDEX-0001-02), the ‘Investissements d’Avenir’ French Government program (M.M.), fellowship no. 0558/12-5 from the Brazilian program for Coordination for the Improvement of Higher Education Personnel (A.J.M.), and a fellowship from the Collège of Aix-Marseille Université (H.Z.) The research leading to these results received funding from LASERLABEUROPE (grant agreement no. 284464, the European Commission's Seventh Framework Programme).

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Author notes

  1. Gerrit Sitters, Andrea Candelli & Stephanie J. Heerema

    Present address: †Present addresses: LUMICKS B.V., 1081 HV Amsterdam, The Netherlands (G.S., A.C.); Kavli Institute of Nanoscience, Delft University of Technology, 2628 CJ Delft, The Netherlands (S.J.H.).,

  2. Ineke Brouwer and Gerrit Sitters: These authors contributed equally to this work.

  3. Mauro Modesti, Erwin J. G. Peterman and Gijs J. L. Wuite: These authors jointly supervised this work.

Authors and Affiliations

  1. Department of Physics and Astronomy and LaserLab, Vrije Universiteit Amsterdam, HV Amsterdam, 1081, The Netherlands

    Ineke Brouwer, Gerrit Sitters, Andrea Candelli, Stephanie J. Heerema, Iddo Heller, Erwin J. G. Peterman & Gijs J. L. Wuite

  2. Cancer Research Center of Marseille, CNRS UMR7258, Inserm U1068, Institut Paoli-Calmettes, Aix-Marseille Université UM105, Marseilles, 13273, France

    Abinadabe J. Melo de, Hongshan Zhang, Davide Normanno & Mauro Modesti

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Contributions

M.M., E.J.G.P. and A.C. conceived the study. I.B., G.S., A.C. and S.J.H. performed the single-molecule experiments. I.H. built the quadruple-trap instrument and advised on the force-fluorescence experiments. A.J.M., H.Z., D.N. and M.M. purified the proteins and performed the biochemical analysis of the protein samples. I.B., G.S., M.M., E.J.G.P. and G.J.L.W. wrote the manuscript. E.J.G.P., M.M. and G.J.L.W. led the research, the analysis and the interpretation of the results. All authors discussed the results and commented on the manuscript.

Corresponding authors

Correspondence to Mauro Modesti or Gijs J. L. Wuite.

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Competing interests

The optical tweezers and fluorescence technology used in this article are patented and licensed to LUMICKS B.V., in which G.S., A.C., I.H., E.J.G.P. and G.J.L.W. have a financial interest.

Additional information

Reviewer Information

Nature thanks M. Morse, M. Nabuan Naufer, M. Williams and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Extended data figures and tables

Extended Data Figure 1 Generation of human XLF ATTO-647N-labelled fluorescent variant by the ‘Cys light’ method.

a, Domain architecture of the XLF polypeptide and amino acid sequences of the wild-type and Cys148 variant obtained by site-directed mutagenesis. All Cys residues were changed to Ser except Cys148, leaving a single solvent accessible Cys residue. Three-dimensional model of the XLF dimer where the position–specific labelling sites are indicated by the black arrows. b, Denaturing and reducing polyacrylamide gel electrophoresis of XLF Cys148 variant after labelling with ATTO 647N maleimide. Left, bright-field image of the gel before staining. Centre, the emission of ATTO 647N. Right, is an image of the gel after staining with Coomassie. M, molecular mass markers. c, Mass spectrometry of the labelled full-length protein. Top, the spectra of XLF Cys148 (red) after labelling with ATTO 647N and compared to the wild-type unlabelled protein (black) giving Δm = 790 Da. Bottom, the calculation of the degree of labelling (D.O.L.), as the relative integrated intensity of the labelled protein (dark grey area) versus the unlabelled protein (light grey area) signals, giving 0.82 dye per monomer. d, DNA bridging activity of the XLF variants. Left, a scheme of the bridging assay in which an end-biotinylated 1,000-bp DNA fragment is coupled to streptavidin-coated magnetic beads. Protein-mediated DNA bridging is assessed by recovery of an unlabelled 500-bp DNA fragment. Right, image of an agarose gel stained with ethidium bromide to detect the bridged 500-bp DNA fragment (indicated by the black arrow) and the corresponding quantification. Asterisk indicates biotinylated 1,000-bp DNA fragment adsorbed non-specifically on the surface of the magnetic beads.

Extended Data Figure 2 Generation of human XLF fluorescent variants by eGFP tagging.

a, Domain architecture of the XLF polypeptide and amino acid sequences of the C- and N-terminal eGFP fusions. b, Denaturing and reducing polyacrylamide gel electrophoresis of the purified eGFP fusions. c, DNA bridging activity of the XLF variants. Top, a scheme of the bridging assay where an end-biotinylated 1,000-bp DNA fragment is coupled to streptavidin-coated magnetic beads. Protein-mediated DNA bridging is assessed by recovery of an unlabelled 500-bp DNA fragment. Bottom, an image of an agarose gel stained with ethidium bromide to detect the bridged 500-bp DNA fragment (indicated by the black arrow). Asterisk indicates biotinylated 1,000-bp DNA fragment adsorbed non-specifically on the surface of the magnetic beads.

Extended Data Figure 3 Generation of human XRCC4 fluorescent variants by the ‘Cys light’ method.

a, Domain architecture of the XRCC4 polypeptide and amino acid sequences of the wild-type, C93 and C218 variants obtained by site-directed mutagenesis. All Cys residues were changed to Ala except, respectively C93 and C218, leaving for each variant a single solvent accessible Cys residue. Three-dimensional models of the XRCC4 dimer where the position-specific labelling sites are indicated by the black arrows. b, Denaturing and reducing polyacrylamide gel electrophoresis of XRCC4-C93 and -C218 variants after labelling with Alexa Fluor 555 maleimide. Left, bright-field image of the gel before staining. Centre, the emission of Alexa Fluor 555. Right, an image of the gel after staining with Coomassie. c, Mass spectrometry of the labelled full-length proteins. Top, the spectra of XRCC4-C93 (orange) and XRCC4-C218 (green) after labelling with Alexa Fluor 555 and compared to the wild-type unlabelled protein (black), giving Δm = 830 Da and Δm = 835 Da for labelled XRCC4-C93 and XRCC4-C218, respectively. Bottom, the calculation of the degree of labelling, as the relative integrated intensity of the labelled protein (dark grey area) versus the unlabelled protein (light grey area) signals, giving 0.91 and 0.94 dye per monomer for XRCC4-C93 and XRCC4-C218, respectively. d, DNA bridging activity of the XRCC4 variants. Top, a scheme of the bridging assay in which an end-biotinylated 1,000-bp DNA fragment is coupled to streptavidin-coated magnetic beads. Protein-mediated DNA bridging is assessed by recovery of an unlabelled 500-bp DNA fragment. Bottom, image of agarose gel stained with ethidium bromide to detect the bridged 500-bp DNA fragment (indicated by the black arrow) and the corresponding quantification (the image is from the same gel as in Extended Data Fig. 1d but only the lanes relevant to the analysis of fluorescently labelled XRCC4 are shown). Asterisk indicates biotinylated 1,000-bp DNA fragment adsorbed non-specifically on the surface of the magnetic beads.

Extended Data Figure 4 Quantification of XRCC4 and XLF binding to dsDNA.

a, Fluorescence image of an overstretched dsDNA molecule in the presence of 100 nM eGFP–XLF. Under the given salt conditions and at this DNA extension, significant DNA melting and formation of ssDNA is expected. XLF shows a high affinity to bind to dsDNA (bright fluorescent signal), whereas it does not appear to bind to ssDNA (dark region). b, c, Size of the DNA-bound protein complexes for XRCC4 (b) and XLF (c) as determined from their fluorescence intensities (after incubation with 50 nM XRCC4 (b) or XLF (c)). d, Sections of a kymograph measured in the protein channel (25 nM eGFP–XLF). Short protein-binding events are visible as brief, local bursts of fluorescence. Events are shorter than the line scan time (10 ms). e, Typical intensity time traces of bound XRCC4 and XLF indicate that the complexes bind as a whole from solution and not monomer by monomer. The decrease of the fluorescence intensity in time is due to photobleaching of the fluorophores. f, Two successive kymographs (separated by white line). Two separate kymographs are recorded owing to technical limitations on the maximum recoding time of XRCC4–Alexa Fluor 555 binding measured using a very low excitation power to reduce the effect of photobleaching. Under these conditions, XRCC4 oligomers stay bound for long periods of time (in the order of several minutes). Scale bars, 5 s and 2 μm. Data are representative examples of 7 (a), 13 (d) and 5 (f) experiments.

Extended Data Figure 5 Properties of protein bridges.

a, b, Bridging also occurs in the absence of free protein in solution. Fluorescence images (XRCC4–Alexa Fluor 555 and XLF–ATTO 647N fluorescence in a, and XRCC4–Alexa Fluor 555 fluorescence in b) acquired in the absence of free protein in solution of two XRCC4–XLF-coated dsDNA molecules before (a) and after (b) wrapping and subsequent unwrapping shows that bridging does not require the presence of free protein in solution. Representative example out of 2 experiments. c, d, DNA–protein bridges also occur in the presence of either XRCC4 or XLF. Fluorescence images of bridges formed by wrapping and subsequent unwrapping in the presence of XRCC4–Alexa Fluor 555 (c) or eGFP–XLF (d). Representative example out of 10 (c) or 19 (d) experiments. e, f, In ~5% of the cases, rupture of protein bridges resulted into two intact dsDNA molecules bound with XRCC4–Alexa Fluor 555 and XLF–ATTO 647N. Fluorescence images before (e) and after (f) rupture of such a protein bridge. Representative example out of 9 experiments. g, h, Fluorescence images taken of an XRCC4–XLF bridge at t = 0 min (g) and t = 95 min (h), showing that the bridge and the protein complexes remain stably bound to the DNA segments. Green, XRCC4; red, XLF, yellow, colocalization. Scale bars, 2 μm.

Extended Data Figure 6 Quantification of diffusion behaviour of XRCC4 and XLF on dsDNA.

a, Typical kymographs showing switching of XLF–ATTO 647N (red signal, top) XRCC4–Alexa Fluor 555 (green signal, middle) and XLF–XRCC4 (yellow signal, bottom) complexes between static and diffusive states. Scale bars, 1 s and 2 μm. b, Typical MSD curve of an individual XRCC4–Alexa Fluor 555 complex. Inset, kymograph of corresponding complex. Red line denotes linear fit to the first three data points; from the slope, a diffusion coefficient of 0.51 μm2 s−1 is determined. See Methods for details on MSD analysis. c, Diffusion coefficients of mobile XRCC4 and XLF complexes at different salt conditions (low salt: 25 mM KCl, high salt: 160 mM KCl) and DNA tensions. d, Diffusion coefficients of mobile XRCC4 protein complexes as a function of complex size. Grey circles, individual data points; black circles, average of 8 successive data points. e, Comparison of the observed diffusion coefficients of XRCC4 (green), XLF (red) and XRCC4–XLF complexes (yellow) to the expected diffusion coefficients based on the helical diffusion model19 (solid black line). To calculate the quantity on the horizontal axis, a hydrodynamic radius of 7–22 nm was used, based on the inner and outer radii of the XRCC4–XLF filament as proposed previously13. f, Average observed dwell times of protein complexes before switching to a different mode. The analysis is performed on 121 events. The shortest dwell time that could be determined with certainty was 1 s. All error bars denote s.e.m.

Extended Data Figure 7 Rupture events in force-extension curves can be caused by DNA–protein bridges or nonspecific sticking of protein-bound DNA to the trapped microspheres.

a, c, Force-extension curves of dsDNA–XRCC4–XLF complexes (red) after incubation at low tension shows rupture events. Black data sets show force-extension curve of bare dsDNA. b, A single, continuous fluorescence kymograph corresponding to red curve in a. At the indicated time (orange arrow in a and b) a protein bridge suddenly ruptures into multiple smaller protein complexes. d, Fluorescence kymograph corresponding to red curve in c reveals that rupture event corresponds to the rupture of a non-specific interaction between a DNA-bound protein complex and the polystyrene microsphere. Scale bars, 1 s and 2 μm.

Extended Data Figure 8 Schematic representation of dual-DNA configurations used in quadruple-trap experiments.

a–c, Wrapped (a), unwrapped (b) and crossed (c) DNA configurations.

Extended Data Figure 9 Quantification of DNA bridging by XRCC4–XLF.

a, b, Normalized length of DNA segments over time during experiments such as shown in Figs 2 and 3. a, The static bridge in the experiment described in Fig. 2 and Supplementary Video 1. b, The mobile bridge in the experiment described in Fig. 3 and Supplementary Video 2.

Extended Data Figure 10 Protein bridges always contain both XRCC4 and XLF.

a, Typical examples of XRCC4–XLF bridges formed by incubating two wrapped DNA molecules in 200 nM eGFP–XLF and 200 nM XRCC4–Alexa Fluor 555 for 2 min. In all 152 bridges that were analysed, clear colocalization of the proteins at the junction is observed.

Supplementary information

Supplementary Figure

This file contains the original source images for all data obtained by electrophoretic separation. (PDF 187 kb)

Bridging by XRCC4-XLF complexes

Video of XRCC4-Alexa Fluor 555 fluorescence (green) and eGFP-XLF fluorescence (red) of the experiment shown in Figure 2. DNA molecules were wrapped, incubated for 2 minutes in a buffer containing 200 nM XRCC4 and 200 nM XLF, brought into a protein-free buffer and subsequently unwrapped. Then individual microspheres were moved to exert tension on the XRCC4-XLF bridge and the DNA ends detach from the microspheres. (MP4 12969 kb)

Mobility of XRCC4-XLF bridges

Video of eGFP-XLF fluorescence bound to two optically trapped DNA molecules bridged by XLF-XRCC4 (individual frames are shown in Figure 3). The movie shows that the bridge slides along one of the DNA molecules when the other DNA molecule is moved. The bridge was formed by incubating the construct for 2 minutes in a buffer containing 200 nM XRCC4 and 200 nM XLF and brought into a protein-free buffer before imaging. (MP4 3292 kb)

XRCC4-XLF complexes keep DNA fragments together after DSBs

Video of XRCC4-Alexa Fluor 555 (green) and eGFP-XLF fluorescence (red) during induction of DSBs in both DNA molecules in an experiment such as shown in Figure 4a-d. A complex of XRCC4 and XLF holds the DNA fragments together in a mobile manner after the occurrence of the DSBs. (MP4 35132 kb)

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Brouwer, I., Sitters, G., Candelli, A. et al. Sliding sleeves of XRCC4–XLF bridge DNA and connect fragments of broken DNA. Nature 535, 566–569 (2016). https://doi.org/10.1038/nature18643

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