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A single XLF dimer bridges DNA ends during nonhomologous end joining

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Nonhomologous end joining (NHEJ) is the primary pathway of DNA double-strand-break repair in vertebrate cells, yet how NHEJ factors assemble a synaptic complex that bridges DNA ends remains unclear. To address the role of XRCC4-like factor (XLF) in synaptic-complex assembly, we used single-molecule fluorescence imaging in Xenopus laevis egg extract, a system that efficiently joins DNA ends. We found that a single XLF dimer binds DNA substrates just before the formation of a ligation-competent synaptic complex between DNA ends. The interaction of both globular head domains of the XLF dimer with XRCC4 is required for efficient formation of this synaptic complex. Our results indicate that, in contrast to a model in which filaments of XLF and XRCC4 bridge DNA ends, binding of a single XLF dimer facilitates the assembly of a stoichiometrically well-defined synaptic complex.

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Fig. 1: XLF–XRCC4 interaction is required for NHEJ in Xenopus egg extract.
Fig. 2: XLF–XRCC4 interaction is required for SR-complex formation.
Fig. 3: Three-color imaging of XLF binding and SR-complex formation.
Fig. 4: End joining in the presence of a synthetic tandem dimer of XLF.
Fig. 5: Characterization of human XLFL115A and Xenopus XLFL117A.

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Data availability

The data that support the findings of this study and the custom-written computer code used to analyze them are available from the corresponding author upon reasonable request. Source data for Figs. 2b,c, 4c, and 5c are available with the paper online.

Change history

  • 24 September 2018

    In the version of this article originally published, Supplementary Tables 1–4 were omitted from the Supplementary Text and Figures file. This error has been corrected.


  1. Walker, J. R., Corpina, R. A. & Goldberg, J. Structure of the Ku heterodimer bound to DNA and its implications for double-strand break repair. Nature 412, 607–614 (2001).

    Article  CAS  PubMed  Google Scholar 

  2. Dobbs, T. A., Tainer, J. A. & Lees-Miller, S. P. A structural model for regulation of NHEJ by DNA-PKcs autophosphorylation. DNA Repair (Amst.) 9, 1307–1314 (2010).

    Article  CAS  PubMed Central  Google Scholar 

  3. Jette, N. & Lees-Miller, S. P. The DNA-dependent protein kinase: a multifunctional protein kinase with roles in DNA double strand break repair and mitosis. Prog. Biophys. Mol. Biol. 117, 194–205 (2015).

    Article  CAS  PubMed  Google Scholar 

  4. Jiang, W. et al. Differential phosphorylation of DNA-PKcs regulates the interplay between end-processing and end-ligation during nonhomologous end-joining. Mol. Cell. 58, 172–185 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Critchlow, S. E., Bowater, R. P. & Jackson, S. P. Mammalian DNA double-strand break repair protein XRCC4 interacts with DNA ligase IV. Curr. Biol. 7, 588–598 (1997).

    Article  CAS  PubMed  Google Scholar 

  6. Grawunder, U. et al. Activity of DNA ligase IV stimulated by complex formation with XRCC4 protein in mammalian cells. Nature 388, 492–495 (1997).

    Article  CAS  PubMed  Google Scholar 

  7. Buck, D. et al. Cernunnos, a novel nonhomologous end-joining factor, is mutated in human immunodeficiency with microcephaly. Cell 124, 287–299 (2006).

    Article  CAS  PubMed  Google Scholar 

  8. Ahnesorg, P., Smith, P. & Jackson, S. P. XLF interacts with the XRCC4-DNA ligase IV complex to promote DNA nonhomologous end-joining. Cell 124, 301–313 (2006).

    Article  CAS  PubMed  Google Scholar 

  9. Junop, M. S. et al. Crystal structure of the Xrcc4 DNA repair protein and implications for end joining. EMBO J. 19, 5962–5970 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Li, Y. et al. Crystal structure of human XLF/Cernunnos reveals unexpected differences from XRCC4 with implications for NHEJ. EMBO J. 27, 290–300 (2008).

    Article  CAS  PubMed  Google Scholar 

  11. Andres, S. N., Modesti, M., Tsai, C. J., Chu, G. & Junop, M. S. Crystal structure of human XLF: a twist in nonhomologous DNA end-joining. Mol. Cell 28, 1093–1101 (2007).

    Article  CAS  PubMed  Google Scholar 

  12. Hammel, M., Yu, Y., Fang, S., Lees-Miller, S. P. & Tainer, J. A. XLF regulates filament architecture of the XRCC4·ligase IV complex. Structure 18, 1431–1442 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Ropars, V. et al. Structural characterization of filaments formed by human Xrcc4-Cernunnos/XLF complex involved in nonhomologous DNA end-joining. Proc. Natl Acad. Sci. USA 108, 12663–12668 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Hammel, M. et al. XRCC4 protein interactions with XRCC4-like factor (XLF) create an extended grooved scaffold for DNA ligation and double strand break repair. J. Biol. Chem. 286, 32638–32650 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Malivert, L. et al. Delineation of the Xrcc4-interacting region in the globular head domain of cernunnos/XLF. J. Biol. Chem. 285, 26475–26483 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Roy, S. et al. XRCC4’s interaction with XLF is required for coding (but not signal) end joining. Nucleic Acids Res. 40, 1684–1694 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Roy, S. et al. XRCC4/XLF interaction is variably required for DNA repair, and is not required for Ligase IV stimulation. Mol. Cell. Biol. 35, 3017–3028 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Lu, H., Pannicke, U., Schwarz, K. & Lieber, M. R. Length-dependent binding of human XLF to DNA and stimulation of XRCC4.DNA ligase IV activity. J. Biol. Chem. 282, 11155–11162 (2007).

    Article  CAS  PubMed  Google Scholar 

  19. Gu, J., Lu, H., Tsai, A. G., Schwarz, K. & Lieber, M. R. Single-stranded DNA ligation and XLF-stimulated incompatible DNA end ligation by the XRCC4-DNA ligase IV complex: influence of terminal DNA sequence. Nucleic Acids Res. 35, 5755–5762 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Tsai, C. J., Kim, S. A. & Chu, G. Cernunnos/XLF promotes the ligation of mismatched and noncohesive DNA ends. Proc. Natl Acad. Sci. USA 104, 7851–7856 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Tadi, S. K. et al. PAXX is an accessory c-NHEJ factor that associates with Ku70 and has overlapping functions with XLF. Cell Rep. 17, 541–555 (2016).

    Article  CAS  PubMed  Google Scholar 

  22. Riballo, E. et al. XLF-Cernunnos promotes DNA ligase IV-XRCC4 re-adenylation following ligation. Nucleic Acids Res. 37, 482–492 (2009).

    Article  CAS  PubMed  Google Scholar 

  23. Mahaney, B. L., Hammel, M., Meek, K., Tainer, J. A. & Lees-Miller, S. P. XRCC4 and XLF form long helical protein filaments suitable for DNA end protection and alignment to facilitate DNA double strand break repair. Biochem. Cell Biol. 91, 31–41 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Wu, Q. et al. Non-homologous end-joining partners in a helical dance: structural studies of XLF-XRCC4 interactions. Biochem. Soc. Trans. 39, 1387–1392 (2011).

    Article  CAS  PubMed  Google Scholar 

  25. Andres, S. N. et al. A human XRCC4-XLF complex bridges DNA. Nucleic Acids Res. 40, 1868–1878 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Brouwer, I. et al. Sliding sleeves of XRCC4-XLF bridge DNA and connect fragments of broken DNA. Nature 535, 566–569 (2016).

    Article  CAS  PubMed  Google Scholar 

  27. Reid, D. A. et al. Organization and dynamics of the nonhomologous end-joining machinery during DNA double-strand break repair. Proc. Natl Acad. Sci. USA 112, E2575–E2584 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Di Virgilio, M. & Gautier, J. Repair of double-strand breaks by nonhomologous end joining in the absence of Mre11. J. Cell Biol. 171, 765–771 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Postow, L. et al. Ku80 removal from DNA through double strand break-induced ubiquitylation. J. Cell Biol. 182, 467–479 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Labhart, P. Nonhomologous DNA end joining in cell-free systems. Eur. J. Biochem. 265, 849–861 (1999).

    Article  CAS  PubMed  Google Scholar 

  31. Labhart, P. Ku-dependent nonhomologous DNA end joining in Xenopus egg extracts. Mol. Cell. Biol. 19, 2585–2593 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Sandoval, A. & Labhart, P. Joining of DNA ends bearing non-matching 3′-overhangs. DNA Repair (Amst.) 1, 397–410 (2002).

    Article  CAS  Google Scholar 

  33. Taylor, E. M. et al. The Mre11/Rad50/Nbs1 complex functions in resection-based DNA end joining in Xenopus laevis. Nucleic Acids Res. 38, 441–454 (2010).

    Article  CAS  PubMed  Google Scholar 

  34. Zhu, S. & Peng, A. Non-homologous end joining repair in Xenopus egg extract. Sci. Rep. 6, 27797 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Thode, S., Schäfer, A., Pfeiffer, P. & Vielmetter, W. A novel pathway of DNA end-to-end joining. Cell 60, 921–928 (1990).

    Article  CAS  PubMed  Google Scholar 

  36. Pfeiffer, P. & Vielmetter, W. Joining of nonhomologous DNA double strand breaks in vitro. Nucleic Acids Res. 16, 907–924 (1988).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Graham, T. G. W., Walter, J. C. & Loparo, J. J. Ensemble and single-molecule analysis of non-homologous end joining in frog egg extracts. Methods Enzymol 591, 233–270 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  38. Graham, T. G. W., Walter, J. C. & Loparo, J. J. Two-stage synapsis of DNA ends during non-homologous end joining. Mol. Cell 61, 850–858 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. DeFazio, L. G., Stansel, R. M., Griffith, J. D. & Chu, G. Synapsis of DNA ends by DNA-dependent protein kinase. EMBO J. 21, 3192–3200 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Hammel, M. et al. Ku and DNA-dependent protein kinase dynamic conformations and assembly regulate DNA binding and the initial non-homologous end joining complex. J. Biol. Chem. 285, 1414–1423 (2010).

    Article  CAS  PubMed  Google Scholar 

  41. Cary, R. B. et al. DNA looping by Ku and the DNA-dependent protein kinase. Proc. Natl Acad. Sci. USA 94, 4267–4272 (1997).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Spagnolo, L., Rivera-Calzada, A., Pearl, L. H. & Llorca, O. Three-dimensional structure of the human DNA-PKcs/Ku70/Ku80 complex assembled on DNA and its implications for DNA DSB repair. Mol. Cell 22, 511–519 (2006).

    Article  CAS  PubMed  Google Scholar 

  43. Cottarel, J. et al. A noncatalytic function of the ligation complex during nonhomologous end joining. J. Cell Biol. 200, 173–186 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Wang, J. L. et al. Dissection of DNA double-strand-break repair using novel single-molecule forceps. Nat. Struct. Mol. Biol. 25, 482–487 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Normanno, D. et al. 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. eLife 6, e22900 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  46. Lilliefors, H. W. On the Kolmogorov-Smirnov test for the exponential distribution with mean unknown. J. Am. Stat. Assoc. 64, 387 (1969).

    Article  Google Scholar 

  47. Fattah, F. J. et al. A role for XLF in DNA repair and recombination in human somatic cells. DNA Repair (Amst.) 15, 39–53 (2014).

    Article  CAS  Google Scholar 

  48. Ochi, T. et al. Structural insights into the role of domain flexibility in human DNA ligase IV. Structure 20, 1212–1222 (2012).

    Article  CAS  PubMed  Google Scholar 

  49. Menon, V. & Povirk, L. F. XLF/Cernunnos: an important but puzzling participant in the nonhomologous end joining DNA repair pathway. DNA Repair (Amst.) 58, 29–37 (2017).

    Article  CAS  Google Scholar 

  50. Hemsley, A., Arnheim, N., Toney, M. D., Cortopassi, G. & Galas, D. J. A simple method for site-directed mutagenesis using the polymerase chain reaction. Nucleic Acids Res. 17, 6545–6551 (1989).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Graham, T. G. W. et al. ParB spreading requires DNA bridging. Genes Dev. 28, 1228–1238 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Lebofsky, R., Takahashi, T. & Walter, J. C. DNA replication in nucleus-free Xenopus egg extracts. Methods Mol. Biol. 521, 229–252 (2009).

    Article  CAS  PubMed  Google Scholar 

  53. Tanner, N. A. et al. Single-molecule studies of fork dynamics in Escherichia coli DNA replication. Nat. Struct. Mol. Biol. 15, 998 (2008).

    Article  CAS  PubMed  Google Scholar 

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We thank E. van Arnam (Harvard Medical School) for assistance with purification of Cy5 Halo ligand, B. Stinson (Harvard Medical School) for providing improved FRET analysis code and generating the pETDuet vector containing XRCC4 and LIG4, K. Schmitz (Massachusetts Institute of Technology) for advice on generating an ‘orthogonal’ codon-optimized XLF sequence, K. Arnett (HMS Center for Macromolecular Interactions) for assistance with biophysical assays, and S. Harrison and S. Jenni (Harvard Medical School) for assistance with dynamic light scattering. We would also like to thank members of the laboratories of J.J.L. and J.C.W. for helpful discussions. This work was funded by a National Institutes of Health grant R01GM115487 (to J.J.L.) and the Howard Hughes Medical Institute (J.C.W.).

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All authors designed experiments and wrote the manuscript. T.G.W.G. and S.M.C. performed experiments and data analysis.

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Correspondence to Joseph J. Loparo.

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Integrated supplementary information

Supplementary Figure 1 Characterization of the Xenopus XLF–XRCC4 interaction.

(A) Bio-layer interferometry (BLI) of full-length X. laevis XLF binding to immobilized X. laevis XRCC4. Full-length wild-type XLF binds XRCC4 during the association phase (white box) and does not completely dissociate when washed with buffer during the dissociation phase (grey box). Full-length XLFL117D does not bind XRCC4. Results are shown from a single representative experiment. (B) Titration series of wild-type XLF1–226 fragment binding to immobilized XRCC4. Time traces during association (white box) and dissociation (gray box) phases were fit to a 1:1 model to determine kon and koff values at each XLF concentration (see panels E-F). (C) Dynamic light scattering (DLS) of X. laevis XLF and XRCC4 mixtures. Average hydrodynamic radius is plotted as a function of time with transparent shading around each trace representing the standard error of the mean from three independent experiments. Wild-type XLF alone forms supra-molecular assemblies hundreds of nanometers in size, consistent with the propensity of the full-length protein to aggregate in BLI experiments (see panel A). When mixed, wild-type XLF and XRCC4 form even larger supra-molecular complexes. Formation of these larger complexes is abolished for XLFL68D, XLFL117D, XRCC4F111E and diminished for XRCC4K104E. (D) XLF-XRCC4 DNA pull-down assay. The details of this assay are described in the online methods and depicted in the schematic on the right. Mutations of either XLF or XRCC4 that ablated their interaction disrupted pull-down of the 500 bp fragment. (E-F) Plots of kon and koff values from BLI titration experiments. Experimental and analysis details can be found in the online methods. Points show the average values, and error bars show the minimum and maximum of two experimental replicates. While the kon values (panel E) were not significantly different between the wild type and K104E mutant (p = 0.41), the koff values (panel F) did differ significantly (p = 0.0051). p-values were calculated using a two tailed, unpaired t-test with unequal variances. (G) Differential scanning fluorimetry of wild-type and mutant XLF and XRCC4 proteins. The average melting temperature of three replicates is shown for each sample with error bars representing the standard error of the mean. The difference between the XRCC4 mutants and wild-type XRCC4 was not statistically significant (p = 0.0251 and 0.3009 for K104E and F111E respectively) while there was a statistically significant difference between the XLF mutants and wild-type XLF (p = 0.004 and 0.0061 for L68D and L117D respectively). p-values were calculated using a two-tailed, unpaired t-test with unequal variances and the Bonferroni correction. However, the range of melting temperatures was less than 1.45 °C for both groups of mutants, implying no major change in protein folding or stability. (H) XLF-XRCC4 DNA pull-down assay. Increasing the buffer KCl concentration from 75 mM to 150 mM inhibits DNA bridging by a mixture of wild-type XLF and XRCC4. Additionally, Halo-XLF is not able to bridge DNA in this assay when mixed with XRCC4, despite the fact that Halo-XLF is functional for end joining (Supplementary Fig. 2A). (I) Dynamic light scattering (DLS) of X. laevis XLF and XRCC4 mixtures. Traces represent the average of three independent experiments with the transparent shading representing the standard error of the mean. Increasing the salt concentration from 75 mM to 150 mM KCl prevented the formation of large supra-molecular complexes by XLF and XRCC4. Data from panel C for the buffer only, XLF, and XLF + XRCC4 conditions at 75 mM are replotted here to permit comparison.

Supplementary Figure 2 Determining the stoichiometry of AF488-XLF in the SR complex.

(A) Rescue of NHEJ by fluorescently labeled Halo-XLF. Radiolabeled substrate DNA was incubated in extract immunodepleted of XLF and supplemented with protein storage buffer, Halo-XLF, or fluorescently labeled Halo-XLF. All labeled proteins rescued end joining in XLF-depleted extract. lin, linear substrate DNA; oc, open-circular products; scc, supercoiled closed-circular products; mult, dimeric and higher-order multimeric products. (B) 2-dimensional histogram of intensity with 488 nm excitation (vertical axis) and intensity with 532 nm excitation (horizontal axis) for Cy3/Cy5-DNA substrates imaged in extract. Direct excitation of Cy3 by 488 nm light produces a weak fluorescence signal whose intensity is linearly proportional to the intensity of emission with 532 nm excitation. Note that variability in Cy3 intensity in this experiment arises from Cy3-Cy5 FRET and variable protein-induced fluorescence enhancement (PIFE). Because Alexa Fluor 488 and Cy3 are imaged in the same channel of our microscope, it is crucial to subtract the contribution of Cy3 emission with 488 nm excitation to obtain the signal due to AF488 emission. This subtraction was accomplished by multiplying the signal with 532 nm excitation by the slope of the blue line and then subtracting this quantity from the total signal with 488 nm excitation (see panels C-D). (C) Plot of apparent AF488 fluorophore number in the absence of AF488-labeled protein in solution, without correction for Cy3 direct excitation by 488 nm light. The non-zero baseline and drop in signal at FRET onset are artifacts due to Cy3 excitation by 488 nm light. (D) Plot of the same data in (C) with correction for Cy3 excitation by 488 nm light, demonstrating effective subtraction of signal due to Cy3. (E) Comparison of AF488 stoichiometry with that predicted from binding of a single, stochastically labeled XLF dimer. Solid blue bars: number of AF488 dyes in the frame prior to FRET onset in 3-color imaging experiments, i.e., the ratio (AF488 intensity)/(AF488-Halo reference spot intensity) rounded to the nearest integer. Red open bars: predicted binomial distribution of number of Alexa Fluor 488 dyes, assuming that 1) a single XLF dimer binds, and 2) 68% of XLF monomers were labeled (as determined by UV-Vis absorbance). The actual distribution is in agreement with the theoretical prediction. (F) AF488 intensity traces in 3-color imaging of AF488-XLF (as in main text Fig. 3A), aligned at the time of FRET onset (red vertical lines; corresponding FRET traces are not shown). Intensity is expressed as number of AF488 fluorophores, which is calculated by dividing intensity in the AF488 channel by the average intensity of AF488-Halo reference spots in the same field-of-view. Horizontal scale bar on the lower right represents 30 seconds.

Supplementary Figure 3 Determining XLF stoichiometry in the SR complex through alternate labeling strategies.

(A) Histogram of Cy5 intensities for substrates exhibiting FRET onset (blue curve), fit to a constrained sum of Gaussians (red curve) to estimate the intensity of a single Cy5 molecule (see Methods). Gray vertical lines show intensity of 1, 2, and 3 Cy5 molecules obtained from the fit. (B) Histogram of Cy5 intensity, expressed as a multiple of the unitary Cy5 intensity determined in panel (A). The black curve includes all substrates, while the red curve includes only the subset of frames within 10 s of the transition to high FRET. (C) Cy5 intensity traces in the presence of Cy5-XLF, aligned at the time of FRET onset (red vertical lines; FRET traces are not shown). Intensity is expressed in number of Cy5 fluorophores, based on the intensity calibration in panel (A). Horizontal scale bar represents 30 seconds. Traces typically start at 1, corresponding to the single Cy5 label on the DNA, and FRET onset is typically preceded by a discrete Cy5-XLF binding event. (D) Rescue of NHEJ by maleimide-labeled XLF. AF488-maleimide-labeled, Cy5-maleimide-labeled, and mock-labeled XLF all rescued end joining in XLF-immunodepleted extract. Maleimide-labeled XLF rescued end joining to a lesser degree than mock-labeled XLF, suggesting that the activity of XLF may be reduced when labeled on some cysteine residues. (E) XLF-XRCC4 DNA pull-down assay with Cy5-maleimide labeled XLF. Upper panel: An agarose gel stained with ethidium bromide shows DNA in the supernatant and precipitate for each condition. Both mock-labeled XLF and Cy5-maleimide-labeled XLF exhibit DNA-bridging activity in combination with XRCC4. Lower panel: Fluorescence image of an SDS-PAGE gel of the same samples, showing that Cy5-mal-XLF is present in DNA bridging complexes.

Supplementary Figure 4 Characterization of synthetic XLF dimer constructs.

(A) SDS-PAGE analysis of XLF constructs. Wild-type XLF, XLFL117D, wild type XLF tandem dimer (tdXLFWT/WT), mutant XLF tandem dimer (tdXLFWT/L68D,L117D), H10-XLFWT:Flag-Avitag-XLFWT heterodimer, and H10-XLFWT:Flag-Avitag-XLFL117D heterodimer separated on an Any kD SDS-PAGE gel (BioRad) and stained with InstantBlue (Expedeon). (B) Size-exclusion chromatography with multi-angle light scattering (SEC-MALS) of tdXLF, showing elution as a single peak of the expected molecular weight. Black curve (y-axis scale on left) represents absorbance at 280 nm with a 1-cm path length, and blue curve (y-axis scale on right) represents calculated molecular weight.. (C-D) Test of monomer exchange between XLF dimers. (C) Flag- and H10-tagged XLF dimers, alone or in combination, were incubated for 3 h at room temperature. Exchange of monomers between H10-XLF and Flag-XLF dimers would yield H10-XLF:Flag-XLF heterodimers in the H10-XLF + Flag-XLF condition. H10-tagged complexes were bound to NiNTA resin, extensively washed with high-salt buffer, and eluted with imidazole. (D) The presence of H10- and Flag-tagged XLF subunits in input (in), supernatant (sup), and eluate (elu) fractions was assessed by western blotting with anti-Flag (αFlag) and anti-His tag (αHis) antibodies. Flag-tagged XLF was not detected in the NiNTA eluate fraction, even upon overexposure of the blot (bottom panel), implying minimal exchange of monomeric subunits between XLF dimers. (E) Dose-dependence of end joining as a function of concentration for H10-XLFWT:Flag-Avitag-XLFWT and H10-XLFWT:Flag-Avitag-XLFL117D heterodimers, following the same protocol as in main text Fig. 4B. Black wedges represent 2-fold serial dilution series from 650 nM to 0.33 nM. Control reaction not supplemented with XLF is labeled "-". lin, linear DNA substrate; oc, open-circular product; multimers, dimeric and multimeric products. (F) Quantification of product formation for the experiment in panel E.

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Graham, T.G.W., Carney, S.M., Walter, J.C. et al. A single XLF dimer bridges DNA ends during nonhomologous end joining. Nat Struct Mol Biol 25, 877–884 (2018).

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