The structural basis of modified nucleosome recognition by 53BP1

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Abstract

DNA double-strand breaks (DSBs) elicit a histone modification cascade that controls DNA repair1,2,3. This pathway involves the sequential ubiquitination of histones H1 and H2A by the E3 ubiquitin ligases RNF8 and RNF168, respectively4,5,6,7,8. RNF168 ubiquitinates H2A on lysine 13 and lysine 15 (refs 7, 8) (yielding H2AK13ub and H2AK15ub, respectively), an event that triggers the recruitment of 53BP1 (also known as TP53BP1) to chromatin flanking DSBs9,10. 53BP1 binds specifically to H2AK15ub-containing nucleosomes through a peptide segment termed the ubiquitination-dependent recruitment motif (UDR), which requires the simultaneous engagement of histone H4 lysine 20 dimethylation (H4K20me2) by its tandem Tudor domain10,11. How 53BP1 interacts with these two histone marks in the nucleosomal context, how it recognizes ubiquitin, and how it discriminates between H2AK13ub and H2AK15ub is unknown. Here we present the electron cryomicroscopy (cryo-EM) structure of a dimerized human 53BP1 fragment bound to a H4K20me2-containing and H2AK15ub-containing nucleosome core particle (NCP-ubme) at 4.5 Å resolution. The structure reveals that H4K20me2 and H2AK15ub recognition involves intimate contacts with multiple nucleosomal elements including the acidic patch. Ubiquitin recognition by 53BP1 is unusual and involves the sandwiching of the UDR segment between ubiquitin and the NCP surface. The selectivity for H2AK15ub is imparted by two arginine fingers in the H2A amino-terminal tail, which straddle the nucleosomal DNA and serve to position ubiquitin over the NCP-bound UDR segment. The structure of the complex between NCP-ubme and 53BP1 reveals the basis of 53BP1 recruitment to DSB sites and illuminates how combinations of histone marks and nucleosomal elements cooperate to produce highly specific chromatin responses, such as those elicited following chromosome breaks.

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Figure 1: Architecture of the NCP-ubme–GST–53BP1 complex.
Figure 2: The molecular basis of H2AK15ub recognition by 53BP1.
Figure 3: Multivalent recognition of NCP-ubme by the 53BP1 UDR.
Figure 4: Flexible association of 53BP1 tandem Tudor domain with NCP-ubme.

Accession codes

Primary accessions

Protein Data Bank

Data deposits

Model coordinates for the NCP-ubme–53BP1 UDR structure are deposited in the Protein Data Bank under accession code 5KGF.

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Acknowledgements

We are grateful to R. Szilard for reading the manuscript. We thank M. Forbes for 1D mass spectrometry analysis, C. Davey for providing the ‘601’ DNA, S. Orlicky and D. Ceccarelli for proteins, R. Guenette and E. Strieter for advice on H2A ubiquitination and T. Sixma for discussions. M.D.W. was supported by a Human Frontiers Science Program fellowship. S.M.N. holds a fellowship of the Dutch Cancer Foundation (KWF). J.L.R. is the Canada Research Chair (Tier 1) in Electron Cryomicroscopy and D.D. is a Canada Research Chair (Tier 1) in the Molecular Mechanisms of Genome Integrity. Work was supported by CIHR grants FDN143343 (to D.D.), FDN143277 (to F.S.) and MOP81294 (to J.L.R.), and a Grant-in-Aid from the Krembil Foundation (to D.D.).

Author information

M.D.W., F.S. and D.D. initiated the project. M.D.W. assembled protein complexes and performed biochemical assays. S.B. and M.D.W. performed cryo-EM and image analysis. A.F.-T. helped with biochemical assays. A.S. performed cellular assays. J.-P.J. supervised bio-layer interferometry assays. A.M. helped to perform molecular biology. S.M.N. identified in vitro ubiquitinated peptides. D.D. supervised the biochemical and cellular experiments and J.L.R. supervised the cryo-EM experiments. M.D.W., F.S., J.L.R. and D.D. wrote the manuscript with input from the other authors.

Correspondence to Frank Sicheri or John L. Rubinstein or Daniel Durocher.

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Competing interests

The authors declare no competing financial interests.

Additional information

Reviewer Information Nature thanks Y. Cheng, G. Stewart and G. G. Wang for their contribution to the peer review of this work.

Extended data figures and tables

Extended Data Figure 1 Generation of homogenously methylated and ubiquitinated NCPs.

a, Schematic of H4 cysteine alkylation to create a dimethyl-lysine analogue. b, 1D intact mass spectra of the alkylated H4KC20me2 protein after desalting and lyophilization. c, Mass spectrum of the identified off-target K36 ubiquitination. Fragmentation spectrum of the 37-K(GlyGly)GNYAER-43 H2A peptide (476.237091 Da, 2+ charge state, Mascot ions score: 45). This spectrum originates from di-ubiquitinated forms of in vitro ubiquitinated H2A that were separated by SDS–PAGE, subjected to limited trypsin digestion and analysed by tandem mass spectrometry. d, Immunoblot analysis of a H2A–H2B dimer ubiquitination reaction. Comparison of K13R, K15R and K36R triple mutated and K13R and K36R double mutated H2A variants using optimized conditions to minimize off-target ubiquitination for large-scale reactions. e, SDS–PAGE analysis of the first step in H2AK15ub purification, cation exchange chromatography (in, input; FT, flow-through; W, wash; M, molecular weight marker). The ubiquitin is tagged with an N-terminal hexa-histidine tag and TEV cleavage site (termed HisTEV-ub). f, SDS–PAGE analysis of the second step in H2AK15ub purification, nickel ion affinity chromatography. g, SDS–PAGE analysis of TEV protease cleavage of HisTEV–H2AK15ub and subsequent nickel column depletion. Cleaved H2AK15ub flows through (Ni FT) the column, while uncleaved product and His-tagged TEV protease binds it (not shown). h, Native polyacrylamide gel analysis of wrapped NCPs. The gel was stained with SYBR green to identify DNA. Wrapping of NCPs results in quenching of the SYBR green signal and a shift in the electrophoretic mobility of the DNA. Ubiquitinated NCPs (H2A-ub) appear as a doublet, which runs higher than solely methylated NCPs (WT H2A). Biotinylation on NCP surface lysines, required for downstream bio-layer interferometry analysis, does not measurable effect migration in the gel (H2A-ub-Bio). WT, wild-type H2A protein.

Extended Data Figure 2 Formation of NCP-ubme and NCP-ubme–GST–53BP1 complexes.

a, Schematic representation of full-length human 53BP1, 53BP1 with the native recruitment region, 53BP1(1220–1631), and GST–53BP1(1484–1631) (termed GST–53BP1). 53BP1(1220–1631) and GST–53BP1 constructs are used throughout this manuscript. Identified domains are highlighted; oligo: oligomerisation domain. b, Pull-down assay of GST–53BP1 variants containing either the 16-residue linker used throughout this study or a longer 34-residue linker. The L1619A UDR mutant was included as a negative control. c, Bio-layer interferometry assays of GST–53BP1 or a native 53BP1(1220–1631) fragment. d, SDS–PAGE of purified 53BP1 proteins used in the pull-down and bio-layer interferometry assays. M, molecular weight markers. e, Isothermal titration calorimetry (ITC) measurement investigating the affinity of GST–53BP1 (syringe) to NCP-ubme (cell). Data reported as the mean ±s.e.m. (n = 2). f, SDS–PAGE analysis of NCP-ubme–GST–53BP1 complex formation by differential PEG precipitation17 (in, input; S, soluble supernatant; P, pellet). An excess of GST–53BP1 was added, which was not precipitated with the NCP-ubme. g, SDS–PAGE analysis of size exclusion chromatography fractions, isolating NCP-ubme–GST–53BP1. In, input; M, size markers. Fractions 8–10 were pooled and used for SEC-MALS analysis (Fig. 1a) and subsequent structure determination. Source data

Extended Data Figure 3 Cryo-EM structure determination and validation of NCP-ubme–GST–53BP1 complex.

a, Representative cryo-EM micrograph of the NCP-ubme–GST–53BP1 complex. Example particle images in different orientations are boxed. b, Power spectrum from a representative micrograph showing Thon rings that extend beyond 7 Å resolution. c, Example particle images, scale bar corresponds to 25 Å. d, Examples of 2D class averages obtained during image processing of the NCPubme–GST–53BP1 complex (CTF corrected, inverted contrast). Scale bar, 25 Å. e, Fourier shell correlation curve after a gold-standard map refinement. f, Euler angle distribution plot of all particles used for the symmetrized final map. Bar length and colour (blue, low; red, high) corresponds to number of particle images contributing to each view. g, Magnified view of the H2B–H4 cleft with clear side chain density observed for H2B Arg89, Gln92 and Arg96 (top left). Magnified view of the H2B αC helix and H2A α1 helix (top right). Densities for aromatic side-chains of H2A Tyr50, H2B Tyr121 and the bulky residue H2A Arg17 are visible. Magnified view of the C terminus of the H2A α2 helix, with density for base of the side chain of Arg71 visible (bottom). h, Schematic of predicted location of flexible GST moiety (green) used for 53BP1 dimerization (Protein Data Bank accession code 1Y6E)47. No cryo-EM density can be attributed to GST, suggesting that it is highly flexible between different particles in the population sampled. Dashed lines indicate the 16 amino acid linker region incorporated in the GST–53BP1 construct (black) and the flexible C-terminal tail of the GST (green). The linker peptide region could span up to ~80 Å, allowing substantial flexibility of the GST dimers, shown here positioned ~50 Å from the N terminus of the modelled Tudor domain.

Extended Data Figure 4 Cryo-EM structure determination and validation of NCP-ubme complex.

a, Surface rendering of the NCP-ubme complex, viewed along the DNA axis and the orthogonal direction. Density corresponding to ubiquitin was segmented, Gaussian filtered and displayed with a threshold of 0.125 (area within dashed line). The rest of the NCP-ubme is displayed with a threshold 0.35. Rigid body fitting of the high resolution structure of histone octamers (Protein Data Bank accession code 1KX5)20, Widom-601 145bp DNA (Protein Data Bank accession code 3LZ0)14 and ubiquitin (Protein Data Bank accession code 1UBI)42 into NCP-ubme density is shown. Ubiquitin could not be readily placed in the attributed density. b, Representative cryo-EM micrograph of the NCP-ubme complex. Example particle images showing different orientations are boxed. c, Power spectrum from a representative micrograph showing Thon rings. d, A selection of particles images after extraction from the data set. Scale bar, 25 Å. e, Examples of 2D class averages obtained during image processing (CTF corrected, inverted contrast); scale bar, 25 Å. f, Fourier shell correlation curve after gold-standard map refinement. g, Euler angle distribution plot of all particle images used for the symmetrized final map. Bar length and colour corresponds to number of particle images in each view that contributed to final 3D map (blue, low; red, high).

Extended Data Figure 5 Chemical ubiquitination of H2A and the constrained conformation of ubiquitin in the NCP-ubme–GST–53BP1 complex.

a, Schematic of cross-linking reaction scheme between an electrophilic acetone (dibromoacetone, DBA) and two engineered cysteine residues in H2A and ubiquitin, respectively. TCEP was added to initially reduce disulfide bonds. b, Pilot reactions of cross-linkable ubiquitin mixed with H2A. Cross-linked products were separated by SDS–PAGE. H2A-only and ubiquitin-only reactions identify non-productive cross-linking in the final reaction, H2A–H2A and ub–ub. Correctly modified H2A is labelled H2AKC15ub. Hexahistadine and TEV consensus sequence-tagged ubiquitin was used (HisTEV-ub) c, Chemically ubiquitinated H2AKC15ub-containing NCPs interact with GST–53BP1. Immunoblot (IB) analysis of GST-53BP1 pull-down (PD) using NCPs assembled with unmodified H2A, catalytically ubiquitinated H2A or chemically ubiquitinated H2A (H2AKC15ub). In this pilot experiment, the chemically ubiquitinated H2A runs with lower mobility due to the retention of the HisTEV tag. The tag was removed in all future experiments. d, Space-filling model of the covalently tethered ubiquitin, in a closed conformation, pulled over the surface of NCP. Key interacting histone residues are labelled. e, Bio-layer interferometry traces of a single concentration of GST–53BP1 association and dissociation with immobilized NCP-ubme containing the indicated mutations in the H2B αC helix. Relative affinities are also shown. WT, wild-type H2B. f, Immunoblot analysis of GST–53BP1 pull-down assay to determine the effect of mutating the αC helix H2B residues that potentially form a hydrogen-bonding network with closed, 53BP1-bound ubiquitin. WT, wild-type H2B protein. g, Stained SDS–PAGE gel of purified reconstituted, nucleosomes used in this figure. H4KC20me2-modified NCPs containing cross-linked ubiquitin at indicated residues in H2A, H2B or both (left). These NCPs were used in assays in panels e and f. Biotinylated NCP-ubme complexes containing H2B variants used in the bio-layer interferometry assays (right). h, Immunoblot analysis of GST–53BP1 pull-down assay investigating the effect of H2BK120ub on GST–53BP1 binding to NCP-ubme.

Extended Data Figure 6 Structural basis of 53BP1 specificity for H2AK15ub.

a, Diagrammatic representation of the arginine-fingers mechanism of 53BP1 recognition of H2AK15ub-containing NCPs. Sequences of H2A mutations are detailed in c. b, Top view of the H2A N-terminal tail, displaying the modelled arginines projecting into the nucleosomal DNA grooves. For clarity, only the DNA phosphodiester backbone is shown. c, Immunoblot analysis of pull-down (PD) assay with RNF168-ubiquitinated NCPs containing the indicated H2A variants detailed at the bottom. GST–53BP1 can recognize H2AK13ub only when arginine 17 has been removed, allowing a shift in the N-terminal tail. Proposed R/KxxxKubxR consensus binding motif is indicated.

Extended Data Figure 7 Specific orientation of the UDR region and H2B–H4 cleft interactions.

a, Schematic of 53BP1 UDR region, indicating sites of engineered cysteines used for BMOE cross-linking (purple arrowheads) (top). Surface representation of modelled NCP with interaction interfaces coloured; tandem Tudor domain (H4 tail: red), UDR (H2B–H4 cleft, H2B αC helix and acidic patch: yellow) and ubiquitin (H2B αC helix and H2AK15: purple) (bottom). Locations of engineered H2B cysteine residues used for cross-linking are indicated (N84C and E105C). b, Immunoblot analysis of covalently cross-linked NCP-ubme–GST–53BP1 variants. BMOE, a bivalent maleimide cross-linker, was first reacted with NCP-ubme-containing H2B single cysteine variants, before incubation with cysteine cross-linkable GST–53BP1 variants. Cross-linking to H2B is visualized by a shift in apparent molecular weight, equivalent to the addition of one GST–53BP1 moiety. The relatively weaker cross-linking of H2B(E105C) and GST–53BP1(K1628C) probably arises from the fact that the lysine is predicted to interact on the other face of the acidic patch. The asterisk denotes a non-specific band due to cross-reactivity of the anti-H2A antibody with non-cross-linked GST–53BP1. c, Magnified view of the H2B–H4 cleft at the rear of the NCP, with modelled UDR chain in the yellow density. Ribbon structure of Sir3 BAH domain (residues 75–83; Protein Data Bank accession code 3TU4)16, also proposed to interact in this region, was overlaid (purple).

Extended Data Figure 8 Validation of the UDR–ubiquitin interaction.

a, Immunoblot analysis of pull-down (PD) assays, immobilizing the indicated GST–53BP1 UDR mutations in residues 1616–1620 and monitoring NCP-ubme interaction. WT, wild-type GST–53BP1 protein. b, SDS–PAGE of purified GST–53BP1 UDR proteins used in the pull-down and bio-layer interferometry assays. M, molecular weight markers. c, Pull-down assays of GST–53BP1 with the indicated NCP-ubme variants. d, Bio-layer interferometry traces of NCP-ubme prepared with the indicated ubiquitin variants chemically ligated to H2A at position 15. NB, no binding detected. e, SDS–PAGE analyses of reconstituted, biotinylated nucleosomes used in f. f, Enlarged view of the 53BP1-bound constrained modelled ubiquitin, with ball and stick representations of ubiquitin lysine residues indicated (left). Note the accessibility of Lys27 and Lys63, both reported to be following DSBs. Model of a Lys-3-linked di-ubiquitin (Protein Data bank accession code 3H7S)48 built on H2AK15 within the NCPubme–GST–53BP1 structure (right). The distal ubiquitin (pink) projects away from the NCP surface towards the tandem Tudor domain of 53BP1, shown in orange. Although we note a minor steric clash between the modelled distal ubiquitin and the tandem Tudor domain, we surmise that the inherent flexibility of ubiquitin chains, coupled with the flexibility of the tethered Tudor domain on the H4 tail, probably enables 53BP1 to bind to NCPs with Lys63-linked ubiquitin chains on H2AK15. Source data

Extended Data Figure 9 53BP1 UDR interactions with the nucleosome acidic patch.

a, Magnified view of a ribbon representation of the NCP-ubme acidic patch with the overlaying density attributed to the UDR. b, Immunoblot analysis of a GST-34–53BP1 pull-down assay performed with RNF168-ubiquitinated NCP-me, in the presence of the acidic-patch-interacting LANA peptide (the GST–53BP1 protein used here has the 34 amino acid linker). The indicated amounts of LANA peptide were added as a competitor during the pull-down (concentration in μM). 8LRS10 peptide has negligible NCP binding19 and used as a control. c, Immunoblot analysis of GST–53BP1 pull-downs (PD) using NCP-ubme incorporating the indicated H2A and H2B mutants, which localize to the acidic patch and adjacent H2B αC helix. d, SDS–PAGE and InstantBlue staining to analyse purified reconstituted, biotinylated nucleosomes containing H2A/H2B mutations used in the bio-layer interferometry assays. Compare to wild-type NCP-ubme in Extended Data Fig. 5g (right panel). e, Immunoblot analysis of GST–53BP1 pull-down assays, using selected 53BP1 UDR basic residue mutations. WT, wild-type GST–53BP1 protein. f, SDS–PAGE and InstantBlue staining to analyse purified GST–53BP1 UDR variant proteins used in the bio-layer interferometry assays in Fig. 3e. M, molecular weight markers. g, Enlarged view of UDR-acidic patch interaction site coloured according to coulombic surface charge, overlaid with the structure of other acidic patch chromatin binding factors: KSHV LANA peptide (Protein Data Bank accession code 1ZLA)19; the Sir3 BAH domain (Protein Data Bank accession code 3TU4)16 and the PRC1 complex (Protein Data Bank accession code 4R8P)15.

Extended Data Figure 10 Flexibility of the 53BP1 tandem Tudor domain in the NCP-ubme structure and comparison of GST–53BP1 to 53BP1(1220–1631).

a, A selection of aligned 3D maps obtained during determination of the NCP-ubme and GST–53BP1 structure, with an enlarged view of the density from the tandem Tudor domain of 53BP1, shown in the lower panels. Note that the position of the tandem Tudor domain density is highly variable, but is always tethered over the H4 N-terminal tail. b, 1D intact mass spectra of biotin-LC-H412–27 (K20C) peptide chemically alkylated to create a lysine mimic. The reaction proceeded to near completion, but some unreacted peptide can be observed at 2,190 Da. c, 1D intact mass spectra of biotin–LC-H412–27 K20C, peptide chemically alkylated to create a dimethyl lysine mimic. The reaction proceeded to near completion, but some unreacted peptide can be observed at 2,190 Da. d, Bio-layer interferometry traces comparing the binding of GST–53BP1 with 53BP1(1220–1631) to NCP-ubme variants. Data from a single 53BP1 protein concentration is plotted. Source data

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Wilson, M., Benlekbir, S., Fradet-Turcotte, A. et al. The structural basis of modified nucleosome recognition by 53BP1. Nature 536, 100–103 (2016) doi:10.1038/nature18951

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