Structural insight into BRCA1-BARD1 complex recruitment to damaged chromatin

•Cryo-EM structure of human BARD1 in complex with H2AK15-ubiquitinated nucleosome•The BUDR motif contacts the nucleosome acidic patch and ubiquitin K63-E64•The ARD domain grips H4 tail by accommodating unmethylated H4K20•Mutations disrupting BARD1-NCPUb association impair homologous recombination The BRCA1-BARD1 directs DNA double-strand break (DSB) repair pathway choice to error-free (HR) during S-G2 stages. Targeting DSB-proximal sites requires BARD1-mediated interaction histone mark recognition. Here, we report cryo-EM bound a ubiquitinated core particle (NCPUb) at 3.1 Å resolution illustrate how simultaneously recognizes damage-induced H2AK15ub replication-associated H4K20me0 on nucleosome. In vitro vivo analyses reveal that is stabilized BARD1-nucleosome interaction, BARD1-ubiquitin domain-BARD1 BRCT abrogating these interactions detrimental HR activity. We further identify multiple disease-causing mutations disrupt hence HR. Together, this study elucidates mechanism recruitment retention DSB-flanking nucleosomes sheds important light cancer therapeutic avenues. breaks (DSBs) are perilous lesions may spawn carcinogenesis or cell death. mammalian cells, non-homologous end joining (NHEJ) work as two main pathways DSBs ensure genome integrity (Ceccaldi et al., 2016Ceccaldi R. Rondinelli B. D’Andrea A.D. Repair choices consequences break.Trends Cell Biol. 2016; 26: 52-64Abstract Full Text PDF PubMed Scopus (768) Google Scholar; Scully 2019Scully Panday A. Elango Willis N.A. repair-pathway somatic cells.Nat. Rev. Mol. 2019; 20: 698-714Crossref (397) Scholar). NHEJ involves direct religation DSB ends, which could cause deletion insertion nucleotides sites. contrast error-prone pathway, accurately restores genetic information suppresses tumorigenesis, it uses sister chromatid template guide process. Hence, deployed only S G2 phases cycle, when available (Daley 2014Daley J.M. Gaines W.A. Kwon Y. Sung P. 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Lukauskas Frimurer al.H4K20me0 chromatids.Nat. 21: 311-318Crossref (77) Recently, ankyrin repeat (ARD) was identified new K20 (H4K20me0) (Nakamura ARD-mediated chromatids explains why restricted more recent revealed contains (Ub)-dependent (BUDR) (residues 704–716), BARD1-BRCT functions novel lysine H2A-type histones (H2AK15ub), RNF168-dependent DSB-specific modification (Becker 2020Becker Bonnet Clifford Groth Chapman links lysine-15 ubiquitination initiation BRCA1-dependent recombination.BioRxiv. 2020; https://doi.org/10.1101/2020.06.01.127951Crossref (0) This targets facilitate Despite all advances, reads nucleosomal context remains missing part our understanding choice. study, cryoelectron microscopy (cryo-EM) resolution. reveals directly also other portions NCP, especially patch. Moreover, H2A-Ub adopts noncanonical surface bridge Interestingly, inter-domain stabilize interplay. Biochemical cell-based assays demonstrate intermolecular intramolecular bindings Our findings address previous questions regarding role BARD1’s point suggest pathogenic cancer-associated BARD1. elucidate DSB-surrounding recruit BRCA1-BARD1, analyzed without ubiquitination. prepared containing both 425–777, hereafter referred BARD1) (Figure 1A). reconstituted NCPs harboring non-hydrolyzable Ub H2AK15 (termed NCPUb), mimics native (see STAR Methods), using 147 bp Widom 601 DNA. performed microscale thermophoresis (MST) examine whether presents preference NCPUb over unmodified 1B). Indeed, NCPUb, yielding dissociation constant (Kd) equal 0.4 ?M. 18-fold tighter than BARD1-unmodified (Kd = 7.1 ?M; Figure 1C). used isothermal titration calorimetry (ITC) probe stoichiometry demonstrated one (N 2.06) S1A). These results agreement finding showing acts H2AK15ub. specific should rely nucleosomes, given displayed no measurable free S1B). Further analysis S1C), implicating cooperation observation supports indispensable activity (Laufer 2007Laufer Nandula S.V. Modi A.P. Jasin Murty V.V.V.S. Ludwig requirements chromosomal stability repair.J. 2007; 282: 34325-34333Abstract (40) decipher determined single-particle overall (Fourier shell correlation [FSC] 0.143 criterion) (Figures S1D–S1J). final reconstruction sufficiently detailed features allowed us build model (Table 1). extra density above attributed 1D). Inspection map well-resolved side-chain majority complex, allowing unambiguously S2). Of note, densities corresponding ends inferior elements S1I), presumably because conformational flexibility ends.Table 1Cryo-EM data collection, refinement, validation statisticsBARD1-NCPUb Complex (EMD-31020) (PDB: 7E8I)Data collection processingMagnification130,000Voltage (kV)200Electron exposure (e–/Å2)50Defocus range (?m)?1.2 1.8Pixel size (Å)1Symmetry imposedC1Initial images (no.)2,783,473Final (no.)168,313Map (Å)3.11FSC threshold0.143Map (Å)2.8–6.0RefinementInitial (PDB code)• 3C5R (ARD), 3FA2 (BRCT)• 5KGF (ubiquitin), 3LZ0 (nucleosome)Model (Å)3.25FSC threshold0.5Map sharpening B (Å2)?81.4Model compositionNon-hydrogen atoms15,280Protein residues1,176Nucleotide290Ligands0B factors (Å2)Protein87.90Nucleotide125.25RMSDsBond lengths (Å)0.005Bond angles (°)0.727ValidationMolProbity score1.63Clashscore7.90Poor rotamers (%)0.00Ramachandran plotFavored (%)96.79Allowed (%)3.21Disallowed (%)0.00RMSD, root-mean-square deviation. Open table tab RMSD, high-resolution single copy wherein NCP classical coin shape sits atop bottom edge plate H2A-H2B 1E 2A ). occupy different areas mediate binding. On side, wedges into cleft formed nucleosome, burying solvent-accessible approximate 437.7 537.4 Å2, respectively 2A). Intriguingly, putative PAR-binding pocket resides far from NCPUb-binding interface 2A), supporting subordinate mediating localization compared BRCT-NCPUb interaction. rests alongside 1E), coinciding ARD’s function H4K20me0. EM linker region (BARD1 residues 546–566) connecting absent 1D 1E). region, will later, crucial Below, describe details each interacting interface. serves hotspot numerous nucleosome-binding (McGinty Tan, 2015McGinty R.K. Tan Nucleosome function.Chem. 115: 2255-2273Crossref (243) Zhou 2019Zhou Gaullier Luger dynamics coming age.Nat. 3-13Crossref (113) takes advantage nexus recognize domain. primary contact centered residue R705, establishes salt network triad E61, D90, E92) 2B). R705-mediated mirror those adopted some well-studied nucleosome-interacting partners (Sundaram Vasudevan, 2020Sundaram Vasudevan modulation.BioEssays. 42: e1900234Crossref (6) reflecting R705 “arginine anchor” S3A). Meanwhile, following K708 points toward E64, rendering charge-charge Besides patch, segment ?C helix H2B. H2B H106 K105 (H109 K108 Homo sapiens) accommodate V713, allows Waals interactions, preceding D712 makes electrostatic S109 K113 well conserved across various species 2F), underscoring key BARD1-NCP interrogated effect MST electrophoretic mobility shift assay (EMSA). R705A D712A induced alanine substitution (R705) nearby reduced 2D, S3B, S3C). Reciprocally, (H106A/K105E) substantially lowered affinity 2E), underlining BUDR-NCP function. line observation, reported E61A, E64S, D90A significantly decreased chromatin-associated proteins, including (Skrajna 2020Skrajna Goldfarb Kedziora K.M. Cousins E.M. Grant G.D. Spangler C.J. Barbour E.H. Yan Hathaway Brown N.G. al.Comprehensive interactome screen fundamental principles binding.Nucleic Acids 48: 9415-9432Crossref (27) Ub. D710 Q715 straddle T66 form hydrogen bonds, followed N718 N60 2C). D729 R751 regions K63 E64 bridges buttressed neighboring K754-Ub S65 Ub-BARD1 unconventional mode, usually exploits hotspots “I44 patch” “I36 Ub-binding engagement (discussed next paragraph) (Dikic 2009Dikic Wakatsuki Walters K.J. Ubiquitin-binding - structures functions.Nat. 2009; 10: 659-671Crossref (598) Komander Rape, 2012Komander Rape code.Annu. Biochem. 2012; 81: 203-229Crossref (2051) Consistent structure, Q715R mutant, abolishes achieved 2D S3D), suggesting defects deficiency. Notably, charge reversal R751E, D729K greatly hindered S3D, S3E). reciprocal dual (K63A/E64A) hampered 2E). Additionally, involved invariably conservation importance BARD1-Ub mode. Collectively, molecular comparison 53BP1-NCPUb (Wilson placed distinct orientation relative S4A), indicating divergent modes 53BP1-UDR binds canonical patch,” do most partners. relocation, masked inaccessible S4B S4C). result indicated conformationally plastic recognized proteins ways. K63-linked polyubiquitin abundant related (Lee 2017Lee B.L. Singh Mark Glover J.N. Hendzel M.J. Spyracopoulos Molecular processes repair: focus kinetics dynamics.J. 2017; 429: 3409-3429Crossref (23) suggested likely incompatible S4D). T12 recently intolerant yet permissive BRCA1/BARD1-mediated (Walser 2020Walser Mulder M.P.C. Bragantini Burger Gubser Gatti Botuyan Villa Altmeyer Neri al.Ubiquitin Thr12 modulates response.Mol. 80: 423-436.e9Abstract (16) conclusion does participate unlikely disturbs S4E). As mentioned above, mainly extensive while I44, H68, V70 T116 K117 (T119 K120 3A). Adjoining Q49 positioned bonding S120 (S123 Consistently, I44R (substitution I44 arginine) H68A H68 alanine) binding, hydrophobic 3B). destabilizing Ub-NCP deleterious interactions. Likewise, T116E (a mimic) K117A substantiated underpin NCPUb. Furthermore, T119 (T116 Xenopus laevis) might affect DSB-neighboring chromatin, emerging meiosis (Baarends 2007Baarends W.M. Wassenaar Hoogerbrugge J.W. Schoenmakers Sun Z.-W. Grootegoed J.A. Increased dimethylation XY body Hr6b-knockout mouse derepression X chromosome.J. Sci. 120: 1841-1851Crossref (50) Within leans composed internal loops BRCT-ARD clusters buries area 444.2 Å2. First, L667 encircled L486, T489, K457 ARD, P669 D673 protrude T490 N494 3C). include bonds contacts. Second, V720 close V523, indicative favorable lack apparent nucleosome-unbound (Fox 2008Fox 3rd, Le Trong Rajagopal Brzovic P.S. Stenkamp Crystal functional consequences.J. 2008; 283: 21179-21186Abstract (32) Choudhary 2017Choudhary Siddiqui M.Q. Thapa Gadewal Nachimuthu S.K. Varma A.K. motion BARD1-ARD CstF50.Sci. Rep. 7: 3849Crossref (8) upon predicted interfacial critical stabilizing complex. assumption, reduce evidenced L486A/L667A T489E/T490E 3D, S5A, S5C). simultaneous replacement anchors, V720, charged (L667K/V720D) seriously disrupted 3D S5A). results, therefore, corroborated peptide (BARD 546–566), invisible drastically S5B). enhance BRCT-ARD-NCPUb individual domains, stabilization context. snakes along surface, traverses pocket, terminates histone-fold 4A). H18 fits four (W462, E467, N470, D500), side chain inserting channel establishing E429, D458, E467 4A 4B). resemble have been H4-TONSL-ARD (Saredi 2016Saredi Hammond C.M. Alabert Bekker-Jensen Forne Reverón-Gómez Mlejnkova Bartke post-replicative recruits TONSL–MMS22L complex.Nature. 534: 714-718Crossref (109) Scholar) ARD-H4K20me0 ARD3A mutant E467A/N470A/D500A) disrupts 4C, 4D, S5D), coincident abrogates accumulation R427 oriented negatively phosphodiester backbone DNA, creating participating int