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Structure of a meiosis-specific complex central to BRCA2 localization at recombination sites

Abstract

Meiotic cells invoke breast cancer susceptibility gene 2 (BRCA2) to repair programmed double-stranded DNA breaks and accomplish homologous recombination. The meiosis-specific protein MEILB2 facilitates BRCA2 recruitment to meiotic recombination sites. Here, we combine crystallography, biochemical analysis and a mouse meiosis model to reveal a robust architecture that ensures meiotic BRCA2 recruitment. The crystal structure of the MEILB2–BRCA2 complex reveals how two MEILB2 homodimers sandwich two chains of BRCA2 to afford a 4:2 architecture. The sandwich lacks close contact between the two MEILB2 dimers or the two BRCA2 chains. Instead, the two halves of each BRCA2 chain bridge two MEILB2 subunits from different homodimers to form the MEILB2–BRCA2–MEILB2 sandwich. Several identical residues from the two MEILB2 subunits are employed to engage the BRCA2 halves, justifying their strict conservation. Mutational analysis of the interface reveals a synergistic mechanism for MEILB2–BRCA2 recruitment during meiosis. Overall, these studies demonstrate how BRCA2 efficiently localizes in the cell to facilitate meiosis.

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Fig. 1: BRCA2 is sandwiched between two MEILB2 homodimers.
Fig. 2: Hydrophobic MEILB2–MEILB2 and ionic MEILB2–BRCA2 interfaces drive formation of the 4:2 complex.
Fig. 3: The MEILB2–BRCA2 assembly falls apart in the presence of mutations at the MEILB2–BRCA2 interface.
Fig. 4: Mutations at the MEILB2–BRCA2 interface compromise recruitment of both proteins to recombination nodules in mouse spermatocytes.
Fig. 5: Model for BRCA2 recruitment to sites of meiotic HR.

Data availability

Coordinates and structure factors of the MEILB283-end–BRCA2MBD crystal structure are deposited in the Protein Data Bank (PDB) under accession code 7LDG. Source data are provided with this paper.

References

  1. 1.

    Kowalczykowski, S. C. & Eggleston, A. K. Homologous pairing and DNA strand-exchange proteins. Annu. Rev. Biochem. 63, 991–1043 (1994).

    CAS  Article  Google Scholar 

  2. 2.

    West, S. C. Molecular views of recombination proteins and their control. Nat. Rev. Mol. Cell Biol. 4, 435–445 (2003).

    CAS  Article  Google Scholar 

  3. 3.

    San Filippo, J., Sung, P. & Klein, H. Mechanism of eukaryotic homologous recombination. Annu. Rev. Biochem. 77, 229–257 (2008).

    CAS  Article  Google Scholar 

  4. 4.

    Pellegrini, L. & Venkitaraman, A. Emerging functions of BRCA2 in DNA recombination. Trends Biochem. Sci. 29, 310–316 (2004).

    CAS  Article  Google Scholar 

  5. 5.

    Moynahan, M. E. & Jasin, M. Mitotic homologous recombination maintains genomic stability and suppresses tumorigenesis. Nat. Rev. Mol. Cell Biol. 11, 196–207 (2010).

    CAS  Article  Google Scholar 

  6. 6.

    Zhao, W., Wiese, C., Kwon, Y., Hromas, R. & Sung, P. The BRCA tumor suppressor network in chromosome damage repair by homologous recombination. Annu. Rev. Biochem. 88, 221–245 (2019).

    CAS  Article  Google Scholar 

  7. 7.

    Li, M. L. & Greenberg, R. A. Links between genome integrity and BRCA1 tumor suppression. Trends Biochem. Sci. 37, 418–424 (2012).

    CAS  Article  Google Scholar 

  8. 8.

    Wooster, R. & Weber, B. L. Breast and ovarian cancer. N. Engl. J. Med. 348, 2339–2347 (2003).

    CAS  Article  Google Scholar 

  9. 9.

    Stracker, T. H. & Petrini, J. H. The MRE11 complex: starting from the ends. Nat. Rev. Mol. Cell Biol. 12, 90–103 (2011).

    CAS  Article  Google Scholar 

  10. 10.

    Syed, A. & Tainer, J. A. The MRE11–RAD50–NBS1 complex conducts the orchestration of damage signaling and outcomes to stress in DNA replication and repair. Annu. Rev. Biochem. 87, 263–294 (2018).

    CAS  Article  Google Scholar 

  11. 11.

    Wold, M. S. Replication protein A: a heterotrimeric, single-stranded DNA-binding protein required for eukaryotic DNA metabolism. Annu. Rev. Biochem. 66, 61–92 (1997).

    CAS  Article  Google Scholar 

  12. 12.

    Neale, M. J. & Keeney, S. Clarifying the mechanics of DNA strand exchange in meiotic recombination. Nature 442, 153–158 (2006).

    CAS  Article  Google Scholar 

  13. 13.

    Scully, R., Panday, A., Elango, R. & Willis, N. A. DNA double-strand break repair-pathway choice in somatic mammalian cells. Nat. Rev. Mol. Cell Biol. 20, 698–714 (2019).

    CAS  Article  Google Scholar 

  14. 14.

    Zickler, D. & Kleckner, N. Recombination, pairing and synapsis of homologs during meiosis. Cold Spring Harb. Perspect. Biol. https://doi.org/10.1101/cshperspect.a016626 (2015).

  15. 15.

    Hassold, T. & Hunt, P. To err (meiotically) is human: the genesis of human aneuploidy. Nat. Rev. Genet. 2, 280–291 (2001).

    CAS  Article  Google Scholar 

  16. 16.

    Keeney, S., Giroux, C. N. & Kleckner, N. Meiosis-specific DNA double-strand breaks are catalyzed by Spo11, a member of a widely conserved protein family. Cell 88, 375–384 (1997).

    CAS  Article  Google Scholar 

  17. 17.

    Lange, J. et al. The landscape of mouse meiotic double-strand break formation, processing and repair. Cell 167, 695–708 (2016).

    CAS  Article  Google Scholar 

  18. 18.

    Robert, T. et al. The TopoVIB-like protein family is required for meiotic DNA double-strand break formation. Science 351, 943–949 (2016).

    CAS  Article  Google Scholar 

  19. 19.

    Zhang, J., Fujiwara, Y., Yamamoto, S. & Shibuya, H. A meiosis-specific BRCA2 binding protein recruits recombinases to DNA double-strand breaks to ensure homologous recombination. Nat. Commun. 10, 722 (2019).

    CAS  Article  Google Scholar 

  20. 20.

    Zhang, J. et al. The BRCA2–MEILB2–BRME1 complex governs meiotic recombination and impairs the mitotic BRCA2–RAD51 function in cancer cells. Nat. Commun. 11, 2055 (2020).

    CAS  Article  Google Scholar 

  21. 21.

    Brandsma, I. et al. HSF2BP interacts with a conserved domain of BRCA2 and is required for mouse spermatogenesis. Cell Rep. 27, 3790–3798 (2019).

    CAS  Article  Google Scholar 

  22. 22.

    Krissinel, E. & Henrick, K. Inference of macromolecular assemblies from crystalline state. J. Mol. Biol. 372, 774–797 (2007).

    CAS  Article  Google Scholar 

  23. 23.

    Shahid, T. et al. Structure and mechanism of action of the BRCA2 breast cancer tumor suppressor. Nat. Struct. Mol. Biol. 21, 962–968 (2014).

    CAS  Article  Google Scholar 

  24. 24.

    Corbett, K. D. et al. The monopolin complex crosslinks kinetochore components to regulate chromosome-microtubule attachments. Cell 142, 556–567 (2010).

    CAS  Article  Google Scholar 

  25. 25.

    Endres, N. F., Barros, T., Cantor, A. J. & Kuriyan, J. Emerging concepts in the regulation of the EGF receptor and other receptor tyrosine kinases. Trends Biochem. Sci. 39, 437–446 (2014).

    CAS  Article  Google Scholar 

  26. 26.

    Wiesmann, C., Ultsch, M. H., Bass, S. H. & de Vos, A. M. Crystal structure of nerve growth factor in complex with the ligand-binding domain of the TrkA receptor. Nature 401, 184–188 (1999).

    CAS  Article  Google Scholar 

  27. 27.

    Liu, M. & Grigoriev, A. Protein domains correlate strongly with exons in multiple eukaryotic genomes—evidence of exon shuffling? Trends Genet. 20, 399–403 (2004).

    Article  Google Scholar 

  28. 28.

    Souquet, B. et al. MEIOB targets single-strand DNA and is necessary for meiotic recombination. PLoS Genet. 9, e1003784 (2013).

    CAS  Article  Google Scholar 

  29. 29.

    Thorslund, T. et al. The breast cancer tumor suppressor BRCA2 promotes the specific targeting of RAD51 to single-stranded DNA. Nat. Struct. Mol. Biol. 17, 1263–1265 (2010).

    CAS  Article  Google Scholar 

  30. 30.

    Luo, M. et al. MEIOB exhibits single-stranded DNA-binding and exonuclease activities and is essential for meiotic recombination. Nat. Commun. 4, 2788 (2013).

    Article  Google Scholar 

  31. 31.

    Ishishita, S., Matsuda, Y. & Kitada, K. Genetic evidence suggests that Spata22 is required for the maintenance of Rad51 foci in mammalian meiosis. Sci. Rep. 4, 6148 (2014).

    CAS  Article  Google Scholar 

  32. 32.

    Mossessova, E. & Lima, C. D. Ulp1-SUMO crystal structure and genetic analysis reveal conserved interactions and a regulatory element essential for cell growth in yeast. Mol. Cell 5, 865–876 (2000).

    CAS  Article  Google Scholar 

  33. 33.

    Leahy, D. J., Hendrickson, W. A., Aukhil, I. & Erickson, H. P. Structure of a fibronectin type III domain from tenascin phased by MAD analysis of the selenomethionyl protein. Science 258, 987–991 (1992).

    CAS  Article  Google Scholar 

  34. 34.

    Kabsch, W. XDS. Acta Crystallogr. D Biol. Crystallogr. 66, 125–132 (2010).

    CAS  Article  Google Scholar 

  35. 35.

    Kabsch, W. Integration, scaling, space-group assignment and post-refinement. Acta Crystallogr. D Biol. Crystallogr. 66, 133–144 (2010).

    CAS  Article  Google Scholar 

  36. 36.

    Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D Biol. Crystallogr. 66, 213–221 (2010).

    CAS  Article  Google Scholar 

  37. 37.

    Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr. 60, 2126–2132 (2004).

    Article  Google Scholar 

  38. 38.

    Afonine, P. V. et al. Towards automated crystallographic structure refinement with phenix.refine. Acta Crystallogr. D Biol. Crystallogr. 68, 352–367 (2012).

    CAS  Article  Google Scholar 

  39. 39.

    Shibuya, H., Morimoto, A. & Watanabe, Y. The dissection of meiotic chromosome movement in mice using an in vivo electroporation technique. PLoS Genet. 10, e1004821 (2014).

    Article  Google Scholar 

Download references

Acknowledgements

We thank J. S. Brunzelle at the Life Sciences Collaborative Access Team (LS-CAT) beamline of the Argonne National Laboratory for help with X-ray diffraction data collection and initial processing, the University of Michigan Structural Biology Supergroup for helpful suggestions, V. M. Tesmer of the Nandakumar laboratory for help with collection of crystals and input on figures, and the members of the Nandakumar laboratory for critical feedback on the manuscript. This work was supported by NIH grants R01-AG050509 (J.N.) and R01-GM120094 (J.N.), American Cancer Society Research Scholar grant RSG-17-037-01-DMC (J.N.), an American Heart Association predoctoral fellowship award ID 830111 (R.A.), Assar Gabrielssons Foundation grant FB-20-57 (J.Z.), European Research Council grant StG-801659 (H.S.), Swedish Research Council grant 2018-03426 (H.S.), Cancerfonden grant 2018/326 (H.S.) and the Knut och Alice Wallensbergs Stiftelse KAW2019.0180 (H.S.).

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Authors

Contributions

J.N., D.F.P. and H.S. designed the study. Crystallography was performed by D.F.P. and J.N. R.A. and D.F.P. performed in vitro validation of the structural interface. J.Z. performed the in vivo validation of the structural interface, with help from H.S. All authors were involved in data analysis. J.N., D.F.P. and H.S. wrote the manuscript, with critical feedback from the remaining authors.

Corresponding authors

Correspondence to Hiroki Shibuya or Jayakrishnan Nandakumar.

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The authors declare no competing interests.

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Peer review information Nature Structural & Molecular Biology thanks Mark Glover, John Weir and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Electron density for BRCA2MBD.

The sandwich between MEILB283-end a and B subunits, and BRCA2MBD is shown in cartoon representation along with 2Fo-Fc electron density map encompassing BRCA2MBD contoured at 1.0 σ.

Extended Data Fig. 2 MEILB2-BRCA2 quaternary structure.

(A) B-factor heat map (blue→red indicates low→high B-factors) for the MEILB283-end-BRCA2MBD complex highlighting the ordered nature of the MEILB2-MEILB2 and MEILB2-BRCA2 interfaces. (B) Back view and (C) side view (right) of the human MEILB283-end-BRCA2MBD complex (relative to depictions in Fig. 1) shown in cartoon representation. Note in panel C how the NCS two-fold axis of MEILB283-end homodimerization is oblique (that is, neither parallel nor perpendicular) with respect to the crystallographic two-fold axis of 4:2 complex formation. (D) Head-to-tail anti-parallel alignments of the N terminus of one BRCA2MBD chain (aa 2276–2282; head) and the C terminus of another BRCA2MBD chain (aa 2335 and upstream residues; tail) in the crystal lattice results in the formation of a triangular 6:3 MEILB283-end-BRCA2MBD assembly from three 2:1 complexes around the three-fold crystallographic axis. (E) Further inclusion of the symmetry mates related by the two-fold crystallographic axis results in a 12:6 assembly in the crystal lattice. (F) SEC-MALS analysis of MEILB283-end - BRCA2MBD ΔPLILVGE strongly suggests formation of a 4:2 complex. (G) Anti-FLAG co-immunoprecipitation (co-IP) analysis of HEK 293T cell lysates containing transiently transfected FLAG-tagged MEILB283-end, myc-tagged MEILB283-end and either eGFP-myc-vector control, eGFP-myc-tagged BRCA2MBD wild type, or eGFP-myc-tagged BRCA2MBD ΔPLILVGE constructs. Data are representative of three replicates.

Source data

Extended Data Fig. 3 MEILB2 ARM repeat domain structure and homodimerization is an adaptation of the extensive ARM repeat structure of β-catenin.

(A) Overlay of 9 different ligand-bound β-catenin structures in the PDB showing the ligands tracing the concave track formed by tandem ARM repeats. β-catenin and its binding partners from the various structures are shown in different shades of green and blue, respectively. (B, C) Closeup of the partner-bound concave surfaces at the N-terminal (B) and C-terminal (C) ARM repeats of β-catenin. PDB IDs: 1qz7, 1jdh, 3ouw, 1jpp, 1g3j, 2gl7, 1i7x, 1i7w, and 1jpw. (D) Alignment of the MEILB283-end B subunit with its top DALI server alignment hit, β-catenin (PDB ID: 1i7w). (E) Alignment of MEILB283-end B with β-catenin as in panel D but also showing the homodimeric partner MEILB283-end A subunit that was not used in the alignment. (F) The hairpin connecting two helices of β-catenin that align with the N-terminal helices of MEILB283-end A and B is structurally replaced by the disulfide linkage between the MEILB283-end subunits. (G) The helices of the N-terminal ARM repeat of MEILB283-end A superimpose with β-catenin regions but the polarity of the helices (n→c) is reversed. Curved lines indicate the superimposed helices of β-catenin and MEILB283-end A. Arrowhead indicates disulfide linkage.

Extended Data Fig. 4 Schematic showing the polar interactions between the BRCA2MBD and its two MEILB2 binding partners.

Ionic (salt-bridge; left) and H-bonding (right) interactions along the entire BRCA2MBD chain is shown schematically. MEILB2 and BRCA2 color-coding is retained from structural depictions in main and other supplementary figures.

Extended Data Fig. 5 Salt-bridges dominate the MEILB2-BRCA2 interface.

(AH) 2Fo-Fc electron density map contoured at 1.0 σ and stick representations for the indicated interactions at the MEILB2-BRCA2 interface. (I, J) Consurf analysis of MEILB283-end (I) and BRCA2MBD (J) showing the high level of conservation of the MEILB2-MEILB2 (blue asterisks) and MEILB2-BRCA2 (red asterisks) interface residues.

Extended Data Fig. 6 The MEILB2-MEILB2 interface is bolstered by BRCA2.

(A) Co-IP analysis of MEILB283-end – BRCA2MBD in the presence of MEILB2 mutations including the D268R/D326H double mutant. (B) Anti-FLAG co-immunoprecipitation (co-IP) analysis of HEK 293T cell lysates containing the indicated transiently transfected with wild type myc-tagged MEILB283-end and indicated FLAG-tagged MEILB283-end constructs harboring mutations in the MEILB283-end homodimerization interface. (C) Co-IP analysis of MEILB2-MEILB2 association in the absence and presence of BRCA2MBD. (D) Co-IP of MEILB283-end homodimerization mutants in the presence of wild type myc-tagged MEILB283-end. WT: wild type. Data are representative of at least three replicates.

Source data

Extended Data Fig. 7 Biochemical analysis of single-mutations in each half of the MEILB2-binding segments of BRCA2MBD.

(A) Size-exclusion profiles of purified human MEILB283-end -BRCA2MBD complexes containing either wild type, D2294R or D2317R BRCA2MBD subunits. (BD) Coomassie-stained SDS-PAGE analysis of fractions for the size-exclusion analysis shown in panel A. (E) SEC-MALS of purified human MEILB283-end-BRCA2MBD D2294R rules out complete dissociation of MEILB283-end -BRCA2MBD or dissociation into smaller (that is, 2:1) complexes; dRI: differential Refractive Index. MEILB283-end-BRCA2MBD D2317R could not be analyzed by SEC-MALS because of closely eluting BRCA2MBD-complexed and free MEILB2 peaks (seen in panel A). WT and mutant complexes were expressed and purified in the same experiment. WT complex has been purified for more than three times while the two mutant complexes were purified once.

Source data

Extended Data Fig. 8 BRCA2MBD and MEILB2 interaction is indispensable for their recombination nodule localization.

(A) Immunoblot of testis extracts after electroporating GFP-BRCA2MBD wild type (WT) or indicated mutants, probed with the indicated antibodies. Asterisks indicate non-specific bands. GFP and actin blots in each lane correspond to the same processed sample run on the same gel but blotted sequentially after stripping. (B) Immunostaining of WT and Meilb2−⁄− mouse spermatocytes expressing GFP-MEILB2 R204D mutant protein. Note that axis-associated foci seen in WT were not detectable in Meilb2−⁄− spermatocytes even after the intensification of the signals. Scale bars: 5 μm in the main panel and 1 μm in the magnified panel. Data are representative of two repeats.

Source data

Extended Data Fig. 9 Protein expression and localization of the MEILB2 dimerization mutants.

(A) and (D) Immunostaining of WT spermatocytes expressing indicated GFP-MEILB2 WT and mutant proteins. (B) and (E) Quantification of GFP foci intensities represented in panel A and panel D, respectively, normalized with the average value of WT. n shows the analyzed foci number pooled from four cells from one electroporated mouse. All analyses used two-tailed t-tests. ****p < 0.0001 (WT vs. Δ1–87: 1.81285 ×10−14). Scale bars: 5 μm. (C) and (F) Immunoblots of testis extracts after electroporating indicated GFP-MEILB2 WT and mutant proteins, blotted with the indicated antibodies (repeated two times). GFP and actin blots in each lane correspond to the same processed sample run on the same gel but blotted sequentially after stripping.

Source data

Supplementary information

Source data

Source Data Fig. 1

Unprocessed gel.

Source Data Fig. 3

Unprocessed western blots.

Source Data Fig. 4

Spreadsheet for quantitation.

Source Data Extended Data Fig. 2

Unprocessed gels and western blots.

Source Data Extended Data Fig. 6

Unprocessed western blots.

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Unprocessed gels.

Source Data Extended Data Fig. 8

Unprocessed western blots.

Source Data Extended Data Fig. 9

Unprocessed western blots.

Source Data Extended Data Fig. 9

Spreadsheet for quantitation.

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Pendlebury, D.F., Zhang, J., Agrawal, R. et al. Structure of a meiosis-specific complex central to BRCA2 localization at recombination sites. Nat Struct Mol Biol 28, 671–680 (2021). https://doi.org/10.1038/s41594-021-00635-0

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