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Structural and functional characterization of the Spo11 core complex

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Abstract

Spo11, which makes DNA double-strand breaks (DSBs) that are essential for meiotic recombination, has long been recalcitrant to biochemical study. We provide molecular analysis of Saccharomyces cerevisiae Spo11 purified with partners Rec102, Rec104 and Ski8. Rec102 and Rec104 jointly resemble the B subunit of archaeal topoisomerase VI, with Rec104 occupying a position similar to the Top6B GHKL-type ATPase domain. Unexpectedly, the Spo11 complex is monomeric (1:1:1:1 stoichiometry), consistent with dimerization controlling DSB formation. Reconstitution of DNA binding reveals topoisomerase-like preferences for duplex–duplex junctions and bent DNA. Spo11 also binds noncovalently but with high affinity to DNA ends mimicking cleavage products, suggesting a mechanism to cap DSB ends. Mutations that reduce DNA binding in vitro attenuate DSB formation, alter DSB processing and reshape the DSB landscape in vivo. Our data reveal structural and functional similarities between the Spo11 core complex and Topo VI, but also highlight differences reflecting their distinct biological roles.

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Fig. 1: The meiotic DNA DSB core complex.
Fig. 2: Protein–protein interactions within the core complex.
Fig. 3: DNA binding by the core complex analyzed by AFM.
Fig. 4: DNA-binding properties of the core complex.
Fig. 5: Mapping DNA-binding surfaces by hydroxyl radical footprinting.
Fig. 6: Mutations that affect Spo11–DNA binding compromise DSB formation.
Fig. 7: Conformational changes upon DNA binding.
Fig. 8: A mutation that affects Spo11–DNA interaction leads to a redistribution of DSBs.

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Data availability

Sequence reads and compiled Spo11–oligo and S1–seq maps were deposited at the Gene Expression Omnibus (accession number GSE150315). Source data are provided with this paper.

Code availability

Code for processing Spo11–oligo reads48,49 is available at http://cbio.mskcc.org/public/Thacker_ZMM_feedback/. Code for processing S1–seq reads47 is available at https://github.com/soccin/S1-seq.

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Acknowledgements

We thank M. Brendel and the Molecular Cytology core facility at MSKCC for performing the AFM experiments. We thank R. Hendrickson, E. Chang and the Microchemistry and Proteomics core facility at MSKCC for assistance with the XL–MS experiments. We thank K. Liu (S.K. laboratory) for discussions. MSKCC core facilities are supported by National Cancer Institute (NCI) Cancer Center support grant no. P30 CA08748. We thank E. Folta-Stogniew and the Biophysics Resource of Keck Facility at Yale University for the SEC–MALS experiments. The SEC–LS/UV/RI instrumentation was supported by NIH Award Number 1S10RR023748-01. E.P.M. was supported in part by a Helen Hay Whitney Foundation fellowship. Work in the S.K. laboratory was supported principally by the Howard Hughes Medical Institute and in part by NIH grant no. R35 GM118092 (S.K.). Work in the J.M.B. laboratory was funded by NCI grant no. R01-CA0777373 (J.M.B.). C.C.B. was supported in part by funding from the European Research Council under the European Union’s Horizon 2020 research and innovation program (European Research Council grant agreement no. 802525) and from the Fonds National de la Recherche Scientifique (FNRS MIS-Ulysse grant no. F.6002.20) (C.C.B.).

Author information

Author notes

  1. Sam E. Tischfield

    Present address: Human Oncology and Pathology Program, Memorial Sloan Kettering Cancer Center (MSKCC), New York, NY, USA

  2. Eleni P. Mimitou

    Present address: New York Genome Center, New York, NY, USA

  3. Ernesto Arias-Palomo

    Present address: Department of Structural & Chemical Biology, CIB Margarita Salas (CSIC), Madrid, Spain

Authors and Affiliations

  1. Molecular Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY, USA

    Corentin Claeys Bouuaert, Sam E. Tischfield, Stephen Pu, Eleni P. Mimitou & Scott Keeney

  2. Howard Hughes Medical Institute, Memorial Sloan Kettering Cancer Center, New York, NY, USA

    Corentin Claeys Bouuaert, Stephen Pu & Scott Keeney

  3. Louvain Institute of Biomolecular Science and Technology, Université Catholique de Louvain, Louvain-La-Neuve, Belgium

    Corentin Claeys Bouuaert

  4. Tri-Institutional Training Program in Computational Biology and Medicine, Cornell University, New York, NY, USA

    Sam E. Tischfield

  5. Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD, USA

    Ernesto Arias-Palomo & James M. Berger

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Contributions

C.C.B. and S.K. designed the study and supervised the research. C.C.B. carried out all experiments except those noted below. S.P. generated expression constructs, prepared viruses and purified proteins under the supervision of C.C.B. and S.K. E.P.M. performed S1–seq. S.E.T. performed Spo11–oligo mapping and other phenotypic studies of the F260A mutant and carried out bioinformatics analyses. E.A.-P. performed nsEM experiments with input from J.M.B. J.M.B. generated structural models. C.C.B. and S.K. wrote the paper with input from the other authors. C.C.B., J.M.B. and S.K. secured funding.

Corresponding authors

Correspondence to Corentin Claeys Bouuaert or Scott Keeney.

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Extended data

Extended Data Fig. 1 Co-expression of core complex subunits, size exclusion chromatography and glycerol gradient sedimentation analyses of the core complex.

a, Silver-stained SDS-PAGE gels (top) and anti-Flag western blot (WB; center) of Spo11 complexes after purification on nickel resin. Absence of Rec102, Rec104, or Ski8 leads to poor solubility of Spo11. Bottom: anti-Flag western blot of lysed Sf9 cells showing Spo11 expression levels. Asterisks: C-terminal truncation of Spo11 that retains the affinity tag and interaction with Ski8. b, Size exclusion chromatography of purified core complex with and without MBP tag on Rec102. Silver-stained SDS-PAGE gels of eluted fractions are shown above, with chromatograms from absorption at 280 nm below. c, Glycerol gradient sedimentation of MBP-tagged Spo11 core complexes. The silver-stained SDS-PAGE gel shows fractions collected from the bottom of the gradient. Quantification of protein signal from two independent experiments is shown together with molecular weight markers run on a separate gradient and quantified by Bradford assay. Note: Material in the void volume (panel b) and at the bottom of the glycerol gradient (panel c) lacks Ski8, which is consistent with Ski8 being required for solubility.

Extended Data Fig. 2 2D class averages of nsEM images with different versions of the core complex.

Core complexes without MBP or with MBP fused at the N-terminus of Rec102, Spo11 or Rec104 are shown. A cartoon of the presumed arrangement of the subunits and the position of the MBP signal is shown. With the MBP-tagged Spo11 construct, the signal from MBP is located at a similar position to the Rec102- or Rec104-tagged constructs. This is consistent with the observation that the N-terminus of Spo11 frequently crosslinks with Rec104 (pink lines in Fig. 2a), suggesting that the N-terminus of Spo11, absent from the structural model, is flexible and perhaps directly contacts Rec102/Rec104. The observation of a single MBP signal for all three subunits tested provides further support for the 1:1:1:1 stoichiometry of the core complex. Complexes with MBP-tagged Ski8 were not well behaved and could not be purified.

Extended Data Fig. 3 The interaction between Ski8 and Spo11 is important for the integrity of the complex.

SDS-PAGE analysis of core complexes purified with wild-type Spo11 or the Ski8-interaction deficient Q376A mutant. Equivalent percentages of the total protein purified from similar amounts of Sf9 extract were loaded in each lane, demonstrating the poor yield when the Spo11–Ski8 interaction is compromised.

Extended Data Fig. 4 Intramolecular crosslinks within Ski8 validate the XL-MS results.

a, Ski8 intramolecular crosslinks modeled on the structure of Ski8. The histogram shows the frequency of XL-MS events as a function of distance between the α-carbons (Cα) of the crosslinked lysines (red spheres). The crosslinkable limit of DSS is 27.4 Å. b, Ski8 intramolecular crosslinks modeled on the core complex show that the crosslinked residues are away from the interaction surface with Spo11.

Extended Data Fig. 5 DNA-binding properties of the core complex.

a, Competition experiment using a labeled 25-bp hairpin substrate with 5′-TA overhang in the presence of unlabeled substrates with various overhang configurations. EMSA gel bands of bound labeled substrate are shown. Mean and ranges from two experiments are plotted. The substrate with a 2-nucleotide 5′ overhang is the most effective competitor. b, Competition experiment using a labeled 25-bp hairpin substrate with 5′-TA overhang in the presence of unlabeled competitor substrates with or without 5’ phosphate. Error bars represent ranges from two experiments. c, EMSA of core complex binding to 400-bp mini-circles in the presence or absence of Mg2+. For the top panel, binding reactions contained 5 mM Mg2+ and the gel and electrophoresis buffer contained 0.5 mM Mg2+. For the bottom panel, the binding reactions, gel, and buffer contained 1 mM EDTA.

Extended Data Fig. 6 Affinity purification of different combinations of tagged complexes and comparison of DNA-binding activities.

a, Purification of core complexes that carry combinations of HisFlag (H) and MBP (M) tags on different subunits. All combinations yielded soluble Spo11 (western blot, bottom panel). While the Coomassie-stained gel shown in Fig. 1a suggests that Rec104 may be sub-stoichiometric, the similar relative intensities between MBP-tagged Rec102 and Rec104 in the silver-stained gel (where the MBP tag makes up the majority of each tagged protein’s mass) and anti-MBP western blot indicate that the two subunits have the same stoichiometry (compare lanes 1 with 2, and 6 with 7). The difficulty in purifying soluble Spo11-containing complexes when Rec104 is absent (Extended Data Fig. 1a) further bolsters the inference that the purified core complexes (nearly) always include Rec104. b, Comparison of the DNA-binding activity of core complexes that carried affinity tags on different subunits. All tagged complexes assayed had similar DNA-binding activities.

Extended Data Fig. 7 In vivo analyses of Spo11 DNA-binding mutants.

a, Southern blot analysis of meiotic DSB formation at the CCT6 hotspot in strains expressing wild-type (WT) Spo11 or the K173A or R344A mutant proteins. b, Quantification of DSB formation at the CCT6 hotspot. Error bars represent the range from two experiments. c, Meiotic progression. MI + MII indicates the fraction of cells that have undergone the first or both meiotic divisions, as scored by DAPI staining.

Extended Data Fig. 8 Genome-wide analyses of DSB formation in the F260A mutant.

a, Relative enrichment of the short Spo11-oligo class in F260A. Deproteinized, labeled oligos were separated by denaturing gel electrophoresis. Lane profiles are shown on the right. A 10-nt ladder is plotted in grey. b, c. Reproducibility of DSB maps. Correlations of Spo11-oligo counts (b) and S1-seq counts (c) within hotspots between two biological replicates of Spo11 wild type and F260A are plotted. Pearson’s r between datasets is indicated. d, Changed DSB distribution in F260A is not correlated with hotspot strength. Spo11 hotspots were binned according to oligo counts in wild type. Boxplots show the distribution of Pearson’s r values comparing within-hotspot Spo11-oligo distributions between wild type and F260A, as in Fig. 8e. The thick horizontal bars are medians, box edges are upper and lower quartiles, whiskers indicate values within 1.5 fold of interquartile range, and points are outliers. e, Base composition in S1-seq maps. The big spike in the G map at +2 is partially because this is the complement of the preferred C 5′ of the scissile phosphate, but it is also the first base of the ligation junction (and end-most base after S1 digestion), so the degree to which there is enrichment to the right but not left of the dyad axis probably reflects a modest end-bias in library prep in S1-seq. f, Spo11 preference at the scissile phosphate (dinucleotide indicated by the red circle).

Extended Data Fig. 9 Possible relation of Rec104 to a GHKL fold.

a, Secondary structure predictions for Rec104 and the GHKL domain of Topo VIB were generated by PsiPred63. b, Rec104 model generated by iTasser (green) overlaid on Topo VI. The transducer domain of Topo VI is yellow, the GHKL domain is grey.

Extended Data Fig. 10 Model of Spo11-induced break formation.

a, AFM experiments suggest a model where the core complex binds a DNA duplex, bends it, then traps a second duplex. Perhaps DNA cleavage happens in the context of a trapped DNA junction, similar to Topo VI. After cleavage, Spo11 remains covalently attached to the DNA end through covalent and non-covalent interactions. b, Model of assembly of the DSB machinery. DNA-driven condensation by Rec114–Mei4–Mer2 is proposed to provide a platform that recruits the core complex, where it engages its DNA substrate58. A hypothetical arrangement is shown where each dimer of core complexes captures a pair of DNA duplexes, which, for example, could be sister chromatids. c, Depletion of long oligos in Spo11 mutants with reduced DNA-binding activity is consistent with a model57 where long oligos arise from occlusion of the DNA substrate by multiple Spo11 complexes that reduce access to MRX/Sae2.

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Crosslinking mass spec (XL–MS) data.

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Claeys Bouuaert, C., Tischfield, S.E., Pu, S. et al. Structural and functional characterization of the Spo11 core complex. Nat Struct Mol Biol 28, 92–102 (2021). https://doi.org/10.1038/s41594-020-00534-w

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