Cryo-EM structure of the Spo11 core complex bound to DNA

The DNA double-strand breaks that initiate meiotic recombination are formed by topoisomerase relative Spo11, supported by conserved auxiliary factors. Because high-resolution structural data are lacking, many questions remain about the architecture of Spo11 and its partners and how they engage with DNA. We report cryo-EM structures at up to 3.3 Å resolution of DNA-bound core complexes of Saccharomyces cerevisiae Spo11 with Rec102, Rec104, and Ski8. In these structures, monomeric core complexes make extensive contacts with the DNA backbone and with the recessed 3’-OH and first 5’ overhanging nucleotide, definitively establishing the molecular determinants of DNA end-binding specificity and providing insight into DNA cleavage preferences in vivo. The structures of individual subunits and their interfaces, supported by functional data in yeast, provide insight into the role of metal ions in DNA binding and uncover unexpected structural variation in homologs of the Top6BL component of the core complex.


SUPPLEMENTAL FIGURES
(A) SDS-PAGE gel of purified core complex (~1 µg), stained with Coomassie.(B,C) EMSA assays and quantification of core complex binding to the hairpin DNA substrate (B) and the gapped DNA substrate (C).In panel C, the position of the second core complex that gives the slowest migrating band is unknown; it could be at the nick location as shown or it could associate with one of the hairpin ends.Error bars indicate mean ± SD of three replicates.(A-D) The secondary structures for Spo11 (panel A), Ski8 (panel B), Rec102 (panel C), and Rec104 (panel D) were determined from the cryo-EM structure of the core complex bound to gapped DNA.Gray-colored regions are not visible in the cryo-EM structure.(A) Multiple sequence alignment of Spo11 and Top6A orthologs at protein-protein and protein-DNA contact sites.(B) Structural alignment of the hypothetical model of a Spo11 core complex pre-DSB dimer and the crystal structure of the dimeric S. shibatae Topo VI holoenzyme (yellow).One copy of the Spo11 core complex is colored as Fig. 1B, and the other copy is colored in gray.Ski8 is not shown for simplicity.

Fig. S2 .
Fig. S2.Cryo-EM reconstruction of the core complex bound to hairpin DNA.(A) Flow chart of cryo-EM image processing.(B) Global Fourier shell correlation (FSC) curves.The overall cryo-EM map resolution is 3.7 Å with FSC set at 0.143.(C, D) Euler angle distribution (panel C) and final 3D reconstructed map colored according to local resolution (panel D). (E) Density map.

Fig. S3 .
Fig. S3.Cryo-EM reconstruction of the core complex bound to gapped DNA.(A) Flow chart of cryo-EM image processing.(B) Global FSC curves.The overall cryo-EM map resolution is 3.3 Å with FSC set at 0.143.(C, D) Euler angle distribution (panel C) and final 3D reconstructed map colored according to local resolution (panel D).

Fig. S4 .
Fig. S4.Examples of protein side chain and DNA base-sugar-phosphate identification in the cryo-EM structure of the core complex bound to gapped DNA.(A-E) The expanded boxes show examples of fitting of protein amino acid side chain (panels A-C) and DNA base-sugar-phosphate and interacting protein side chains (panels D and E) into the density map.

Fig. S6 .
Fig. S6.Interactions between Ski8 and Spo11.All images are from the cryo-EM structure of the core complex bound to gapped DNA.(A) QREIFF motif from Spo11 (magenta) and the WD40 repeats from Ski8 (cyan).(B) Hydrophobic interactions between Ski8 WD40 repeats and Spo11.(C) Hydrogen bond interactions between Ski8 and Spo11.(D) Additional hydrogen bonding interactions between Spo11 and the extended loop in Ski8.

(
A) Top6B and Rec102/Top6BL architectures.Strands, helices, and switch loops are colored as in Fig. 3B and strands are labeled as in Fig. 3A.All structures show truncated proteins, starting at the first β strand (strand A) and ending with the switch loop before the start of the stalk.The switch loop is truncated for the N. crassa and S. macrospora proteins for clarity.(B) Secondary structure and sequence information for models shown in (A).The conserved WKxY motif is highlighted in red and β strands are labeled as in (A).(C) Packing for S. shibatae Top6B (PDB: 2zbk) in comparison to Rec102 for the W in the WKxY motif.Ribbons and sticks are colored as in Fig. 3B.(D) Detail view of the hydrophobic interface between the amphipathic stalk and the β strands; related residues in Top6B and Rec102 are indicated.