Structural and functional characterization of the Spo11 core complex

Abstract

Spo11, which makes DNA double-strand breaks (DSBs) essential for meiotic recombination, is poorly understood mechanistically because it has been recalcitrant to biochemical study. Here, we provide a molecular analysis of S. cerevisiae Spo11 purified with partners Rec102, Rec104 and Ski8. Rec102 and Rec104 jointly resemble the B subunit of archaeal Topoisomerase VI, with Rec104 similar to a GHKL domain but without conserved ATPase motifs. Unexpectedly, the Spo11 complex is monomeric (1:1:1:1 stoichiometry), indicating that dimerization may control DSB formation. Reconstitution of DNA binding reveals topoisomeraselike 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.

Structures of core eukaryotic protein complexes

“…2). The predicted Spo11-Ski8 complex structure is supported by crosslinking data and by the disruption of Spo11-Ski8 interactions in vitro and in vivo by mutations in Spo11 in the predicted interface regions (24) (25). Our model resembles a previous model based on the Ski3-Ski8 complex, with Ski8 contacting a sequence in Ski3 that is similar to the sequence QREIF 380 in Spo11 (25)(26) (fig.…”

C. elegansDSB-3 Reveals Conservation and Divergence among Protein Complexes Promoting Meiotic Double-Strand Breaks

“…Consistent with this view, chromatin immunoprecipitation experiments in both S. cerevisiae and S. pombe have shown that these proteins interact with both axis-enriched DNA sequences and with DSB sites (25,(28)(29)(30). Additionally, S. cerevisiae Rec114 and Mei4 have been found to interact with the Rec102 and Rec104 subunits that together comprise the TopVIB-like component of the Spo11 core complex (9,23). Together these findings implicate Rec114-Mei4 in recruiting Spo11 to the meiotic chromosome axis.…”

“…The mechanism of DNA breakage involves formation of a covalent linkage between Spo11 protein DNA, analogous to a key intermediate in the topisomerase reaction (1). Despite identification of structural and mechanistic conservation between Spo11 and TopVIA more than 20 years ago, however, counterparts of the archaeal TopVIB subunit that partner with Spo11 in "Spo11 core complexes" were not recognized until much later, reflecting substantial divergence both from TopVIB and among their eukaryotic orthologs (7)(8)(9). DSB formation also depends on multiple additional factors that play critical roles in determining the location, timing, levels, and regulation of DSB formation (2).…”

Topoisomerase VI is a chirally-selective, preferential DNA decatenase

“…It was later demonstrated to be present throughout the archaeal domain, in some bacteria, and in certain eukaryotes, including plants and algae (10). In addition to the eukaryotic encoding of topo VI, the Top6A subunit is highly homologous to the eukaryotic meiotic factor, Spo11, crucial for the generation of DSBs during recombination (28,29). Top6B structural homologues have also been identified in higher eukaryotes including mouse and A. thaliana, and shown to interact with Spo11 (30,31).…”

“…Top6B structural homologues have also been identified in higher eukaryotes including mouse and A. thaliana, and shown to interact with Spo11 (30,31). Recently, the purification of the Spo11 core complex was achieved, and structural data indicated a high degree of similarity to the topo VI heterotetramer (29).…”