The double-stranded break-forming activity of plant SPO11s and a novel rice SPO11 revealed by a Drosophila bioassay

To determine whether SPO11 can cleave DNA by itself, and to identify which plant SPO11 homologue cleaves DNA, we developed a Drosophila bioassay system that detects the DSB signals generated by a plant SPO11 homologue expressed ectopically. We cytologically and genetically demonstrated the DSB activities of Arabidopsis AtSPO11-1 and AtSPO11-2, which are required for meiosis, in the absence of other plant proteins. Using this bioassay, we further found that a novel SPO11-homologue, OsSPO11D, which has no counterpart in Arabidopsis, displays prominent DSB-forming activity. Quantitative analyses of the rice SPO11 transcripts revealed the specific increase in OsSPO11D mRNA in the anthers containing meiotic pollen mother cells.The Drosophila bioassay system successfully demonstrated that some plant SPO11 orthologues have intrinsic DSB activities. Furthermore, we identified a novel SPO11 homologue, OsSPO11D, with robust DSB activity and a possible meiotic function.Homologous genetic recombination plays critical roles in meiosis, for genetic diversification and precise disjunction of homologous chromosomes. In the budding yeast Saccharomyces cerevisiae, meiotic recombination is initiated by chromatin locus- and timing-specific DNA double-strand breaks [1-3], which require the function of the SPO11 protein [4,5]. Saccharomyces cerevisiae SPO11 shares amino acid sequence homology with subunit A of the type II DNA topoisomerase (topoisomerase VI) from the archaeon Sulfolobus shibatae [5]. Type II topoisomerases generate double-strand breaks (DSBs) at specified sequences in the cleavage complex, an intermediate for topological DNA reactions, composed of the covalent complex of a topoisomerase subunit and the terminus of each DSB strand. As in the cleavage complex, SPO11 covalently attaches to the 5' termini of a nascent meiotic DSB. SPO11 is then removed from the DNA by the endonuclease activity of Mre11 [6-9], and by the functions of the SAE2/CPT1 [10,11] and RAD50 proteins [