Investigation of SPO11 protein complexes which are essential for meiotic double strand break induction in plants

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The meiotic recombination process depends in nearly all eukaryotes on the initiation of double strand breaks (DSBs) by SPO11, a meiosis specific transesterase. In animals and fungi a single SPO11 gene is present and sufficient whereas in plants and other eukaryotes at least two of them, SPO11-1 and 2 are essential for the initiation of meiotic DSBs. Both SPO11 proteins are quite different and necessary to be present in a catalytic active form. Our new results indicate that they build up a heterodimeric SPO11-complex in Arabidopsis thaliana but also other so far unknown proteins might be involved. Furthermore, we identified different regions of both SPO11 proteins from Arabidopsis which are specific and essential for the respective SPO11 protein. We now want to mutate several conserved single amino acids (three of approx. 20 aa) in a small region of the protein (swap no. 2) which exhibited a strong dominant negative effect causing near sterility in the case in which we exchanged this region between AthSPO11-1 and -2. In addition to these experiments we want to replace the same region by the corresponding heterologous sequences from Carica papaya and other plants to elucidate whether it is functionally conserved in evolution. Using the new antibodies which we produced recently, we now can investigate for the first time which other proteins bind to or interact with SPO11-2 during meiotic DSB induction by coimmunoprecipitation and fluorescent staining of meiotic figures. We want to isolate and sequence the interacting proteins by pull down assays followed up by MALDI TOF/MS and native 2D PAGE analysis. Considering the specificity and high binding affinity of the SPO11-2 antibody we also will try to isolate the SPO11/DNA complexes directly and analyze it by next generation sequencing.