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

在几乎所有的真核生物中，减数分裂重组过程都依赖于一种减数分裂特异性转移酶SPO11启动双链断裂（DSBs）。在动物和真菌中，一个SPO11基因是足够的，而在植物和其他真核生物中，至少有两个SPO11-1和SPO11- 2基因对于减数分裂dsb的起始是必不可少的。这两种SPO11蛋白完全不同，必须以催化活性形式存在。我们的新结果表明，他们在拟南芥中建立了一个异二聚体spo11复合物，但其他迄今未知的蛋白质可能也参与其中。此外，我们从拟南芥中鉴定出SPO11蛋白的不同区域，这些区域对各自的SPO11蛋白是特异性和必需的。我们现在想要突变几个保守的单个氨基酸（大约三分之二）。20aa)在蛋白质的一个小区域（交换号20aa）。2)当我们在AthSPO11-1和-2之间交换该区域时，表现出强烈的显性负作用，导致接近不育。除了这些实验外，我们还想用来自番木瓜和其他植物的相应异源序列替换相同的区域，以阐明它在进化中是否具有功能保守性。利用我们最近制备的新抗体，我们现在可以通过共免疫沉淀和减数分裂图的荧光染色首次研究在减数分裂DSB诱导过程中其他蛋白质与SPO11-2结合或相互作用。我们希望通过拉下实验分离和测序相互作用蛋白，随后使用MALDI TOF/MS和原生2D PAGE分析。考虑到SPO11-2抗体的特异性和高结合亲和力，我们也将尝试直接分离SPO11/DNA复合物，并通过下一代测序进行分析。

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.