Transition of DNA double-strand breaks to genetic crossovers

Since more than 10 000 years humans are growing crop plants, selecting for local adaption, high yields and further traits to deliver food security. These efforts are still ongoing, now framed by well controlled plant breeding programs. Though, it has long been recognized that not all beneficial traits of a certain crop species, present in various sub-species, can be combined in the same individual plant. Only recently, the molecular base for this impediment starts to emerge, relating to specific processes during meiosis. Meiosis is a specialised cell division that ensures the reduction of the genome prior to the formation of generative cells. During meiosis, novel combinations between parts of paternal and maternal chromosomes are generated through the process of homologous recombination (HR). A pre-requisite for HR are DNA double strand breaks (DSBs). Previously, a lot of effort has been dedicated towards understanding the determinants and the mechanisms of meiotic DSB formation and members of the given MEIOREC consortium contributed significantly. While meiotic DSBs are essential for subsequent exchange of parental genetic information, less than 10% of meiotic DSBs actually constitute such an exchange point. It is not understood, how the fate of a DSB at a certain genomic locus is determined and hence what limits the maturation of a meiotic DSB into a genetic exchange point, a cross-over (CO). The MEIOREC researchers have harnessed a research program to remedy this shortcoming of understanding and will experimentally address various aspect of the DSB-to-CO transition. In the frame of the proposed project, the group of Karl Mechtler based at the Research Institute of Molecular Pathology in Vienna will analyse certain characteristics of protein complexes by mass spectrometry (MS), which play crucial roles during the meiotic cell cycle in relation to recombination. MS techniques will be used to map sites of protein-protein interactions within protein complexes, which will be provided by the consortium partners. New reagents and MS methods will be tested and evaluated to improve established protocols. In addition to MS, the group of Karl Mechtler will provide the expertise and the technology (peptide array) for very detailed studies of protein interacting domains in vitro. Additionally, MS will be applied to study dynamic changes in the modifications of meiotic proteins involved in chromosome axis formation during different phases of the meiotic cell cycle. Here, the kind of modification will be identified, localized on the protein and the changes in their occurrence will be relatively quantified. These analyses will allow to pinpoint modifications, that potentially have an influence on the programmed remodeling of the chromosome axis during meiosis.

Meiosis is a specialized type of cell division required for sexual reproduction. It ensures the reduction of the genome and the recombination of maternal and paternal chromosomal segments prior to the formation of generative cells. The process of meiotic recombination is initiated by programmed DNA double-strand breaks (DSBs), introduced by the conserved Spo11 protein. In a single meiotic cell only a small subset of DSBs is destined to form genetic crossovers (COs), which leads to mutual genetic exchange, while the remainder are repaired via non-CO pathways. In plants more than 90% of the DSBs are repaired as non-COs and the distribution of COs, notably in cereal crops is skewed towards specific chromosomal regions. This limits the genetic variation that is generated in each meiotic division. How the transition of a DSB to yield a CO is controlled at a particular genomic locus and how this can be optimized is not fully understood. Deciphering the factors that control DSB formation and processing to form COs is of fundamental scientific interest, moreover this knowledge will have important implications for manipulating meiotic recombination in crop plants. This collaborative project (Meiorec) focused on the identification of factors controlling DSB formation, analysis of the transitional steps of DSB processing, CO resolution, and the evaluation of strategies to manipulate CO formation using model species. In the frame of the joint project, the group of Karl Mechtler based at the Research Institute of Molecular Pathology in Vienna contributed to different objectives of the collaborative project by performing mass spectrometric experiments. MS was applied to identify protein-interaction partners and study modifications of meiotic protein complexes. Furthermore, the method of protein cross-linking coupled to MS (XL-MS) was further developed for investigation of protein-protein interactions and dynamic changes in the conformation of protein complexes of factors involved in meiotic recombination. Finally, a substantial effort was made to improve MS sample preparation and analysis methods for ultra-low input samples, which can be applied to analyze meiotic cells, that are available at limited amounts.