Mechanisms and regulation of meiotic recombination

Recombination is the only error free way to repair DNA double strand breaks (DSBs) and is therefore crucial to maintain genome integrity. Further, in diploid organisms, recombination is utilized to ensure chromosome segregation during meiosis, the specialized cell division program that generates haploid gametes. Thus, perturbations in the process of recombination can lead to cancer, infertility and birth defects. During meiotic recombination, DSBs are deliberately introduced and channeled to be repaired using the homologous chromosome as a repair template to yield either crossover (CO) or non-crossover (NCO) recombination products. Both CO and NCO repair outcomes are crucial for successful reproduction, as COs form the basis of physical connections between homologs that enable proper meiotic chromosome segregation in anaphase I, whereas repair of the remaining DSBs via NCO recombination is required to restore genome integrity prior to cell division. Using C. elegans as a model, this proposal will address how distinct recombination pathways are utilized and regulated to achieve a successful outcome of meiosis. I will employ a combination of genetic, genomic, biochemical and cytological approaches to investigate the mechanisms that promote both CO and NCO repair of DSBs during meiosis and to reveal how these distinct repair processes are regulated to ensure restoration of genome integrity and faithful segregation of chromosomes. First, I will investigate mechanisms of inter homologous repair (IHR) and CO/NCO differentiation by analysis of DNA associated with recombination sites. My approach will include both: A) Assessment of repair outcomes for DSBs introduced in a controlled manner at defined genomics sites, and B) Sequencing-based genome wide detection of meiotic DSB repair signatures. I will identify the patterns of gene conversion tracts associated with different IHR repair outcomes, assess the contributions of different components of the meiotic recombination machinery, and determine whether the relative position of a repair event along the chromosome biases the outcome. Second, I will examine the role of the COSA-1 protein, which localizes specifically at CO- designated recombination sites, in repair pathway choice and execution. I will target COSA-1 to sites of artificially introduced DSBs to test whether local enrichment of COSA-1 is sufficient to alter repair pathway decisions. I will use IP/mas spec to identify COSA-1 binding partners and post- transcriptional modifications of CO factors to gain new insights into the regulation and mechanism of CO establishment. Finally, I will sequence COSA-1-associated DNA to generate a map of CO sites and to test for specific sequence features favoring CO formation. Overall, this work will elucidate how meiotic chromosomes achieve the critical balance of CO and NCO outcomes of DSB repair needed to enable proper chromosome segregation and maintenance of genome integrity.

In order to prevent genomic duplication at each generation, during meiosis, the last cell division before chromosomes are packaged into sperms and oocytes, the genomic contend is halved byone round of chromosome duplication, followed by two rounds of chromosome segregation. The first meiotic division separates the homologous chromosomes, the second sister chromatids. To be reliably partitioned from each other, homologous chromosomes need to pair and transiently connect. This vital connection between homologous chromosomes is provided by a cross-over (CO), a physical exchange of chromosome arms between the two homologous chromosomes. CO arise from repair of a DNA break on one chromosome, using the intact homologous partner as a repair template. To ensure that every pair of homologous chromosomes receives at least one CO,many breaks are deliberately introduced during meiosis. Strikingly, most of the time only a single CO is formed per chromosome arm. The excess breaks are repaired by alternative mechanisms, not yielding COs, but restoring genome integrity. Failure in this process are the major driver for miscarriages, de novo mutations and birth defects, such as Down syndrome. How one DNA break is reliably turned into a CO, but at the same time, in the same cell and even on the same chromosome arm, all the other DNA breaks are repaired in a fashion not yielding CO, remains mysterious. Over the course of this project, we developed and employed novel cytological approaches to establish a comprehensive time course of the localization of evolutionary conserved meiotic DNA repair factors at sites of meiotic DNA repair. Furthermore, using essential super-resolution microscopy, we determined the dynamic architecture of these DNA repair machineries. This approach enabled us to infer 1) the nature of the underlying DNA repair intermediates 2) the timing of CO and Non-CO formation and 3) the total number of DNA breaks processed during meiosis. Also, we found that a meiosis specific structure, the Synaptonemal Complex (SC), which decorates paired homologous chromosomes along their interface, generates a compartment that transiently engulfs future COs. In contrast, non-CO repair takes place outside of these compartments. These findings provided novel insight into how distinct repair sites can be treated differently at the same time. Additionally, biochemical analysis demonstrated that Polo kinase 1 and 2 physically interact with the DNA repair machinery. PLK-2 progressively decorates the interface of paired homologous chromosome, starting from DNA repair events, which involve the homologous partner. Using advanced live cell microscopy, we could demonstrate that local Polo kinase activity, exerted between homologous chromosomes, stabilizes/enriches the SC. Consistently, we found Polo and the SC to be required for both, (stable) homologous repair intermediates and CO formation. Thus, we propose that local Polo kinase activity alters the SC in response DNA repair to control the number of COs on a given pair of homologous chromosome.