Meiotic crossovers, necessary for gamete viability, are generated by the cleavage of a precursor recombination intermediate: the double Holliday junction (dHJ), by an uncharacterized meiotic “resolvase”. A key question is to identify all actors directly involved in dHJ resolution, a reaction that requires two cleavage reactions each of two DNA strands, and to understand why the cleavage is made only asymmetrically, such that only crossovers are produced. This differs from the mechanism proposed for the canonical resolvases operating to repair recombination intermediates in somatic cells, where the resolution produces both crossover and non-crossover products. A wealth of converging genetic evidence points to the conserved MutLg complex, Mlh1-Mlh3, for being a key actor in the resolution of meiotic dHJ, but this complex shows no resolution activity in vitro, suggesting the need for additional partners. MutLg complex being a mismatch repair factor, another key issue is to determine how it is recruited to dHJ. Our project is to explore the mechanism of crossover resolution and to identify the meiotic resolvase complex. For this, we will use a highly multidisciplinary approach combining yeast genetics, structural biochemistry, biochemistry and mouse genetics. We will study in deep the localization of the MutL? complex in yeast meiotic cells, providing for the first time a high resolution map of meiotic crossover sites. Next, we will combine efforts to decipher the mechanism of dHJ resolution by MutLg. First, we will determine in yeast meiotic cells the protein partners associated with MutL?. Next, we will determine the crystal structure of the whole MutLg together with a dHJ DNA substrate, and use biochemical and biophysical assays with various types of DNA substrates and study the dHJ cleavage reaction in vitro by MutLg. Finally, we will use mouse genetics and genome engineering to test the functional conservation of our findings regarding the role of MutLg partners by building separation of function mutants in mice. Together, this ambitious and comprehensive study of the MutLg complex in meiosis should uncover how the Mutlg complex promotes double Holliday junction resolution in meiosis, which guarantees gamete viability, fertility and diversity of the progeny. A major outcome of this project will be to rationalize meiosis dysfunctions observed in humans and associated with sterility or chromosomal abnormalities such as Trisomy 21.