DNA double strand breaks are repaired through homologous recombination in mitotically dividing cells and germ line cells undergoing meiosis. Intermediates formed through homologous recombination are processed by structure-specific resolvases resulting in crossovers and noncrossovers. There are several known eukaryotic resolvases with a number of functions, but there are many aspects of the complexes yet to be uncovered. Specifically, I aim to elucidate the multiple complexes that function as resolvases and in what contexts they operate. Three main resolvases being studied include GEN1/Yen1, and the complexes MUS81-EME1 and SLX1-SLX4. In Drosophila, we believe MEI-9- ERCC1 in conjunction with MUS312 (SLX4) is the major meiotic resolvase. However, there are residual crossovers present in a mus312 mutant and MUS312 may interact with other proteins in a mitotic context. Therefore, I hypothesize SLX4 assembles multiple complexes and that there are other proteins yet to be discovered forming resolvase complexes. MEI-9 and MUS81 are both nucleases that belong to the ERCC4 nuclease family of proteins, and both MEI-9 and MUS81 have binding partners that contain inactive nuclease domains. It is suspected that MUS81-EME1 work in concert with SLX1-SLX4 to resolve junctions, so due to the similarities between MUS81 and MEI-9, I hypothesize that there is another protein similar to SLX1 working in concert with the MUS312-MEI9-ERCC1 resolvase complex. We have identified a candidate gene, Ankle1 that may act in a complex through interactions with the scaffolding protein MUS312 in concert with MEI-9 – ERCC1. Ankle1 is a GIY-YIG nuclease domain protein, similar to SLX1. In humans, Ankle1 has been shown to possess endonuclease activities both in vitro and in vivo. I am interested in determining if the Drosophila Ankle1 protein has the same endonuclease properties. My goal is to study the in vitro activities of Ankle1 and correlate these data with genetics studies. Components of the enzyme complexes will be determined by direct protein-protein interactions and proteomic studies. Preliminary protein-protein interaction studies using the yeast two-hybrid assay have shown an interaction of Ankle1 with MUS312 and MEI-9. I have used the CRISPR/Cas9 gene editing tool to create a mutant of Ankle1 in Drosophila, and I plan to use this mutant for further genetic characterization. Mitotic and meiotic repair assays will be done with single and multiple mutants in Drosophila melanogaster to deduce functions and epistatic relationships. Through this research, I will elucidate the interactions of Ankle1, while uncovering the roles of resolvases in recombination.