Protein structure-function relationships are critical for understanding molecular mechanisms and the impacts of genetic variation. Mutational scanning approaches can deliver scalable analysis, usually through the study of loss-of-function variants. Rarer dominant negative and gain-of-function variants can be more information rich, as they retain a stable proteoform and can be used to dissect molecular function while retaining biological context. Dominant variant proteoforms can still engage substrates and interact with binding partners. Here, we probe the structure-function relationships of the Mus81 endonuclease by ectopic expression of deep mutational scanning libraries to find amino acid variants that confer dominant sensitivity to genotoxic stress and dominant synthetic lethality. Screening more than 2,200 variants at 100 positions identified 13 amino acids that can be altered to elicit a dominant phenotype. The dominant phenotype of these variants required the presence of the obligate Mus81 binding protein, Mms4. The dominant variants affect amino acids in a contiguous surface on Mus81 and fall into two distinct classes: residues that bind the catalytic magnesium atoms and residues that form the hydrophobic wedge. Most of the variant amino acids were conserved across species and cognate variants expressed in human cell lines resulted in dominant sensitivity to replication stress and synthetic growth defects in cells lacking BLM helicase. The dominant variants in both yeast and human resulted in phenotypes distinct from a knockout. These data demonstrate the utility of dominant genetics using ectopic expression of amino acid site saturation variant libraries to link function to protein structure providing insight into molecular mechanisms.