Abstract The DNA mismatch repair (MMR) system is responsible for correcting base substitution and insertion-deletion mutations (IDLs) generated during erroneous replication in bacteria, yeast and mammals. MMR complexes also recognize damaged-base mispairs resulting from environmental exposure to DNA damaging agents. The loss of MMR causes a “mutator” phenotype, resulting in the genome-wide accumulation of mutations, and is the underlying cause for Hereditary Non-polyposis Colorectal Cancer / Lynch Syndrome (HNPCC/LS) and a significant proportion of sporadic colorectal cancer (CRC). MMR-deficient tumors also display resistance to DNA damaging agents and, as a consequence, are resistant to a variety of commonly chemotherapeutic agents. Thus, the development of novel therapeutic strategies that more efficiently target MMR-deficient cancer cells would be highly desirable. A promising new direction to identify potential targets for anticancer treatment is the harnessing of synthetic lethality. Here, we have identified conserved synthetic lethal / sick genetic interactions (GIs) for MMR factors between two distant yeast species, S. pombe and S. cerevisiae. We have confirmed several of these negative GIs in mammalian cells, indicating that they are evolutionarily conserved. Specifically, we have identified a negative genetic relationship between Msh2, a major MMR component mutated in colorectal cancer, and Senp6 protease, which regulates the SUMOylation of a range of genome maintenance proteins. Our findings suggest that Senp6 is a promising new target for the treatment of MMR-deficient CRCs. Citation Format: Elena Tosti, Joseph A. Katakowski, Sonja Schaetzlein, Hyun-Soo Kim, Colm J. Ryan, Michael Shales, Assen Roguev, Nevan J. Krogan, Deborah Palliser, Michael-Christopher Keogh, Winfried Edelmann. Identification of evolutionarily conserved genetic interactions in DNA mismatch repair. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Synthetic Lethal Approaches to Cancer Vulnerabilities; May 17-20, 2013; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(5 Suppl):Abstract nr A13.