Determining mutation signatures is standard for understanding the etiology of human tumors and informing cancer treatment. Multiple determinants of DNA replication fidelity prevent mutagenesis that leads to carcinogenesis, including regulation of free deoxyribonucleoside triphosphate (dNTP) pools and the repair of base mismatches. We utilized a targeted deep-sequencing approach to determine mutational signatures associated with mismatch repair (MMR) pathway defects. By combining rnr1 and msh mutations to increase dNTP levels and alter the mutational load, we observed previously unreported specificities of Msh2-Msh3 and Msh2-Msh6. Msh2-Msh3 is uniquely able to direct repair of G/C single base deletions in GC runs, while Msh2-Msh6 specifically directs repair of substitutions at G/C dinucleotides. We also identified broader sequence contexts that influence variant profiles in different genetic backgrounds and found that there was not necessarily a simple additive relationship of mutation profiles in double mutants. Our results have implications for interpreting mutation signatures from human tumors, particularly when MMR is defective.