AbstractATM and ATR are conserved regulators of the DNA damage response linked to cancer. Comprehensive DNA sequencing efforts identified ~4,000 cancer-associated mutations in ATM/ATR; however, their cancer implications remain largely unknown. To gain insights, we identify functionally important conserved residues in ATM, ATR and budding yeast Mec1ATR via cancer genome datamining and a functional genetic analysis, respectively. Surprisingly, only a small fraction of the critical residues is in the active site of the respective enzyme complexes, implying that loss of the intrinsic kinase activity is infrequent in carcinogenesis. A number of residues are solvent accessible, suggestive of their involvement in interacting with a protein-partner(s). The majority, buried inside the respective enzyme complexes, might play a structural or regulatory role. Together, these findings identify evolutionarily conserved ATM, ATR, and Mec1ATR residues involved in diverse aspects of the enzyme function and provide fresh insights into the elusive genotype-phenotype relationships in ATM/ATR and their cancer-associated variants.