The ATR and ATM kinases are at the apex of the DNA damage response signaling pathway. These large protein kinases are members of an atypical protein kinase family that includes DNA protein kinase (DNA-PK) and target of rapamycin (TOR). Mutations in ATR cause the rare disease Seckel syndrome and mutations in ATM cause the childhood neurodegenerative and cancer-predisposition disorder ataxia-telangiectasia. We have an ongoing project to understand the regulation of these kinases in response to DNA damage. ATM is primarily activated in response to double strand breaks while ATR is activated by agents that cause replication stress such as ultraviolet radiation and the chemotherapeutic agents cisplatinum, hydroxyurea, and etoposide. Activation of these kinases involves at least two steps. The kinases are recruited to the sites of DNA damage and then biochemical changes in the proteins allow them to phosphorylate substrates. Key substrates include p53, Nbs1, Mre11, Chk2, and Chk1. Many of these substrates are themselves encoded by tumor suppressor genes. p53, for example, may be the most widely mutated genes in human cancer. We are using biochemical, genetic, and cell biological techniques to determine how these kinases are initially activated in response to genotoxic stress.