Cells respond to genotoxic stress by activating signaling pathways that regulate the cell cycle, DNA repair, and apoptosis. The ataxia-telangiectasia mutated (ATM) and ATM and Rad3-related (ATR) protein kinases are at the apex of these signaling pathways. These proteins phosphorylate the checkpoint kinases Chk1 and Chk2 and regulate numerous tumor suppressor proteins including p53 and the breast and ovarian cancer susceptibility protein BRCA1. Defects in these pathways lead to genomic instability and cause several cancer syndromes. Studies in our laboratory aim to identify components of these DNA damage response pathways and to understand how they work in a coordinated manner to prevent cancer by regulating the cell cycle, promoting DNA repair or initiating apoptosis. We use a variety of biochemical and genetic techniques to achieve these goals. For example, we recently created a somatic cell knockout of the ATR gene and used a proteomic approach to identify a subunit of the ATR kinase. We are currently searching for novel ATM/ATR substrates that are involved in regulating DNA replication. ATM/ATR-dependent regulation of DNA replication is essential to preserve genomic integrity. DNA replication is also a target for many cancer drugs, and the effects of checkpoint signaling may modify their efficacy. Therefore, these studies will significantly improve our understanding of cancer and facilitate the search for better cancer therapies.