The DNA damage response (DDR) is a critical process in eukaryotic cells that maintains genomic stability by coordinating DNA repair, cell-cycle control, and other processes. The DDR is primarily regulated by the ATM and ATR kinases, which are activated by DNA damage and replication stress. While both kinases are activated by DNA damage, they have distinct specificities and functions. ATM is mainly activated by double-stranded DNA breaks (DSBs), whereas ATR responds to a broader range of DNA damage, including DSBs and replication stress. Together, ATM and ATR work to sense and respond to DNA damage, activating downstream signaling pathways that repair DNA and regulate the cell cycle. ATM and ATR are part of a signaling pathway that includes sensors, transducers, and effectors. The sensors recognize DNA damage structures, while the transducers (ATM, ATR, and their downstream kinases) amplify the signal. Effectors then carry out the cellular responses, such as DNA repair and cell-cycle arrest. The structural organization of ATM and ATR includes multiple domains, including HEAT repeats, FAT, and FATC domains, which are involved in their activation and regulation. ATM is activated by DSBs through the MRN complex, which recognizes DNA ends and activates ATM. This leads to the phosphorylation of substrates such as Brca1, Chk2, and p53, which mediate DNA repair, cell-cycle arrest, and apoptosis. ATR is activated by single-stranded DNA (ssDNA) structures, often generated during DNA replication stress. ATR-ATRIP recognizes ssDNA coated by RPA, leading to ATR activation and downstream signaling. ATM and ATR also interact with other proteins, such as TopBP1, to regulate DNA repair and cell-cycle checkpoints. The activation of ATR is a multi-step process involving the recognition of ssDNA and ssDNA/dsDNA junctions, which ensures that ATR is only activated in the presence of specific DNA damage. This "fail-safe" mechanism ensures that ATR signaling is accurate and efficient. ATM and ATR have distinct but overlapping functions in the DDR. They can cross-talk at multiple levels, including through the phosphorylation of each other and the regulation of their respective substrates. Despite their functional overlap, genetic evidence suggests that ATM and ATR have non-redundant roles in the DDR. Understanding the mechanisms of ATM and ATR activation and their interactions is crucial for developing targeted therapies for cancer, as these kinases are essential for the survival of cells under genomic stress. Recent advances in the development of specific inhibitors for ATM, ATR, and related kinases offer promising avenues for cancer treatment.The DNA damage response (DDR) is a critical process in eukaryotic cells that maintains genomic stability by coordinating DNA repair, cell-cycle control, and other processes. The DDR is primarily regulated by the ATM and ATR kinases, which are activated by DNA damage and replication stress. While both kinases are activated by DNA damage, they have distinct specificities and functions. ATM is mainly activated by double-stranded DNA breaks (DSBs), whereas ATR responds to a broader range of DNA damage, including DSBs and replication stress. Together, ATM and ATR work to sense and respond to DNA damage, activating downstream signaling pathways that repair DNA and regulate the cell cycle. ATM and ATR are part of a signaling pathway that includes sensors, transducers, and effectors. The sensors recognize DNA damage structures, while the transducers (ATM, ATR, and their downstream kinases) amplify the signal. Effectors then carry out the cellular responses, such as DNA repair and cell-cycle arrest. The structural organization of ATM and ATR includes multiple domains, including HEAT repeats, FAT, and FATC domains, which are involved in their activation and regulation. ATM is activated by DSBs through the MRN complex, which recognizes DNA ends and activates ATM. This leads to the phosphorylation of substrates such as Brca1, Chk2, and p53, which mediate DNA repair, cell-cycle arrest, and apoptosis. ATR is activated by single-stranded DNA (ssDNA) structures, often generated during DNA replication stress. ATR-ATRIP recognizes ssDNA coated by RPA, leading to ATR activation and downstream signaling. ATM and ATR also interact with other proteins, such as TopBP1, to regulate DNA repair and cell-cycle checkpoints. The activation of ATR is a multi-step process involving the recognition of ssDNA and ssDNA/dsDNA junctions, which ensures that ATR is only activated in the presence of specific DNA damage. This "fail-safe" mechanism ensures that ATR signaling is accurate and efficient. ATM and ATR have distinct but overlapping functions in the DDR. They can cross-talk at multiple levels, including through the phosphorylation of each other and the regulation of their respective substrates. Despite their functional overlap, genetic evidence suggests that ATM and ATR have non-redundant roles in the DDR. Understanding the mechanisms of ATM and ATR activation and their interactions is crucial for developing targeted therapies for cancer, as these kinases are essential for the survival of cells under genomic stress. Recent advances in the development of specific inhibitors for ATM, ATR, and related kinases offer promising avenues for cancer treatment.