ATR is a critical regulator of genome integrity, functioning alongside ATM in the DNA damage response (DDR) to maintain cellular viability and prevent disease. ATR and ATM are phosphoinositide 3-kinase-related kinases (PIKKs) that control cell cycle transitions, DNA replication, DNA repair, and apoptosis. While both kinases share biochemical and functional similarities, ATR is essential for the viability of replicating human and mouse cells, whereas ATM is not. ATR is activated during S-phase to regulate replication origins, repair damaged replication forks, and prevent premature mitosis, whereas ATM responds to rare double-strand breaks. ATR activation is primarily triggered by single-stranded DNA (ssDNA) coated with replication protein A (RPA), and requires the interaction with ATRIP, a obligate subunit of the ATR kinase. The 9-1-1 checkpoint complex also plays a role in recruiting TOPBP1, which activates ATR-ATRIP complexes. ATR signaling is crucial for DNA repair, cell cycle control, and DNA replication, and is a promising target for cancer therapy. ATR substrates include CHK1, which regulates cell cycle transitions, and the MCM helicase complex, which is involved in DNA replication. ATR also regulates DNA repair by phosphorylating proteins such as BRCA1, WRN, and BLM. ATR and ATM have overlapping but non-redundant functions in the DDR, with crosstalk occurring due to the interconversion of DNA lesions. ATR is essential for the survival of replicating cells due to the ubiquity of DNA lesions and replication stress. ATR signaling is important for maintaining genome stability and preventing cancer, and is a target for drug development. The PIK kinase family, including ATR, ATM, DNA-PKcs, mTOR, and SMG-1, shares common regulatory themes, such as localization, protein activators, and post-translational modifications. Understanding ATR signaling is crucial for developing therapies for cancer and other diseases linked to genome instability.ATR is a critical regulator of genome integrity, functioning alongside ATM in the DNA damage response (DDR) to maintain cellular viability and prevent disease. ATR and ATM are phosphoinositide 3-kinase-related kinases (PIKKs) that control cell cycle transitions, DNA replication, DNA repair, and apoptosis. While both kinases share biochemical and functional similarities, ATR is essential for the viability of replicating human and mouse cells, whereas ATM is not. ATR is activated during S-phase to regulate replication origins, repair damaged replication forks, and prevent premature mitosis, whereas ATM responds to rare double-strand breaks. ATR activation is primarily triggered by single-stranded DNA (ssDNA) coated with replication protein A (RPA), and requires the interaction with ATRIP, a obligate subunit of the ATR kinase. The 9-1-1 checkpoint complex also plays a role in recruiting TOPBP1, which activates ATR-ATRIP complexes. ATR signaling is crucial for DNA repair, cell cycle control, and DNA replication, and is a promising target for cancer therapy. ATR substrates include CHK1, which regulates cell cycle transitions, and the MCM helicase complex, which is involved in DNA replication. ATR also regulates DNA repair by phosphorylating proteins such as BRCA1, WRN, and BLM. ATR and ATM have overlapping but non-redundant functions in the DDR, with crosstalk occurring due to the interconversion of DNA lesions. ATR is essential for the survival of replicating cells due to the ubiquity of DNA lesions and replication stress. ATR signaling is important for maintaining genome stability and preventing cancer, and is a target for drug development. The PIK kinase family, including ATR, ATM, DNA-PKcs, mTOR, and SMG-1, shares common regulatory themes, such as localization, protein activators, and post-translational modifications. Understanding ATR signaling is crucial for developing therapies for cancer and other diseases linked to genome instability.