The human CtIP protein plays a critical role in DNA double-strand break (DSB) resection, a process essential for homologous recombination (HR) and ATR-dependent checkpoint signaling. CtIP is recruited to DSBs exclusively during the S and G2 phases of the cell cycle and is required for the resection of DSBs, which facilitates the recruitment of RPA and ATR to the damage sites. CtIP physically and functionally interacts with the MRE11 complex, which is also involved in DSB processing. Together, CtIP and MRE11 are necessary for efficient HR. Additionally, CtIP shares sequence homology with Sae2, a protein involved in MRE11-dependent DSB processing in yeast, suggesting that CtIP and Sae2 are functional homologs. CtIP is essential for the repair of DSBs generated during DNA replication, particularly in S-phase. Depletion of CtIP leads to hypersensitivity to DSB-inducing agents such as camptothecin and etoposide, but not to bleocin, which generates DSBs at all cell cycle stages. CtIP depletion impairs ATR activation and HR, as evidenced by reduced Chk1 phosphorylation and RPA recruitment to DSBs. CtIP is also required for the formation of ssDNA, which is necessary for ATR activation and HR. CtIP promotes ATR recruitment to DSBs, which is essential for checkpoint signaling. CtIP interacts with the MRN complex, which is involved in DSB resection and HR. The CtIP-MRN interaction is stable and not DNA-mediated. CtIP and MRN work together to facilitate DSB resection, and both are required for efficient HR. CtIP also interacts with BRCA1, which is involved in DNA repair and checkpoint control. The C-terminal region of CtIP is highly conserved and is required for its function. This region shows homology with the C-terminal region of Sae2, a yeast protein involved in DSB processing. These findings suggest that CtIP and Sae2 are functional homologs that control DSB resection, checkpoint signaling, and HR in eukaryotes. CtIP is phosphorylated by ATM and Mec1, and its phosphorylation is most effective in S-phase. CtIP is also ubiquitylated by BRCA1 and interacts with the RB protein. These interactions may regulate the fate of DSBs during the cell cycle. CtIP is a potential target for drug discovery, as its alterations have been reported in some cancer cells. Overall, CtIP is a critical regulator of DNA repair and checkpoint signaling, working in concert with the MRN complex and other proteins to ensure genomic stability.The human CtIP protein plays a critical role in DNA double-strand break (DSB) resection, a process essential for homologous recombination (HR) and ATR-dependent checkpoint signaling. CtIP is recruited to DSBs exclusively during the S and G2 phases of the cell cycle and is required for the resection of DSBs, which facilitates the recruitment of RPA and ATR to the damage sites. CtIP physically and functionally interacts with the MRE11 complex, which is also involved in DSB processing. Together, CtIP and MRE11 are necessary for efficient HR. Additionally, CtIP shares sequence homology with Sae2, a protein involved in MRE11-dependent DSB processing in yeast, suggesting that CtIP and Sae2 are functional homologs. CtIP is essential for the repair of DSBs generated during DNA replication, particularly in S-phase. Depletion of CtIP leads to hypersensitivity to DSB-inducing agents such as camptothecin and etoposide, but not to bleocin, which generates DSBs at all cell cycle stages. CtIP depletion impairs ATR activation and HR, as evidenced by reduced Chk1 phosphorylation and RPA recruitment to DSBs. CtIP is also required for the formation of ssDNA, which is necessary for ATR activation and HR. CtIP promotes ATR recruitment to DSBs, which is essential for checkpoint signaling. CtIP interacts with the MRN complex, which is involved in DSB resection and HR. The CtIP-MRN interaction is stable and not DNA-mediated. CtIP and MRN work together to facilitate DSB resection, and both are required for efficient HR. CtIP also interacts with BRCA1, which is involved in DNA repair and checkpoint control. The C-terminal region of CtIP is highly conserved and is required for its function. This region shows homology with the C-terminal region of Sae2, a yeast protein involved in DSB processing. These findings suggest that CtIP and Sae2 are functional homologs that control DSB resection, checkpoint signaling, and HR in eukaryotes. CtIP is phosphorylated by ATM and Mec1, and its phosphorylation is most effective in S-phase. CtIP is also ubiquitylated by BRCA1 and interacts with the RB protein. These interactions may regulate the fate of DSBs during the cell cycle. CtIP is a potential target for drug discovery, as its alterations have been reported in some cancer cells. Overall, CtIP is a critical regulator of DNA repair and checkpoint signaling, working in concert with the MRN complex and other proteins to ensure genomic stability.