biostudies-literatureBiller MTakeda Bioscience ResearchNaito ResearchNational Institutes of HealthMitsubishi ResearchAstellas Research FoundationNIH HHSJapan Society for the Promotion of Sciencegkaf1527https://www.ebi.ac.uk/biostudies/studies/S-EPMC12820532biostudies-literatureUnknown54(2)Ataxia-telangiectasia mutated and RAD3-related (ATR) and its partner ATR-interacting protein (ATRIP) function as a critical proximal sensor and transducer of the DNA damage response (DDR). Several ATR substrates, including p53 and CHK1, are crucial for the coordination of cell cycle phase transitions, transcription, and DNA repair when cells sustain DNA damage. While much is known about ATR activation mechanisms, it is less clear how ATR signaling is negatively regulated in cells. Here, we identify the DNA repair protein REV7 as a novel direct binding partner of ATRIP. We define a REV7-interaction motif in ATRIP, which, when mutated, abrogates the REV7-ATRIP interaction in vitro and in intact cells. Using in vitro kinase assays, we show that REV7 inhibits ATR-mediated phosphorylation of itNucleic acids researchREV7 associates with ATRIP and inhibits ATR kinase activity.PMC128205321R15CA263784CA229530R01 ES00955819H042671R15CA305114CA215347Tomida JSasanuma HZhou PKabir SBiller MNipper SVaziri CAllen SKayali YKuncik SfalseREV7 associates with ATRIP and inhibits ATR kinase activity.Ataxia-telangiectasia mutated and RAD3-related (ATR) and its partner ATR-interacting protein (ATRIP) function as a critical proximal sensor and transducer of the DNA damage response (DDR). Several ATR substrates, including p53 and CHK1, are crucial for the coordination of cell cycle phase transitions, transcription, and DNA repair when cells sustain DNA damage. While much is known about ATR activation mechanisms, it is less clear how ATR signaling is negatively regulated in cells. Here, we identify the DNA repair protein REV7 as a novel direct binding partner of ATRIP. We define a REV7-interaction motif in ATRIP, which, when mutated, abrogates the REV7-ATRIP interaction in vitro and in intact cells. Using in vitro kinase assays, we show that REV7 inhibits ATR-mediated phosphorylation of it2026-01-01T00:00:00Z2026 Jan2026-06-06T22:35:38.854Z2026-06-06T03:10:43.808ZS-EPMC128205324156225810.1093/nar/gkaf1527