DNA replication stress triggers the activation of Checkpoint Kinase 1 (Chk1) in a pathway that requires the independent chromatin loading of the ATRIP–ATR (ATR-interacting protein/ATM [ataxia-telangiectasia mutated]–Rad3-related kinase) complex and the Rad9–Hus1–Rad1 (9–1–1) clamp. We show that Rad9’s role in Chk1 activation is to bind TopBP1, which stimulates ATR-mediated Chk1 phosphorylation via TopBP1’s activation domain (AD), a domain that binds and activates ATR. Notably, fusion of the...