Mechanisms of loading and release of the 9-1-1 checkpoint clamp

5 single-stranded/double-stranded DNA serve as loading sites for the checkpoint clamp, 9-1-1, which mediates activation of the apical checkpoint kinase, ATRMec1. However, the basis for 9-1-1s recruitment to 5 junctions is unclear. Here, we present structures of the yeast checkpoint clamp loader, Rad24-RFC, in complex with 9-1-1 and a 5 junction and in a post-ATP-hydrolysis state. Unexpectedly, 9-1-1 adopts both closed and planar open states in the presence of Rad24-RFC and DNA. Moreover, Rad24-RFC associates with the DNA junction in the opposite orientation of processivity clamp loaders with Rad24 exclusively coordinating the double-stranded region. ATP hydrolysis stimulates conformational changes in Rad24-RFC, leading to disengagement of DNA-loaded 9-1-1. Together, these structures explain 9-1-1s recruitment to 5 junctions and reveal new principles of sliding clamp loading.