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

Single-stranded or double-stranded DNA junctions with recessed 5 ' ends serve as loading sites for the checkpoint clamp, 9-1-1, which mediates activation of the apical checkpoint kinase, ATR(Mec1). However, the basis for 9-1-1's recruitment to 5 ' junctions is unclear. Here, we present structures of the yeast checkpoint clamp loader, Rad24-replication factor C (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 9-1-1's recruitment to 5 ' junctions and reveal new principles of sliding clamp loading. Cryo-EM structures of the yeast 9-1-1 checkpoint clamp in complex with the Rad24-RFC clamp loader and a DNA substrate explain how 9-1-1 is recruited to DNA junctions with recessed 5 ' ends and reveal the mechanism of sliding clamp loading.

Automated summary

The study finds that the 9-1-1 checkpoint clamp can adopt both closed and planar open states, and it can bind to DNA junctions to form a loading site. Furthermore, the study reveals the mechanism of sliding clamp loading.