The Rad9–Hus1–Rad1 (9–1–1) clamp activates checkpoint signaling via TopBP1

Abstract

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 AD to proliferating cell nuclear antigen (PCNA) or histone H2B bypasses the requirement for the 9–1–1 clamp, indicating that the 9–1–1 clamp’s primary role in activating Chk1 is to localize the AD to a stalled replication fork.