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.Supplemental material is available at http://www.genesdev.org.

Mcm10 interacts with Rad4/Cut5TopBP1 and its association with origins of DNA replication is dependent on Rad4/Cut5TopBP1

“…However origin association is Rad9 independent whilst accumulation distal to the origin, in the presence of HU, is Rad9 dependent. The Rad9-dependency of Rad4 TopBP1 accumulation at stalled replisomes is consistent with earlier data demonstrating that Rad4 TopBP1 acts downstream of Rad9 and Hus1 as judged by phosphorylation of these two checkpoint proteins, and the fact that interaction between Rad4 TopBP1 and Rad9 requires DNA damage-dependent phosphorylation of Rad9 (30,31,33). In addition it bears upon Rad4 TopBP1 replisome association.…”

“…This association is required for checkpointdependent Chk1 activation, as well as a number of other checkpoint proteins (29,31). Human 6 TopBP1 has also been shown to function in both DNA replication and the response to DNA damage and recent data mirror the requirement for interaction between hRad9 and TopBP1 for checkpoint signalling (32,33). In Xenopus, human and budding yeast systems TopBP1 has also been shown to directly interact with and activate the ATR-ATRIP protein kinase complex and in Xenopus regulation of this process is 9-1-1 dependent (34,35,36,37).…”

Activation and regulation of ATM kinase activity in response to DNA double-strand breaks

“…ATR phosphorylation of Chk1 after hydroxyurea (HU) treatment was found to be stimulated by the interaction of ATR with the small active domain (AD) of TopBP1, which is recruited to chromatin through Rad9 phosphorylation (Delacroix et al, 2007;Lee et al, 2007). They also showed that fusion of the TopBP1 AD with other chromatin-binding proteins including PCNA and H2B induces Chk1 phosphorylation after HU treatment.…”

Human FEM1B is required for Rad9 recruitment and CHK1 activation in response to replication stress

“…Decreased ATR activity in FEM1B-knockdown cells Upon replication stress, activation of ATR is coupled to the Rad9-Hus1-Rad1 complex through Top-BP1 (Delacroix et al, 2007;Lee et al, 2007). Because loading of Rad9 onto chromatin was impaired in FEM1B-downregulated cells, we wondered if ATR activity may also be similarly affected, thus contributing to reduced CHK1 Ser345 and Rad17 Ser645 phosphorylation.…”

“…The reason why two independent-sensor complexes are needed is not entirely clear. However, evidence has emerged indicating that interaction between ATR and Rad9 through TopBP1 is required to sustain ATR activation (Delacroix et al, 2007;Lee et al, 2007). Moreover, ATR-dependent phosphorylation of Rad17 and Claspin facilitates activation of CHK1 (Kumagai and Dunphy, 2003;Wang et al, 2006), a crucial downstream effector kinase of ATR.…”