Recruitment of the 9-1-1 complex to DNA Loading of claspin onto DNA during replication origin firing Binding of ATR-ATRIP to the RPA-ssDNA complex Recruitment of Rad17-RFC complex to DNA Stalling of DNA replication fork and RPA binding Phosphorylation of Cdc25A at Ser-123 by Chk1 Recruitment and activation of Chk1 PathwayStep4489 PathwayStep4484 PathwayStep4483 PathwayStep4488 PathwayStep4487 PathwayStep4486 PathwayStep4485 PathwayStep4467 PathwayStep4472 Activation of claspin Phosphorylation of Cdc25C at Ser 216 by Chk1 Activation of ATR in response to replication stress Authored: Borowiec, JA, 2006-02-25 17:40:15 Edited: D'Eustachio, P, 2006-02-25 17:41:28 GENE ONTOLOGYGO:0006260 Genotoxic stress caused by DNA damage or stalled replication forks can lead to genomic instability. To guard against such instability, genotoxically-stressed cells activate checkpoint factors that halt or slow cell cycle progression. Among the pathways affected are DNA replication by reduction of replication origin firing, and mitosis by inhibiting activation of cyclin-dependent kinases (Cdks). A key factor involved in the response to stalled replication forks is the ATM- and rad3-related (ATR) kinase, a member of the phosphoinositide-3-kinase-related kinase (PIKK) family. Rather than responding to particular lesions in DNA, ATR and its binding partner ATRIP (ATR-interacting protein) sense replication fork stalling indirectly by associating with persistent ssDNA bound by RPA. These structures would be formed, for example, by dissociation of the replicative helicase from the leading or lagging strand DNA polymerase when the polymerase encounters a DNA lesion that blocks DNA synthesis. Along with phosphorylating the downstream transducer kinase Chk1 and the tumor suppressor p53, activated ATR modifies numerous factors that regulate cell cycle progression or the repair of DNA damage. The persistent ssDNA also stimulates recruitment of the RFC-like Rad17–Rfc2-5 alternative clamp-loading complex, which subsequently loads the Rad9-Hus1-Rad1 complex onto the DNA. The latter '9-1-1' complex serves to facilitate Chk1 binding to the stalled replication fork, where Chk1 is phosphorylated by ATR and thereby activated. Upon activation, Chk1 can phosphorylate additional substrates including the Cdc25 family of phosphatases (Cdc25A, Cdc25B, and Cdc25C). These enzymes catalyze the removal of inhibitory phosphate residues from cyclin-dependent kinases (Cdks), allowing their activation. In particular, Cdc25A primarily functions at the G1/S transition to dephosphorylate Cdk2 at Thr 14 and Tyr 15, thus positively regulating the Cdk2-cyclin E complex for S-phase entry. Cdc25A also has mitotic functions. Phosphorylation of Cdc25A at Ser125 by Chk1 leads to Cdc25A ubiquitination and degradation, thus inhibiting DNA replication origin firing. In contrast, Cdc25B and Cdc25C regulate the onset of mitosis through dephosphorylation and activation of Cdk1-cyclin B complexes. In response to replication stress, Chk1 phosphorylates Cdc25B and Cdc25C leading to Cdc25B/C complex formation with 14-3-3 proteins. As these complexes are sequestered in the cytoplasm, they are unable to activate the nuclear Cdk1-cyclin B complex for mitotic entry.<p>These events are outlined in the figure. Persistent single-stranded DNA associated with RPA binds claspin (A) and ATR:ATRIP (B), leading to claspin phosphorylation (C). In parallel, the same single-stranded DNA:RPA complex binds RAD17:RFC (D), enabling the loading of RAD9:HUS1:RAD1 (9-1-1) complex onto the DNA (E). The resulting complex of proteins can then repeatedly bind (F) and phosphorylate (G) CHK1, activating multiple copies of CHK1. Pubmed12781359 Pubmed12791985 Pubmed15530773 Pubmed16314342 Reactome Database ID Release 43176187 Reactome, http://www.reactome.org ReactomeREACT_6769