Direct Kinase-to-Kinase Signaling Mediated by the FHA Phosphoprotein Recognition Domain of the Dun1 DNA Damage Checkpoint Kinase

Abstract

. trans phosphorylation by Rad53 does not require the Dun1 kinase activity and is likely to involve only a transient interaction between the two kinases. The checkpoint functions of Dun1 kinase in DNA damage-induced transcription, G 2 /M cell cycle arrest, and Rad55 phosphorylation are severely compromised in an FHA domain mutant of Dun1. As a consequence, the Dun1 FHA domain mutant displays enhanced sensitivity to genotoxic stress induced by UV, methyl methanesulfonate, and the replication inhibitor hydroxyurea. We show that the Dun1 FHA domain is critical for direct kinase-to-kinase signaling from Rad53 to Dun1 in the DNA damage checkpoint pathway.DNA damage checkpoints coordinate the cellular responses to genotoxic stress and ensure genomic integrity (31,40,55,60). Besides cell cycle transitions, DNA damage checkpoints in the yeast Saccharomyces cerevisiae control damage-induced transcription; DNA replication; DNA repair and genomic stability; deoxynucleoside triphosphate metabolism; the relocalization of the Sir3/4, Ku80, and Rap1 proteins; and possibly other physiological responses to genotoxic stress (5,20,35,55,58,60,61).Central to the DNA damage checkpoints in S. cerevisiae is a branched kinase cascade consisting of five protein kinases (Mec1, Tel1, Rad53, Chk1, and Dun1) (55, 60). Mec1 and Tel1 are both high-molecular-weight phosphoinositide 3-kinase-related protein kinases that are activated by unknown mechanisms. Their human counterparts, ATM and ATR, are also essential for the human DNA damage checkpoints. Rad53 and Dun1 are related forkhead-associated (FHA) domain kinases (see Fig. 1) and have counterparts in other organisms, including fission yeast Cds1 and human Chk2 (40, 60). Finally, Chk1 kinase, as well as its fission yeast and human homologs, is critical for the G 2 cell cycle arrest in response to DNA damage (41). Genetic analysis of S. cerevisiae established that Mec1 controls the activities of the three downstream kinases Rad53, Dun1, and Chk1 (4,38,41,42,61). Under certain conditions, Tel1 controls the activation of Rad53 kinase in a Mec1-independent fashion (52). The exact mechanisms of how DNA damage checkpoints are activated and how the checkpoint kinases transmit and possibly amplify the signal, as well as control the effector pathways, are only beginning to be understood.Dun1 kinase functions in DNA damage-induced transcription of a subset of damage-inducible genes, including the RNR genes, by controlling the inactivating phosphorylation of the Crt1 transcriptional repressor (26,27,61). Mutations in DUN1 cause sensitivity to DNA damaging-agents and the replication inhibitor hydroxyurea (HU). This sensitivity can be partly suppressed by elevating the deoxynucleoside triphosphate pools through deletion of the ribonucleotide reductase inhibitor Sml1 or by overexpression of RNR1, the gene encoding the large subunit of ribonucleotide reductase (58, 61). In addition, Dun1 functions in one pathway with Rad53 kinase to cause a G 2 /M arrest in response to DNA damage by negatively regulating ...

Proteome-wide identification of in vivo targets of DNA damage checkpoint kinases

“…It is possible that Kap95 and Srp1 may function as adaptors to mediate Rad53 phosphorylation of these NPC components in cells. Considering the rather limited substrate sequence specificity of Rad53, it appears that its interaction with potential substrates in cells is important for substrate recognition, analogous to the situation of Dun1 being a substrate of Rad53 (31,32). The DNA damage checkpoint kinases were known to regulate nuclear export of Rnr2 and Rnr4 (40,41) and have genetic interactions with NPC components (42,43).…”

“…Because Mec1 and Tel1 control the activities of Rad53, Dun1 and possibly other kinases (12,13,31,32), Mec1-and Tel1-dependent phosphorylation should include both direct targets of Mec1 and Tel1 and those of their downstream kinases. Phosphorylation of the S/T-motif ( denotes a hydrophobic residue: F, I, L, or V) was also found to be a relatively common Mec1/Tel1-dependent phosphorylation, accounting for 23% of these phosphopeptides (see Fig.…”

Phosphorylation of Sae2 Mediates Forkhead-associated (FHA) Domain-specific Interaction and Regulates Its DNA Repair Function

“…Additionally, many other ligands of the Rad53 FHA1 domain have been identified (22). Similarly, the FHA domain of Dun1 is known to be critical for its interaction with Rad53 and trans-activation by Rad53 (42,43). The Xrs2 FHA domain has also been shown to interact with Lif1 to promote nonhomologous end joining (44).…”

Saccharomyces cerevisiae chromatin-assembly factors that act during DNA replication function in the maintenance of genome stability

“…because it lies downstream of RAD53 in regard to some checkpoint responses (36,51,58). Thus, an alternative explanation for the lack of an interaction between asf1 and dun1 mutations is that asf1 mutations significantly inactivate the RAD53 DUN1 checkpoint branch.…”