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

Understanding the role of DNA damage checkpoint kinases in the cellular response to genotoxic stress requires the knowledge of their substrates. Here, we report the use of quantitative phosphoproteomics to identify in vivo kinase substrates of the yeast DNA damage checkpoint kinases Mec1, Tel1, and Rad53 (orthologs of human ATR, ATM, and CHK2, respectively). By analyzing 2,689 phosphorylation sites in wild-type and various kinase-null cells, 62 phosphorylation sites from 55 proteins were found to be controlled by the DNA damage checkpoint. Examination of the dependency of each phosphorylation on Mec1 and Tel1 or Rad53, combined with sequence and biochemical analysis, revealed that many of the identified targets are likely direct substrates of these kinases. In addition to several known targets, 50 previously undescribed targets of the DNA damage checkpoint were identified, suggesting that a wide range of cellular processes is likely regulated by Mec1, Tel1, and Rad53.     

A dominant-negative MEC3 mutant uncovers new functions for the Rad17 complex and Tel1

The Rad17-Mec3-Ddc1 complex is essential for the cellular response to genotoxic agents and is thought to be important for sensing DNA lesions. Deletion of any of the RAD17, MEC3 or DDC1 genes abolishes the G(1) and G(2) and impairs the intra-S DNA-damage checkpoints. We characterize a dominant-negative mec3-dn mutation that has an unexpected phenotype. It inactivates the G(1) checkpoint while it leaves the G(2) response functional, thus revealing a difference in the requirements of the DNA-damage response in different phases of the cell cycle. In an attempt to identify the molecular defect imparted by the mutation, we dissected step-by-step the signaling cascade, which is triggered by DNA lesions and requires the activity of Mec1 and Rad53 kinases. The analysis of the phosphorylation state of checkpoint factors and critical protein interactions showed that, in mec3-dn cells, the signal transduction cascade is triggered normally, and the central kinase Mec1 can be activated. In G(1) cells expressing the mutation, the signaling cannot proceed any further along the pathway, indicating that the Rad17 complex acts after the activation of Mec1, possibly recruiting targets for the kinase. We also show that the function of the G(2) checkpoint in mutant cells is maintained by an uncharacterized activity of Tel1, the yeast homologue of ATM. This work thus reports a previously undiscovered role for Tel1 in checkpoint control.     

Histone chaperone Anp32e removes H2A.Z from DNA double-strand breaks and promotes nucleosome reorganization and DNA repair

The repair of DNA double-strand breaks (DSBs) requires open, flexible chromatin domains. The NuA4–Tip60 complex creates these flexible chromatin structures by exchanging histone H2A.Z onto nucleosomes and promoting acetylation of histone H4. Here, we demonstrate that the accumulation of H2A.Z on nucleosomes at DSBs is transient, and that rapid eviction of H2A.Z is required for DSB repair. Anp32e, an H2A.Z chaperone that interacts with the C-terminal docking domain of H2A.Z, is rapidly recruited to DSBs. Anp32e functions to remove H2A.Z from nucleosomes, so that H2A.Z levels return to basal within 10 min of DNA damage. Further, H2A.Z removal by Anp32e disrupts inhibitory interactions between the histone H4 tail and the nucleosome surface, facilitating increased acetylation of histone H4 following DNA damage. When H2A.Z removal by Anp32e is blocked, nucleosomes at DSBs retain elevated levels of H2A.Z, and assume a more stable, hypoacetylated conformation. Further, loss of Anp32e leads to increased CtIP-dependent end resection, accumulation of single-stranded DNA, and an increase in repair by the alternative nonhomologous end joining pathway. Exchange of H2A.Z onto the chromatin and subsequent rapid removal by Anp32e are therefore critical for creating open, acetylated nucleosome structures and for controlling end resection by CtIP. Dynamic modulation of H2A.Z exchange and removal by Anp32e reveals the importance of the nucleosome surface and nucleosome dynamics in processing the damaged chromatin template during DSB repair.The repair of DNA double-strand breaks (DSBs), which cleave the DNA backbone, requires remodeling of the local chromatin architecture. This reorganization of the chromatin is important for promoting access to the site of damage, for creating a template for the repair machinery, and for repackaging the chromatin and resetting the epigenetic landscape following repair. Chromatin remodeling at DSBs is linked to changes in posttranslational modification of histones. DSBs activate the ataxia-telangiectasia mutated (ATM) and DNA–PKcs kinases, which phosphorylate multiple DNA repair proteins, including histone H2AX. Phosphorylated H2AX (γH2AX) provides a binding site for mdc1, which promotes spreading of γH2AX for hundreds of kilobases either side of the break (1–3). DSBs also promote complex patterns of chromatin ubiquitination, including ubiquitination of H2A/H2AX by the RNF8/RNF168 ubiquitin ligases, which, in turn, creates binding sites for repair proteins such as 53BP1 and brca1 (4–7). DSBs also lead to increased methylation of histone H3 on lysine 36, which can regulate DNA repair pathway choice (8, 9) and methylation of H3 on lysine 9 (10), which drives activation of the Tip60 acetyltransferase and the ATM kinase (11, 12). Further, the NuA4–Tip60 complex (5, 13–15) promotes acetylation of histone H4 at DSBs and drives the formation of open, flexible chromatin domains at DSBs (5, 13, 14). The repair of DSBs is therefore fundamentally a chromatin-driven process requiring dynamic changes in histone modification and chromatin reorganization, which directly promote recruitment of DSB repair proteins (16).The NuA4–Tip60 remodeling complex plays a central role in nucleosome reorganization at DSBs (16). NuA4–Tip60 is a 16 subunit complex containing 2 key subunits—the p400 SWI/SNF ATPase and the Tip60 acetyltransferase. The p400 ATPase promotes exchange of H2A for the histone variant H2A.Z (13, 17). This increase in H2A.Z at DSBs then promotes acetylation of histone H4 by the Tip60, creating open, flexible chromatin at sites of DNA damage (5, 11, 14). Inactivation of NuA4–Tip60 blocks both H2A.Z exchange and H4 acetylation, leading to a reduction in chromatin mobility at DSBs. Consequently, loss of H2A.Z exchange leads to defective DSB repair, increased sensitivity to DNA damage, and genomic instability (13, 18, 19).Here, we demonstrate that H2A.Z exchange at DSBs is dynamic, with H2A.Z accumulating at DSBs within minutes of damage, followed by rapid H2A.Z eviction. Further, we show that Anp32e, an H2A.Z-specific histone chaperone, binds specifically to the docking domain of H2A.Z and is required to remove H2A.Z from the damaged chromatin template. Failure to remove H2A.Z leads to defects in DSB repair, including a loss of H4 acetylation, defects in nonhomologous end joining (NHEJ), and increased end resection of DSBs.     

Checkpoint genes and Exo1 regulate nearby inverted repeat fusions that form dicentric chromosomes in Saccharomyces cerevisiae

Genomic rearrangements are common, occur by largely unknown mechanisms, and can lead to human diseases. We previously demonstrated that some genome rearrangements occur in budding yeast through the fusion of two DNA sequences that contain limited sequence homology, lie in inverted orientation, and are within 5 kb of one another. This inverted repeat fusion reaction forms dicentric chromosomes, which are well-known intermediates to additional rearrangements. We have previously provided evidence indicating that an error of stalled or disrupted DNA replication forks can cause inverted repeat fusion. Here we analyze how checkpoint protein regulatory pathways known to stabilize stalled forks affect this form of instability. We find that two checkpoint pathways suppress inverted repeat fusion, and that their activities are distinguishable by their interactions with exonuclease 1 (Exo1). The checkpoint kinase Rad53 (Chk2) and recombination protein complex MRX(MRN) inhibit Exo1 in one pathway, whereas in a second pathway the ATR-like kinases Mec1 and Tel1, adaptor protein Rad9, and effector kinases Chk1 and Dun1 act independently of Exo1 to prevent inverted repeat fusion. We provide a model that indicates how in Rad53 or MRX mutants, an inappropriately active Exo1 may facilitate faulty template switching between nearby inverted repeats to form dicentric chromosomes. We further investigate the role of Rad53, using hypomorphic alleles of Rad53 and null mutations in Rad9 and Mrc1, and provide evidence that only local, as opposed to global, activity of Rad53 is sufficient to prevent inverted repeat fusion.     

Pph3-Psy2 is a phosphatase complex required for Rad53 dephosphorylation and replication fork restart during recovery from DNA damage

Activation of the checkpoint kinase Rad53 is a critical response to DNA damage that results in stabilization of stalled replication forks, inhibition of late-origin initiation, up-regulation of dNTP levels, and delayed entry to mitosis. Activation of Rad53 is well understood and involves phosphorylation by the protein kinases Mec1 and Tel1 as well as in trans autophosphorylation by Rad53 itself. However, deactivation of Rad53, which must occur to allow the cell to recover from checkpoint arrest, is not well understood. Here, we present genetic and biochemical evidence that the type 2A-like protein phosphatase Pph3 forms a complex with Psy2 (Pph3-Psy2) that binds and dephosphorylates activated Rad53 during treatment with, and recovery from, methylmethane sulfonate-mediated DNA damage. In the absence of Pph3-Psy2, Rad53 dephosphorylation and the resumption of DNA synthesis are delayed during recovery from DNA damage. This delay in DNA synthesis reflects a failure to restart stalled replication forks, whereas, remarkably, genome replication is eventually completed by initiating late origins of replication despite the presence of hyperphosphorylated Rad53. These findings suggest that Rad53 regulates replication fork restart and initiation of late firing origins independently and that regulation of these processes is mediated by specific Rad53 phosphatases.     

The Saccharomyces cerevisiae 14-3-3 proteins Bmh1 and Bmh2 directly influence the DNA damage-dependent functions of Rad53

In this study, we mutated autophosphorylation sites in Rad53 based on their conservation with previously identified autophosphorylation sites in the mammalian Rad53 ortholog, Chk2. As with wild-type Rad53, the autophosphorylation mutant, rad53-TA, undergoes Mec1/Tel1-dependent interactions with Rad9 and Dun1 in response to genotoxic stress. Whereas rad53-TA in vitro kinase activity is severely impaired, the rad53-TA strains are not completely deficient for cell-cycle checkpoint functions, indicating that the mutant kinase retains a basal level of function. We describe a genetic interaction among Rad53, Dun1, and the 14-3-3 proteins Bmh1 and Bmh2 and present evidence that 14-3-3 proteins directly facilitate Rad53 function in vivo. The data presented account for the previously observed checkpoint defects associated with 14-3-3 mutants in Saccharomyces pombe and Saccharomyces cerevisiae. The 14-3-3 functional interaction appears to modulate Rad53 activity, reminiscent of 14-3-3's effect on human Raf1 kinase and distinct from the indirect mode of regulation by 14-3-3 observed for Chk1 or Cdc25.     

Surprising complexity of the Asf1 histone chaperone-Rad53 kinase interaction

The histone chaperone Asf1 and the checkpoint kinase Rad53 are found in a complex in budding yeast cells in the absence of genotoxic stress. Our data suggest that this complex involves at least three interaction sites. One site involves the H3-binding surface of Asf11 with an as yet undefined surface of Rad53. A second site is formed by the Rad53-FHA1 domain binding to Asf1-T₂₇₀ phosphorylated by casein kinase II. The third site involves the C-terminal 21 amino acids of Rad53 bound to the conserved Asf1 N-terminal domain. The structure of this site showed that the Rad53 C-terminus binds Asf1 in a remarkably similar manner to peptides derived from the histone cochaperones HirA and CAF-I. We call this binding motif, (R/K)R(I/A/V) × (L/P), the AIP box for Asf1-Interacting Protein box. Furthermore, C-terminal Rad53-F₈₂₀ binds the same pocket of Asf1 as does histone H4-F₁₀₀. Thus Rad53 competes with histones H3-H4 and cochaperones HirA/CAF-I for binding to Asf1. Rad53 is phosphorylated and activated upon genotoxic stress. The Asf1-Rad53 complex dissociated when cells were treated with hydroxyurea but not methyl-methane-sulfonate, suggesting a regulation of the complex as a function of the stress. We identified a rad53 mutation that destabilized the Asf1-Rad53 complex and increased the viability of rad9 and rad24 mutants in conditions of genotoxic stress, suggesting that complex stability impacts the DNA damage response.     

DNA double-strand breaks promote methylation of histone H3 on lysine 9 and transient formation of repressive chromatin

Dynamic changes in histone modification are critical for regulating DNA double-strand break (DSB) repair. Activation of the Tip60 acetyltransferase by DSBs requires interaction of Tip60 with histone H3 methylated on lysine 9 (H3K9me3). However, how H3K9 methylation is regulated during DSB repair is not known. Here, we demonstrate that a complex containing kap-1, HP1, and the H3K9 methyltransferase suv39h1 is rapidly loaded onto the chromatin at DSBs. Suv39h1 methylates H3K9, facilitating loading of additional kap-1/HP1/suv39h1 through binding of HP1’s chromodomain to the nascent H3K9me3. This process initiates cycles of kap-1/HP1/suv39h1 loading and H3K9 methylation that facilitate spreading of H3K9me3 and kap-1/HP1/suv39h1 complexes for tens of kilobases away from the DSB. These domains of H3K9me3 function to activate the Tip60 acetyltransferase, allowing Tip60 to acetylate both ataxia telangiectasia-mutated (ATM) kinase and histone H4. Consequently, cells lacking suv39h1 display defective activation of Tip60 and ATM, decreased DSB repair, and increased radiosensitivity. Importantly, activated ATM rapidly phosphorylates kap-1, leading to release of the repressive kap-1/HP1/suv39h1 complex from the chromatin. ATM activation therefore functions as a negative feedback loop to remove repressive suv39h1 complexes at DSBs, which may limit DSB repair. Recruitment of kap-1/HP1/suv39h1 to DSBs therefore provides a mechanism for transiently increasing the levels of H3K9me3 in open chromatin domains that lack H3K9me3 and thereby promoting efficient activation of Tip60 and ATM in these regions. Further, transient formation of repressive chromatin may be critical for stabilizing the damaged chromatin and for remodeling the chromatin to create an efficient template for the DNA repair machinery.DNA double-strand breaks (DSBs) are toxic and must be repaired to maintain genomic stability. Detection of DSBs requires recruitment of the mre11–rad50–nbs1 (MRN) complex to the DNA ends (1). MRN then recruits and activates the ataxia telangiectasia-mutated (ATM) kinase (2, 3) through a mechanism that also requires the Tip60 acetyltransferase (3). Tip60 directly acetylates and activates ATM’s kinase activity (4–6) and functions, in combination with MRN, to promote ATM-dependent phosphorylation of DSB repair proteins (3), including histone H2AX. This process creates domains of phosphorylated H2AX (γH2AX) extending for hundreds of kilobases along the chromatin (7, 8). Mdc1 then binds to γH2AX, providing a landing pad for other DSB repair proteins, including the RNF8/RNF168 ubiquitin ligases (1, 3, 9, 10). Tip60 also plays a critical role in regulating chromatin structure at DSBs as part of the NuA4–Tip60 complex (11). NuA4-Tip60 catalyzes histone exchange (via the p400 ATPase subunit) and acetylation of histone H4 (by Tip60) at DSBs (12–15), leading to the formation of open, flexible chromatin domains adjacent to the break (12, 13). These open chromatin structures then facilitate histone ubiquitination, the loading of brca1 and 53BP1, and repair of the DSB (13, 16). The ordered acetylation and ubiquitination of the chromatin and loading of DNA repair proteins is therefore critical for DSB repair.Activation of Tip60’s acetyltransferase activity requires interaction between Tip60’s chromodomain and histone H3 methylated on lysine 9 (H3K9me3) on nucleosomes at the break (4, 6). This interaction, in combination with tyrosine phosphorylation of Tip60 (17), increases Tip60’s acetyltransferase activity and promotes acetylation of both the ATM kinase and histone H4 (4–6, 17). Consequently, loss of H3K9me2/3 leads to failure to activate the ATM signaling pathway, loss of H4 acetylation during DSB repair, disruption of heterochromatin, genomic instability, and defective DSB repair (4, 17–19). H3K9me3s therefore play a critical role in linking chromatin structure at DSBs to the activation of DSB signaling proteins such as Tip60 and ATM.How Tip60 gains access to H3K9me3 and how H3K9me3 levels at DSBs are regulated is not known. H3K9me3 is concentrated in heterochromatin domains, where it recruits HP1, kap-1, and H3K9 methyltransferases (20, 21) to maintain the silent, compact conformation of heterochromatin (20). This implies that Tip60’s acetyltransferase activity can only be activated at DSBs in regions of high H3K9me3 density, such as heterochromatin. Alternatively, H3K9 methylation may be actively increased at DSBs in regions of low H3K9me3 density to allow for Tip60 activation and efficient DSB repair in euchromatin. Understanding the dynamics of H3K9 methylation at DSBs is therefore critical to understanding how Tip60 activity is regulated by the local chromatin architecture. Here, we show that the suv39h1 methyltransferase is recruited to DSBs in euchromatin as part of a larger kap-1/HP1/suv39h1 complex. Suv39h1 increases H3K9me3 at DSBs, activating Tip60’s acetyltransferase activity and promoting the subsequent acetylation and activation of ATM. Further, loss of inducible H3K9me3 at DSBs leads to defective repair and increased radiosensitivity. Finally, loading of the kap-1/HP1/suv39h1 complex is transient, and the complex is rapidly released from the chromatin through a negative feedback loop driven by ATM-dependent phosphorylation of the kap-1 protein.     

RAD9-dependent G1 arrest defines a second checkpoint for damaged DNA in the cell cycle of Saccharomyces cerevisiae.

Exposure of the yeast Saccharomyces cerevisiae to ultraviolet (UV) light, the UV-mimetic chemical 4-nitroquinoline-1-oxide (4NQO), or gamma radiation after release from G1 arrest induced by alpha factor results in delayed resumption of the cell cycle. As is the case with G2 arrest following ionizing radiation damage [Weinert, T. A. & Hartwell, L. H. (1988) Science 241, 317-322], the normal execution of DNA damage-induced G1 arrest depends on a functional yeast RAD9 gene. We suggest that the RAD9 gene product may interact with cellular components common to the G1/S and G2/M transition points in the cell cycle of this yeast. These observations define a checkpoint in the eukaryotic cell cycle that may facilitate the repair of lesions that are otherwise processed to lethal and/or mutagenic damage during DNA replication. This checkpoint apparently operates after the mating pheromone-induced G1 arrest point but prior to replicative DNA synthesis, S phase-associated maximal induction of histone H2A mRNA, and bud emergence.     

Serine-345 is required for Rad3-dependent phosphorylation and function of checkpoint kinase Chk1 in fission yeast

Genome integrity is monitored by a checkpoint that delays mitosis in response to DNA damage. This checkpoint is enforced by Chk1, a protein kinase that inhibits the mitotic inducer Cdc25. In fission yeast, Chk1 is regulated by a group of proteins that includes Rad3, a protein kinase related to human ATM and ATR. These kinases phosphorylate serine or threonine followed by glutamine (SQ/TQ). Fission yeast and human Chk1 proteins share two conserved SQ motifs at serine-345 and serine-367. Serine-345 of human Chk1 is phosphorylated in response to DNA damage. Here we report that Rad3 and ATM phosphorylate serine-345 of fission yeast Chk1. Mutation of serine-345 (chk1-S345A) abrogates Rad3-dependent phosphorylation of Chk1 in vivo. The chk1-S345A cells are sensitive to DNA damage and are checkpoint defective. In contrast, mutations of serine-367 and other SQ/TQ sites do not substantially impair the checkpoint or cause damage sensitivity. These findings attest to the importance of serine-345 phosphorylation for Chk1 function and strengthen evidence that transduction of the DNA damage checkpoint signal requires direct phosphorylation of Chk1 by Rad3.     

The anaphase inhibitor of Saccharomyces cerevisiae Pds1p is a target of the DNA damage checkpoint pathway

Inhibition of DNA replication and physical DNA damage induce checkpoint responses that arrest cell cycle progression at two different stages. In Saccharomyces cerevisiae, the execution of both checkpoint responses requires the Mec1 and Rad53 proteins. This observation led to the suggestion that these checkpoint responses are mediated through a common signal transduction pathway. However, because the checkpoint-induced arrests occur at different cell cycle stages, the downstream effectors mediating these arrests are likely to be distinct. We have previously shown that the S. cerevisiae protein Pds1p is an anaphase inhibitor and is essential for cell cycle arrest in mitosis in the presence DNA damage. Herein we show that DNA damage, but not inhibition of DNA replication, induces the phosphorylation of Pds1p. Analyses of Pds1p phosphorylation in different checkpoint mutants reveal that in the presence of DNA damage, Pds1p is phosphorylated in a Mec1p- and Rad9p-dependent but Rad53p-independent manner. Our data place Pds1p and Rad53p on parallel branches of the DNA damage checkpoint pathway. We suggest that Pds1p is a downstream target of the DNA damage checkpoint pathway and that it is involved in implementing the DNA damage checkpoint arrest specifically in mitosis.