Rad53 kinase activation-independent replication checkpoint function of the N-terminal forkhead-associated (FHA1) domain

Saccharomyces cerevisiae Rad53 has crucial functions in many aspects of the cellular response to DNA damage and replication blocks. To coordinate these diverse roles, Rad53 has two forkhead-associated (FHA) phosphothreonine-binding domains in addition to a kinase domain. Here, we show that the conserved N-terminal FHA1 domain is essential for the function of Rad53 to prevent the firing of late replication origins in response to replication blocks. However, the FHA1 domain is not required for Rad53 activation during S phase, and as a consequence of defective downstream signaling, Rad53 containing an inactive FHA1 domain is hyperphosphorylated in response to replication blocks. The FHA1 mutation dramatically hypersensitizes strains with defects in the cell cycle-wide checkpoint pathways (rad9Delta and rad17Delta) to DNA damage, but it is largely epistatic with defects in the replication checkpoint (mrc1Delta). Altogether, our data indicate that the FHA1 domain links activated Rad53 to downstream effectors in the replication checkpoint. The results reveal an important mechanistic difference to the homologous Schizosaccharomyces pombe FHA domain that is required for Mrc1-dependent activation of the corresponding Cds1 kinase. Surprisingly, despite the severely impaired replication checkpoint and also G(2)/M checkpoint functions, the FHA1 mutation by itself leads to only moderate viability defects in response to DNA damage, highlighting the importance of functionally redundant pathways.     

Location-specific functions of the two forkhead-associated domains in Rad53 checkpoint kinase signaling

Signaling proteins often contain multiple modular protein-protein interaction domains of the same type. The Saccharomyces cerevisiae checkpoint kinase Rad53 contains two phosphothreonine-binding forkhead-associated (FHA) domains. To investigate if the precise position of these domains relative to each other is important, we created three rad53 alleles in which FHA1 and FHA2 domains were individually or simultaneously transposed to the opposite location. All three mutants were approximately 100-fold hypersensitive to DNA lesions whose survival requires intact Rad53 FHA domain functions, but they were not hypersensitive to DNA damage that is addressed in an FHA domain-independent manner. FHA domain-transposed Rad53 could still be recruited for activation by upstream kinases but then failed to autophosphorylate and activate FHA domain-dependent downstream functions. The results indicate that precise FHA domain positions are important for their roles in Rad53, possibly via regulation of the topology of oligomeric Rad53 signaling complexes.     

Role of the Saccharomyces cerevisiae Rad53 checkpoint kinase in signaling double-strand breaks during the meiotic cell cycle

DNA double-strand breaks (DSBs) can arise at unpredictable locations after DNA damage or in a programmed manner during meiosis. DNA damage checkpoint response to accidental DSBs during mitosis requires the Rad53 effector kinase, whereas the meiosis-specific Mek1 kinase, together with Red1 and Hop1, mediates the recombination checkpoint in response to programmed meiotic DSBs. Here we provide evidence that exogenous DSBs lead to Rad53 phosphorylation during the meiotic cell cycle, whereas programmed meiotic DSBs do not. However, the latter can trigger phosphorylation of a protein fusion between Rad53 and the Mec1-interacting protein Ddc2, suggesting that the inability of Rad53 to transduce the meiosis-specific DSB signals might be due to its failure to access the meiotic recombination sites. Rad53 phosphorylation/activation is elicited when unrepaired meiosis-specific DSBs escape the recombination checkpoint. This activation requires homologous chromosome segregation and delays the second meiotic division. Altogether, these data indicate that Rad53 prevents sister chromatid segregation in the presence of unrepaired programmed meiotic DSBs, thus providing a salvage mechanism ensuring genetic integrity in the gametes even in the absence of the recombination checkpoint.     

Characterization of the activation domain of the Rad53 checkpoint kinase

Rad53 protein, the yeast orthologue of the human checkpoint kinase Chk2, presents two highly conserved phosphorylatable threonine residues (T354 and T358) in the activation domain, whose phosphorylation is critical to allow the activation of the kinase. In this study we found that Rad53 protein variants in which alanine and/or aspartate replace the threonine residues 354 and/or 358 do not retain kinase activity and do not undergo auto-phosphorylation, leading to defect in the checkpoint response and iper-sensitivity to DNA damage and DNA replication stress agents. Interestingly, we found that the rad53-T358D mutation severely affects the kinase activity and causes accumulation of the S129-phosphorylated isoform of histone H2A, even during an unperturbed cell cycle, thus indicating the accumulation of spontaneous DNA breaks. We further found that the protein level of Sml1, which is the physiological inhibitor of ribonucleotide reductase, remains high during DNA replication in rad53-T358D cells, suggesting that an inadequate pool of dNTPs in checkpoint defective cells causes the accumulation of spontaneous DNA breaks.

In conclusion, our results indicate that phosphorylation of both T354 and T358 residues strongly influences the catalytic activity of Rad53 also in unperturbed cell cycles, and support the notion that Rad53 is essential to preserve genome integrity, by controlling the level of Sml1 and the functionality of ribonucleotide reductase.     

Diverse but overlapping functions of the two forkhead-associated (FHA) domains in Rad53 checkpoint kinase activation

Forkhead-associated (FHA) domains are phosphothreonine-binding modules prevalent in proteins with important cell cycle and DNA damage response functions. The yeast checkpoint kinase Rad53 is unique in containing two FHA domains. We have generated novel recessive rad53 alleles with abolished FHA domain functions resulting from Ala substitution of the critical phosphothreonine-binding residues Arg70 and Arg605. In asynchronous cells, inactivation of the N-terminal FHA1 domain did not impair Rad53 activation and downstream functions, whereas inactivation of the C-terminal FHA2 domain led to reduced Rad53 activation and significantly increased DNA damage sensitivity. Simultaneous inactivation of both FHA domains abolished Rad53 activation and all downstream functions and dramatically increased the sensitivity to DNA damage and replication blocks similar to kinase-defective and rad53 null alleles, but did not compromise the essential viability function of Rad53. Interestingly, in G2/M synchronized cells, mutation of either FHA domain prevented Rad53 activation and impaired the cell cycle arrest checkpoint. Our data demonstrate that both FHA domains are required for normal Rad53 functions and indicate that the two FHA domains have differential but partially overlapping roles in Rad53 activation and downstream signaling.     

Diminished S-phase cyclin-dependent kinase function elicits vital Rad53-dependent checkpoint responses in Saccharomyces cerevisiae

Cyclin-dependent kinase (CDK) is required for the initiation of chromosomal DNA replication in eukaryotes. In Saccharomyces cerevisiae, the Clb5 and Clb6 cyclins activate Cdk1 and drive replication origin firing. Deletion of CLB5 reduces initiation of DNA synthesis from late-firing origins. We have examined whether checkpoints are activated by loss of Clb5 function and whether checkpoints are responsible for the DNA replication defects associated with loss of Clb5 function. We present evidence for activation of Rad53 and Ddc2 functions with characteristics suggesting the presence of DNA damage. Deficient late origin firing in clb5Delta cells is not due to checkpoint regulation, but instead, directly reflects the decreased abundance of S-phase CDK, as Clb6 activates late origins when its dosage is increased. Moreover, the viability of clb5Delta cells depends on Rad53. Activation of Rad53 by either Mrc1 or Rad9 contributes to the survival of clb5Delta cells, suggesting that both DNA replication and damage pathways are responsive to the decreased origin usage. These results suggest that reduced origin usage leads to stress or DNA damage at replication forks, necessitating the function of Rad53 in fork stabilization. Consistent with the notion that decreased S-CDK function creates stress at replication forks, deletion of RRM3 helicase, which facilitates replisome progression, greatly diminished the growth of clb5Delta cells. Together, our findings indicate that deregulation of S-CDK function has the potential to exacerbate genomic instability by reducing replication origin usage.     

Activation of the checkpoint kinase Rad53 by the phosphatidyl inositol kinase-like kinase Mec1

Saccharomyces cerevisiae Rad53, the ortholog of mammalian Chk2, is an essential protein kinase in DNA damage and DNA replication checkpoint pathways. Consecutive phosphatidyl inositol kinase-like kinase (PIKK)-dependent and PIKK-independent steps in activation of Rad53 are key steps for controlling and transmitting diverse downstream responses to DNA damage. However, these activities have not been demonstrated in vitro in defined systems. Here, we have shown that enzymatically dephosphorylated purified Rad53 autoactivates in vitro through a phosphorylation-dependent mechanism. Kinetic analysis demonstrated that autophosphorylation results in a more than 9-fold increase in protein kinase activity. Autophosphorylation was Rad53 concentration-dependent, indicating that the reaction follows an intermolecular mechanism. DNA damage induced oligomerization of a subset of Rad53 molecules in vivo. At low concentrations of Rad53, preincubation of Rad53 with immune complexes containing the Mec1/Ddc2 complex can activate Rad53 kinase activity. Our findings showed that Mec1/Ddc2 complexes can directly activate Rad53 through a phosphorylation-dependent mechanism, and more generally, supported the hypothesis that PIKKs regulate Chk2 orthologs through phosphorylation. Moreover, this work has substantiated a model for PIKK-independent amplification of Rad53 activation (and by extension, activation of other Chk2 orthologs) mediated by inter-Rad53 phosphorylation.     

Distinct phosphatases mediate the deactivation of the DNA damage checkpoint kinase Rad53

The DNA damage checkpoint regulates DNA replication and arrests cell cycle progression in response to genotoxic stress. In Saccharomyces cerevisiae, the protein kinase Rad53 plays a central role in preventing genomic instability and maintaining viability in the presence of replication stress and DNA damage. Activation of Rad53 depends on phosphorylation by the upstream kinase Mec1, followed by autophosphorylation on multiple residues. Also critical for cell viability, the molecular mechanism of Rad53 deactivation remains incompletely understood. Rad53 dephosphorylation after repair of a persistent double strand break in G(2)/M has been shown to depend on the presence of the PP2C-type phosphatases Ptc2 and Ptc3. More recently, the PP2A-like protein phosphatase Pph3 has been shown to be required to dephosphorylate Rad53 after DNA methylation damage in S phase. However, we show here that Ptc2/3 are dispensable for Rad53 deactivation after replication stress or DNA methylation damage. Pph3 is also dispensable for the deactivation of Rad53 after replication stress. In addition, Rad53 kinase activity is still deactivated in pph3 null cells after DNA methylation damage, despite persistent Rad53 hyperphosphorylation. Finally, a strain in which the three phosphatases are deleted shows a severe defect in Rad53 kinase deactivation after DNA methylation damage but not after replication stress. In all, our results suggest that distinct phosphatases operate to return Rad53 to its basal state after different genotoxic stresses and that a yet unidentified phosphatase may be responsible for the deactivation of Rad53 after replication stress.     

Mechanisms of checkpoint kinase Rad53 inactivation after a double-strand break in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, double-strand breaks (DSBs) activate DNA checkpoint pathways that trigger several responses including a strong G(2)/M arrest. We have previously provided evidence that the phosphatases Ptc2 and Ptc3 of the protein phosphatase 2C type are required for DNA checkpoint inactivation after a DSB and probably dephosphorylate the checkpoint kinase Rad53. In this article we have investigated further the interactions between Ptc2 and Rad53. We showed that forkhead-associated domain 1 (FHA1) of Rad53 interacts with a specific threonine of Ptc2, T376, located outside its catalytic domain in a TXXD motif which constitutes an optimal FHA1 binding sequence in vitro. Mutating T376 abolishes Ptc2 interaction with the Rad53 FHA1 domain and results in adaptation and recovery defects following a DSB. We found that Ckb1 and Ckb2, the regulatory subunits of the protein kinase CK2, are necessary for the in vivo interaction between Ptc2 and the Rad53 FHA1 domain, that Ckb1 binds Ptc2 in vitro and that ckb1Delta and ckb2Delta mutants are defective in adaptation and recovery after a DSB. Our data thus strongly suggest that CK2 is the kinase responsible for the in vivo phosphorylation of Ptc2 T376.     

Cds1 phosphorylation by Rad3-Rad26 kinase is mediated by forkhead-associated domain interaction with Mrc1

The protein kinase Cds1 is an effector of the replication checkpoint in the fission yeast Schizosaccharomyces pombe. Cds1 is required to stabilize stalled replication forks, and it helps to prevent the onset of mitosis until the genome is fully replicated. Mrc1 (mediator of the replication checkpoint-1) and Rad3-Rad26 kinase are required for Cds1 activation, but exactly how Mrc1 mediates Cds1 activation is unknown. Here we show that Mrc1 is required for the initial threonine 11 phosphorylation of Cds1 by Rad3-Rad26. Mrc1 specifically interacts with the forkhead-associated (FHA) domain of Cds1 in yeast two-hybrid assays. Mutations in the FHA domain that abolish this interaction also eliminate Thr-11 phosphorylation of Cds1. Weak Thr-11 phosphorylation of a "kinase-dead" mutant of Cds1 is rescued by co-expression of wild type Cds1. The requirement for Mrc1 in the replication checkpoint can be partially eliminated by expression of a Rad26-Cds1 fusion protein. These findings suggest that recognition of Mrc1 by the FHA domain of Cds1 serves to recruit Cds1 to Rad3-Rad26. This interaction mediates the initial Thr-11 phosphorylation of Cds1 by Rad3-Rad26 with subsequent intermolecular phosphorylation events leading to full activation of Cds1.     

Remodelling the Rad9 checkpoint complex: preparing Rad53 for action

DNA damage checkpoints are signal transduction pathways that are activated after genotoxic insults to protect genomic integrity. The Rad9 protein functions in the DNA damage checkpoint pathway in Saccharomyces cerevisiae and is essential for the Mec1-dependent activation of the effector kinase Rad53. We recently described the purification of two soluble distinct Rad9 complexes. The large 850 kDa complex consists of hypophosphorylated Rad9 and the chaperone proteins Ssa1/2. This complex is found both in undamaged cells as well as in cells treated with DNA damaging agents. The smaller 560 kDa complex contains hyperphosphorylated Rad9, Ssa1/2 and, in addition, Rad53. This complex forms only in cells with compromised DNA integrity. Once bound to the smaller complex, Rad53 can be activated by in trans autophosphorylation. Here, we propose a model in which the large Rad9 complex is remodelled after a genomic insult by chaperone activity to a smaller Rad53 activating complex.     

Dissection of Rad9 BRCT domain function in the mitotic checkpoint response to telomere uncapping

In Saccharomyces cerevisiae, destabilizing telomeres, via inactivation of telomeric repeat binding factor Cdc13, induces a cell cycle checkpoint that arrests cells at the metaphase to anaphase transition—much like the response to an unrepaired DNA double strand break (DSB). Throughout the cell cycle, the multi-domain adaptor protein Rad9 is required for the activation of checkpoint effector kinase Rad53 in response to DSBs and is similarly necessary for checkpoint signaling in response to telomere uncapping. Rad53 activation in G1 and S phase depends on Rad9 association with modified chromatin adjacent to DSBs, which is mediated by Tudor domains binding histone H3 di-methylated at K79 and BRCT domains to histone H2A phosphorylated at S129. Nonetheless, Rad9 Tudor or BRCT mutants can initiate a checkpoint response to DNA damage in nocodazole-treated cells. Mutations affecting di-methylation of H3 K79, or its recognition by Rad9 enhance 5′ strand resection upon telomere uncapping, and potentially implicate Rad9 chromatin binding in the checkpoint response to telomere uncapping. Indeed, we report that Rad9 binds to sub-telomeric chromatin, upon telomere uncapping, up to 10 kb from the telomere. Rad9 binding occurred within 30 min after inactivating Cdc13, preceding Rad53 phosphorylation. In turn, Rad9 Tudor and BRCT domain mutations blocked chromatin binding and led to attenuated checkpoint signaling as evidenced by decreased Rad53 phosphorylation and impaired cell cycle arrest. Our work identifies a role for Rad9 chromatin association, during mitosis, in the DNA damage checkpoint response to telomere uncapping, suggesting that chromatin binding may be an initiating event for checkpoints throughout the cell cycle.     

FHA domain-mediated DNA checkpoint regulation of Rad53

Saccharomyces cerevisiae Rad53 is a protein kinase central to the DNA damage and DNA replication checkpoint signaling pathways. In addition to its catalytic domain, Rad53 contains two forkhead homology-associated (FHA) domains (FHA1 and FHA2), which are phosphopeptide binding domains. The Rad53 FHA domains are proposed to mediate the interaction of Rad53 with both upstream and downstream branches of the DNA checkpoint signaling pathways. Here we show that concurrent mutation of Rad53 FHA1 and FHA2 causes DNA checkpoint defects approaching that of inactivation or loss of RAD53 itself. Both FHA1 and FHA2 are required for the robust activation of Rad53 by the RAD9-dependent DNA damage checkpoint pathway, while an intact FHA1 or FHA2 allows the activation of Rad53 in response to replication block. Mutation of Rad53 FHA1 causes the persistent activation of the RAD9-dependent DNA damage checkpoint pathway in response to replicational stress, suggesting that the RAD53-dependent stabilization of stalled replication forks functions through FHA1. Rad53 FHA1 is also required for the phosphorylation-dependent association of Rad53 with the chromatin assembly factor Asf1, although Asf1 itself is apparently not required for the prevention of DNA damage in response to replication block.     

Role of c-Abl kinase in DNA mismatch repair-dependent G2 cell cycle checkpoint arrest responses

Current published data suggest that DNA mismatch repair (MMR) triggers prolonged G(2) cell cycle checkpoint arrest after alkylation damage from N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) by activating ATR (ataxia telangiectasia-Rad3-related kinase). However, analyses of isogenic MMR-proficient and MMR-deficient human RKO colon cancer cells revealed that although ATR/Chk1 signaling controlled G(2) arrest in MMR-deficient cells, ATR/Chk1 activation was not involved in MMR-dependent G(2) arrest. Instead, we discovered that disrupting c-Abl activity using STI571 (Gleevec, a c-Abl inhibitor) or stable c-Abl knockdown abolished MMR-dependent p73alpha stabilization, induction of GADD45alpha protein expression, and G(2) arrest. In addition, inhibition of c-Abl also increased the survival of MNNG-exposed MMR-proficient cells to a level comparable with MMR-deficient cells. Furthermore, knocking down GADD45alpha (but not p73alpha) protein levels affected MMR-dependent G(2) arrest responses. Thus, MMR-dependent G(2) arrest responses triggered by MNNG are dependent on a human MLH1/c-Abl/GADD45alpha signaling pathway and activity. Furthermore, our data suggest that caution should be taken with therapies targeting c-Abl kinase because increased survival of mutator phenotypes may be an unwanted consequence.     

Oxidative stress and cell cycle checkpoint function

Oxidative stress and the damage that results from it have been implicated in a wide number of disease processes including atherosclerosis, autoimmune disorders, neuronal degeneration, and cancer. Reactive oxygen species (ROS) are ubiquitous and occur naturally in all aerobic species, coming from both exogenous and endogenous sources. ROS are quite reactive and readily damage biological molecules, including DNA. While the damaging effects of ROS on DNA have been intensively studied, the effects of oxidative damage on cell cycle checkpoint function have not. Here will we review several biologically important ROS and their sources, the cell cycle, checkpoints, and current knowledge about the effects of ROS on initiating checkpoint responses.     

Gain of function properties of mutant p53 proteins at the mitotic spindle cell cycle checkpoint

Mutations in the p53 tumor suppressor gene locus predispose human cells to chromosomal instability. This is due in part to interference of mutant p53 proteins with the activity of the mitotic spindle and postmitotic cell cycle checkpoints. Recent data demonstrates that wild type p53 is required for postmitotic checkpoint activity, but plays no role at the mitotic spindle checkpoint. Likewise, structural dominant p53 mutants demonstrate gain-of-function properties at the mitotic spindle checkpoint and dominant negative properties at the postmitotic checkpoint. At mitosis, mutant p53 proteins interfere with the control of the metaphase-to-anaphase progression by up-regulating the expression of CKs1, a protein that mediates activatory phosphorylation of the anaphase promoting complex (APC) by Cdc2. Cells that carry mutant p53 proteins overexpress CKs1 and are unable to sustain APC inactivation and mitotic arrest. Thus, mutant p53 gain-of-function at mitosis constitutes a key component to the origin of chromosomal instability in mutant p53 cells.