Genetic interaction of RAD53 protein kinase with histones is important for DNA replication

Studies in budding yeast suggest the protein kinase Rad53 plays novel roles in controlling initiation of DNA replication and in maintaining cellular histone levels, and these roles are independent of Rad53-mediated regulation of the checkpoint and of nucleotide levels. In order to elucidate the role of Rad53 in replication initiation, we isolated a novel allele of RAD53, rad53-rep,that separates the checkpoint function of RAD53 from the DNA replication function. rad53-rep mutants display a chromosome loss phenotype that is suppressed by increased origin dosage, providing further evidence that Rad53 plays a role in the initiation of DNA replication. Deletion of the major histone H3-H4 pair suppresses rad53-rep-cdc7-1 synthetic lethality, suggesting Rad53's functions in degradation of excess cellular histone and in replication initiation are related. Rad53-rep is active as a protein kinase yet fails to interact with origins of replication and like the rad53D mutant, the rad53-rep mutant accumulates excess soluble histones, and it is sensitive to histone dosage. In contrast, a checkpoint defective allele of RAD53 with mutations in both FHA domains, binds origins, and growth of a rad53-FHA mutant is unaffected by histone dosage. Based on these observations, we hypothesize that the origin binding and the histone degradation activities of Rad53 are central to its function in DNA replication and are independent of its checkpoint functions. We propose a model in which Rad53 acts as a "nucleosome buffer," interacting with origins of replication to prevent the binding of excess histones to origin DNA and to maintain proper chromatin configuration.     

Genetic interaction of RAD53 protein kinase with histones is important for DNA replication

Studies in budding yeast suggest the protein kinase Rad53 plays novel roles in controlling initiation of DNA replication and in maintaining cellular histone levels, and these roles are independent of Rad53-mediated regulation of the checkpoint and of nucleotide levels. In order to elucidate the role of Rad53 in replication initiation, we isolated a novel allele of RAD53, rad53-rep, that separates the checkpoint function of RAD53 from the DNA replication function. rad53-rep mutants display a chromosome loss phenotype that is suppressed by increased origin dosage, providing further evidence that Rad53 plays a role in the initiation of DNA replication. Deletion of the major histone H3–H4 pair suppresses rad53-rep-cdc7-1 synthetic lethality, suggesting Rad53''s functions in degradation of excess cellular histone and in replication initiation are related. Rad53-rep is active as a protein kinase yet fails to interact with origins of replication and like the rad53Δ mutant, the rad53-rep mutant accumulates excess soluble histones, and it is sensitive to histone dosage. In contrast, a checkpoint defective allele of RAD53 with mutations in both FHA domains, binds origins and growth of this mutant is unaffected by histone dosage. Based on these observations, we hypothesize that the origin binding and the histone degradation activities of Rad53 are central to its function in DNA replication and are independent of its checkpoint functions. We propose a model in which Rad53 acts as a “nucleosome buffer”, interacting with origins of replication to prevent the binding of excess histones to origin DNA and to maintain proper chromatin configuration.Key words: DNA replication, Rad53, histones, checkpoint, origins of replication     

DDK phosphorylates checkpoint clamp component Rad9 and promotes its release from damaged chromatin

When inappropriate DNA structures arise, they are sensed by DNA structure-dependent checkpoint pathways and subsequently repaired. Recruitment of checkpoint proteins to such structures precedes recruitment of proteins involved in DNA metabolism. Thus, checkpoints can regulate DNA metabolism. We show that fission yeast Rad9, a 9-1-1 heterotrimeric checkpoint-clamp component, is phosphorylated by Hsk1(Cdc7), the Schizosaccharomyces pombe?Dbf4-dependent kinase (DDK) homolog, in response to replication-induced DNA damage. Phosphorylation of Rad9 disrupts its interaction with replication protein A (RPA) and is dependent on 9-1-1 chromatin loading, the Rad9-associated protein Rad4/Cut5(TopBP1), and prior phosphorylation by Rad3(ATR). rad9 mutants defective in DDK phosphorylation show wild-type checkpoint responses but abnormal DNA repair protein foci and decreased viability after replication stress. We propose that Rad9 phosphorylation by DDK releases Rad9 from DNA damage sites to facilitate DNA repair.     

The Dbf4 motif C zinc finger promotes DNA replication and mediates resistance to genotoxic stress

The Dbf4/Cdc7 kinase (DDK) plays an essential role in stimulating DNA replication by phosphorylating subunits of the Mcm2-7 helicase complex at origins. This kinase complex is itself phosphorylated and removed from chromatin in a Rad53-dependent manner when an S phase checkpoint is triggered. Comparison of Dbf4 sequence across a variety of eukaryotic species has revealed three conserved regions that have been termed motifs N, M and C. The most highly conserved of the three, motif C, encodes a zinc finger, which are known to mediate protein-protein and protein-DNA interactions. Mutation of conserved motif C cysteines and histidines disrupted the association of Dbf4 with ARS1 origin DNA and Mcm2, but not other known ligands including Cdc7, Rad53 or the origin recognition complex subunit Orc2. Furthermore, these mutations impaired the ability of Dbf4 to phosphorylate Mcm2. Budding yeast strains for which the single genomic DBF4 copy was replaced with these motif C mutant alleles were compromised for entry into and progression through S phase, indicating that the observed weakening of the Mcm2 interaction prevents DDK from efficiently stimulating the initiation of DNA replication. Following initiation, Mcm2-7 migrates with the replication fork. Interestingly, the motif C mutants were sensitive to long-term, but not short-term exposure to the genotoxic agents hydroxyurea and methyl methanesulfonate. These results support a model whereby DDK interaction with Mcm2 is important to stabilize and/or restart replication forks during conditions where a prolonged S-phase checkpoint is triggered.     

Saccharomyces cerevisiae Dbf4 has unique fold necessary for interaction with Rad53 kinase

Dbf4 is a conserved eukaryotic protein that functions as the regulatory subunit of the Dbf4-dependent kinase (DDK) complex. DDK plays essential roles in DNA replication initiation and checkpoint activation. During the replication checkpoint, Saccharomyces cerevisiae Dbf4 is phosphorylated in a Rad53-dependent manner, and this, in turn, inhibits initiation of replication at late origins. We have determined the minimal region of Dbf4 required for the interaction with the checkpoint kinase Rad53 and solved its crystal structure. The core of this fragment of Dbf4 folds as a BRCT domain, but it includes an additional N-terminal helix unique to Dbf4. Mutation of the residues that anchor this helix to the domain core abolish the interaction between Dbf4 and Rad53, indicating that this helix is an integral element of the domain. The structure also reveals that previously characterized Dbf4 mutants with checkpoint phenotypes destabilize the domain, indicating that its structural integrity is essential for the interaction with Rad53. Collectively, these results allow us to propose a model for the association between Dbf4 and Rad53.     

Analysis of Rad3 and Chk1 protein kinases defines different checkpoint responses.

Eukaryotic cells respond to DNA damage and S phase replication blocks by arresting cell-cycle progression through the DNA structure checkpoint pathways. In Schizosaccharomyces pombe, the Chk1 kinase is essential for mitotic arrest and is phosphorylated after DNA damage. During S phase, the Cds1 kinase is activated in response to DNA damage and DNA replication blocks. The response of both Chk1 and Cds1 requires the six 'checkpoint Rad' proteins (Rad1, Rad3, Rad9, Rad17, Rad26 and Hus1). We demonstrate that DNA damage-dependent phosphorylation of Chk1 is also cell-cycle specific, occurring primarily in late S phase and G2, but not during M/G1 or early S phase. We have also isolated and characterized a temperature-sensitive allele of rad3. Rad3 functions differently depending on which checkpoint pathway is activated. Following DNA damage, rad3 is required to initiate but not maintain the Chk1 response. When DNA replication is inhibited, rad3 is required for both initiation and maintenance of the Cds1 response. We have identified a strong genetic interaction between rad3 and cds1, and biochemical evidence shows a physical interaction is possible between Rad3 and Cds1, and between Rad3 and Chk1 in vitro. Together, our results highlight the cell-cycle specificity of the DNA structure-dependent checkpoint response and identify distinct roles for Rad3 in the different checkpoint responses. Keywords: ATM/ATR/cell-cycle checkpoints/Chk1/Rad3     

Phosphopeptide binding by Sld3 links Dbf4‐dependent kinase to MCM replicative helicase activation

The initiation of eukaryotic DNA replication requires the assembly of active CMG (Cdc45‐MCM‐GINS) helicases at replication origins by a set of conserved and essential firing factors. This process is controlled during the cell cycle by cyclin‐dependent kinase (CDK) and Dbf4‐dependent kinase (DDK), and in response to DNA damage by the checkpoint kinase Rad53/Chk1. Here we show that Sld3, previously shown to be an essential CDK and Rad53 substrate, is recruited to the inactive MCM double hexamer in a DDK‐dependent manner. Sld3 binds specifically to DDK‐phosphorylated peptides from two MCM subunits (Mcm4, 6) and then recruits Cdc45. MCM mutants that cannot bind Sld3 or Sld3 mutants that cannot bind phospho‐MCM or Cdc45 do not support replication. Moreover, phosphomimicking mutants in Mcm4 and Mcm6 bind Sld3 without DDK and facilitate DDK‐independent replication. Thus, Sld3 is an essential “reader” of DDK phosphorylation, integrating signals from three distinct protein kinase pathways to coordinate DNA replication during S phase.     

Dbf4 is direct downstream target of ataxia telangiectasia mutated (ATM) and ataxia telangiectasia and Rad3-related (ATR) protein to regulate intra-S-phase checkpoint

Dbf4/Cdc7 (Dbf4-dependent kinase (DDK)) is activated at the onset of S-phase, and its kinase activity is required for DNA replication initiation from each origin. We showed that DDK is an important target for the S-phase checkpoint in mammalian cells to suppress replication initiation and to protect replication forks. We demonstrated that ataxia telangiectasia mutated (ATM) and ataxia telangiectasia and Rad3-related (ATR) proteins directly phosphorylate Dbf4 in response to ionizing radiation and replication stress. We identified novel ATM/ATR phosphorylation sites on Dbf4 and showed that ATM/ATR-mediated phosphorylation of Dbf4 is critical for the intra-S-phase checkpoint to inhibit DNA replication. The kinase activity of DDK, which is not suppressed upon DNA damage, is required for fork protection under replication stress. We further demonstrated that ATM/ATR-mediated phosphorylation of Dbf4 is important for preventing DNA rereplication upon loss of replication licensing through the activation of the S-phase checkpoint. These studies indicate that DDK is a direct substrate of ATM and ATR to mediate the intra-S-phase checkpoint in mammalian cells.     

RAD53 regulates DBF4 independently of checkpoint function in Saccharomyces cerevisiae

The Cdc7p and Dbf4p proteins form an active kinase complex in Saccharomyces cerevisiae that is essential for the initiation of DNA replication. A genetic screen for mutations that are lethal in combination with cdc7-1 led to the isolation of seven lsd (lethal with seven defect) complementation groups. The lsd7 complementation group contained two temperature-sensitive dbf4 alleles. The lsd1 complementation group contained a new allele of RAD53, which was designated rad53-31. RAD53 encodes an essential protein kinase that is required for the activation of DNA damage and DNA replication checkpoint pathways, and that is implicated as a positive regulator of S phase. Unlike other RAD53 alleles, we demonstrate that the rad53-31 allele retains an intact checkpoint function. Thus, the checkpoint function and the DNA replication function of RAD53 can be functionally separated. The activation of DNA replication through RAD53 most likely occurs through DBF4. Two-hybrid analysis indicates that the Rad53p protein binds to Dbf4p. Furthermore, the steady-state level of DBF4 message and Dbf4p protein is reduced in several rad53 mutant strains, indicating that RAD53 positively regulates DBF4. These results suggest that two different functions of the cell cycle, initiation of DNA replication and the checkpoint function, can be coordinately regulated through the common intermediate RAD53.     

Molecular Basis of the Essential S Phase Function of the Rad53 Checkpoint Kinase

The essential yeast kinases Mec1 and Rad53, or human ATR and Chk1, are crucial for checkpoint responses to exogenous genotoxic agents, but why they are also required for DNA replication in unperturbed cells remains poorly understood. Here we report that even in the absence of DNA-damaging agents, the rad53-4AQ mutant, lacking the N-terminal Mec1 phosphorylation site cluster, is synthetic lethal with a deletion of the RAD9 DNA damage checkpoint adaptor. This phenotype is caused by an inability of rad53-4AQ to activate the downstream kinase Dun1, which then leads to reduced basal deoxynucleoside triphosphate (dNTP) levels, spontaneous replication fork stalling, and constitutive activation of and dependence on S phase DNA damage checkpoints. Surprisingly, the kinase-deficient rad53-K227A mutant does not share these phenotypes but is rendered inviable by additional phosphosite mutations that prevent its binding to Dun1. The results demonstrate that ultralow Rad53 catalytic activity is sufficient for normal replication of undamaged chromosomes as long as it is targeted toward activation of the effector kinase Dun1. Our findings indicate that the essential S phase function of Rad53 is comprised by the combination of its role in regulating basal dNTP levels and its compensatory kinase function if dNTP levels are perturbed.     

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.     

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.     

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.     

Regulation of initiation of S phase, replication checkpoint signaling, and maintenance of mitotic chromosome structures during S phase by Hsk1 kinase in the fission yeast

Hsk1, Saccharomyces cerevisiae Cdc7-related kinase in Shizosaccharomyces pombe, is required for G1/S transition and its kinase activity is controlled by the regulatory subunit Dfp1/Him1. Analyses of a newly isolated temperature-sensitive mutant, hsk1-89, reveal that Hsk1 plays crucial roles in DNA replication checkpoint signaling and maintenance of proper chromatin structures during mitotic S phase through regulating the functions of Rad3 (ATM)-Cds1 and Rad21 (cohesin), respectively, in addition to expected essential roles for initiation of mitotic DNA replication through phosphorylating Cdc19 (Mcm2). Checkpoint defect in hsk1-89 is indicated by accumulation of cut cells at 30 degrees C. hsk1-89 displays synthetic lethality in combination with rad3 deletion, indicating that survival of hsk1-89 depends on Rad3-dependent checkpoint pathway. Cds1 kinase activation, which normally occurs in response to early S phase arrest by nucleotide deprivation, is largely impaired in hsk1-89. Furthermore, Cds1-dependent hyperphosphorylation of Dfp1 in response to hydroxyurea arrest is eliminated in hsk1-89, suggesting that sufficient activation of Hsk1-Dfp1 kinase is required for S phase entry and replication checkpoint signaling. hsk1-89 displays apparent defect in mitosis at 37 degrees C leading to accumulation of cells with near 2C DNA content and with aberrant nuclear structures. These phenotypes are similar to those of rad21-K1 and are significantly enhanced in a hsk1-89 rad21-K1 double mutant. Consistent with essential roles of Rad21 as a component for the cohesin complex, sister chromatid cohesion is partially impaired in hsk1-89, suggesting a possibility that infrequent origin firing of the mutant may affect the cohesin functions during S phase.     

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.     

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.     

Saccharomyces CDK1 Phosphorylates Rad53 Kinase in Metaphase, Influencing Cellular Morphogenesis

Rad53 is an essential protein kinase governing DNA damage and replication stress checkpoints in budding yeast. It also appears to be involved in cellular morphogenesis processes. Mass spectrometry analyses revealed that Rad53 is phosphorylated at multiple SQ/TQ and at SP/TP residues, which are typical consensus sites for phosphatidylinositol 3-kinase-related kinases and CDKs, respectively. Here we show that Clb-CDK1 phosphorylates Rad53 at Ser⁷⁷⁴ in metaphase. This phosphorylation event does not influence the DNA damage and replication checkpoint roles of Rad53, and it is independent of the spindle assembly checkpoint network. Moreover, the Ser-to-Asp mutation, mimicking a constitutive phosphorylation state at site 774, causes sensitivity to calcofluor, supporting a functional linkage between Rad53 and cellular morphogenesis.