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.     

Rad4TopBP1, a scaffold protein, plays separate roles in DNA damage and replication checkpoints and DNA replication

Rad4TopBP1, a BRCT domain protein, is required for both DNA replication and checkpoint responses. Little is known about how the multiple roles of Rad4TopBP1 are coordinated in maintaining genome integrity. We show here that Rad4TopBP1 of fission yeast physically interacts with the checkpoint sensor proteins, the replicative DNA polymerases, and a WD-repeat protein, Crb3. We identified four novel mutants to investigate how Rad4TopBP1 could have multiple roles in maintaining genomic integrity. A novel mutation in the third BRCT domain of rad4+TopBP1 abolishes DNA damage checkpoint response, but not DNA replication, replication checkpoint, and cell cycle progression. This mutant protein is able to associate with all three replicative polymerases and checkpoint proteins Rad3ATR-Rad26ATRIP, Hus1, Rad9, and Rad17 but has a compromised association with Crb3. Furthermore, the damaged-induced Rad9 phosphorylation is significantly reduced in this rad4TopBP1 mutant. Genetic and biochemical analyses suggest that Crb3 has a role in the maintenance of DNA damage checkpoint and influences the Rad4TopBP1 damage checkpoint function. Taken together, our data suggest that Rad4TopBP1 provides a scaffold to a large complex containing checkpoint and replication proteins thereby separately enforcing checkpoint responses to DNA damage and replication perturbations during the cell cycle.     

The phosphorylation network for efficient activation of the DNA replication checkpoint in fission yeast

Protein phosphorylation is the hallmark of checkpoint activation. Hundreds of targets of checkpoint kinases have been identified recently by genome-wide investigations. However, the complete picture of a phosphorylation network required for activation of a checkpoint pathway has not been available. The DNA replication checkpoint in Schizosaccharomyces pombe contains two major protein kinases, the sensor kinase Rad3 and the effector kinase Cds1, with the latter mediating most of the checkpoint functions. We show here that when DNA replication is arrested, efficient activation of Cds1 requires five phosphorylations that cooperate in a parallel or a sequential manner. Phosphorylation of a threonine residue (Thr(11)) in Cds1 by Rad3 occurs at a basal level in the absence of three other parallel Rad3-dependent phosphorylations on the mediator Mrc1 and Rad9 in the checkpoint clamp complex. However, the three parallel Rad3-dependent phosphorylations are all required for efficient phosphorylation of Thr(11) in Cds1 by Rad3. Phosphorylation of Thr(11) has been shown previously to promote autophosphorylation of Thr(328) in the kinase domain of Cds1, which directly activates the enzyme, leading to full activation of the checkpoint pathway. Interestingly, phosphorylation of Mrc1 by Rad3 does not require the phosphorylation of Rad9, suggesting that activation of the sensor kinase Rad3 in the replication checkpoint of fission yeast may involve a different mechanism.     

Basis for the Checkpoint Signal Specificity That Regulates Chk1 and Cds1 Protein Kinases

Six checkpoint Rad proteins (Rad1, Rad3, Rad9, Rad17, Rad26, and Hus1) are needed to regulate checkpoint protein kinases Chk1 and Cds1 in fission yeast. Chk1 is required to prevent mitosis when DNA is damaged by ionizing radiation (IR), whereas either kinase is sufficient to prevent mitosis when DNA replication is inhibited by hydroxyurea (HU). Checkpoint Rad proteins are required for IR-induced phosphorylation of Chk1 and HU-induced activation of Cds1. IR activates Cds1 only during the DNA synthesis (S) phase, whereas HU induces Chk1 phosphorylation only in cds1 mutants. Here, we investigate the basis of the checkpoint signal specificity of Chk1 phosphorylation and Cds1 activation. We show that IR fails to induce Chk1 phosphorylation in HU-arrested cells. Release from the HU arrest following IR causes substantial Chk1 phosphorylation. These and other data indicate that Cds1 prevents Chk1 phosphorylation in HU-arrested cells, which suggests that Cds1 actively suppresses a repair process that leads to Chk1 phosphorylation. Cds1 becomes more highly concentrated in the nucleus only during the S phase of the cell cycle. This finding correlates with S-phase specificity of IR-induced activation of Cds1. However, constitutive nuclear localization of Cds1 does not enhance IR-induced activation of Cds1. This result suggests that Cds1 activation requires DNA structures or protein activities that are present only during S phase. These findings help to explain how Chk1 and Cds1 respond to different checkpoint signals.     

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     

Activation of Rad53 kinase in response to DNA damage and its effect in modulating phosphorylation of the lagging strand DNA polymerase

The Saccharomyces cerevisiae Rad53 protein kinase is required for the execution of checkpoint arrest at multiple stages of the cell cycle. We found that Rad53 autophosphorylation activity depends on in trans phosphorylation mediated by Mec1 and does not require physical association with other proteins. Uncoupling in trans phosphorylation from autophosphorylation using a rad53 kinase-defective mutant results in a dominant-negative checkpoint defect. Activation of Rad53 in response to DNA damage in G(1) requires the Rad9, Mec3, Ddc1, Rad17 and Rad24 checkpoint factors, while this dependence is greatly reduced in S phase cells. Furthermore, during recovery from checkpoint activation, Rad53 activity decreases through a process that does not require protein synthesis. We also found that Rad53 modulates the lagging strand replication apparatus by controlling phosphorylation of the DNA polymerase alpha-primase complex in response to intra-S DNA damage.     

Fission yeast Rad26 is a regulatory subunit of the Rad3 checkpoint kinase

Fission yeast Rad3 is a member of a family of phosphoinositide 3-kinase -related kinases required for the maintenance of genomic stability in all eukaryotic cells. In fission yeast, Rad3 regulates the cell cycle arrest and recovery activities associated with the G2/M checkpoint. We have developed an assay that directly measures Rad3 kinase activity in cells expressing physiological levels of the protein. Using the assay, we demonstrate directly that Rad3 kinase activity is stimulated by checkpoint signals. Of the five other G2/M checkpoint proteins (Hus1, Rad1, Rad9, Rad17, and Rad26), only Rad26 was required for Rad3 kinase activity. Because Rad26 has previously been shown to interact constitutively with Rad3, our results demonstrate that Rad26 is a regulatory subunit, and Rad3 is the catalytic subunit, of the Rad3/Rad26 kinase complex. Analysis of Rad26/Rad3 kinase activation in rad26.T12, a mutant that is proficient for cell cycle arrest, but defective in recovery, suggests that these two responses to checkpoint signals require quantitatively different levels of kinase activity from the Rad3/Rad26 complex.     

Retention but not recruitment of Crb2 at double-strand breaks requires Rad1 and Rad3 complexes

The fission yeast checkpoint protein Crb2, related to budding yeast Rad9 and human 53BP1 and BRCA1, has been suggested to act as an adapter protein facilitating the phosphorylation of specific substrates by Rad3-Rad26 kinase. To further understand its role in checkpoint signaling, we examined its localization in live cells by using fluorescence microscopy. In response to DNA damage, Crb2 localizes to distinct nuclear foci, which represent sites of DNA double-strand breaks (DSBs). Crb2 colocalizes with Rad22 at persistent foci, suggesting that Crb2 is retained at sites of DNA damage during repair. Damage-induced Crb2 foci still form in cells defective in Rad1, Rad3, and Rad17 complexes, but these foci do not persist as long as in wild-type cells. Our results suggest that Crb2 functions at the sites of DNA damage, and its regulated persistent localization at damage sites may be involved in facilitating DNA repair and/or maintaining the checkpoint arrest while DNA repair is under way.     

Mec1p is essential for phosphorylation of the yeast DNA damage checkpoint protein Ddc1p, which physically interacts with Mec3p.

Checkpoints prevent DNA replication or nuclear division when chromosomes are damaged. The Saccharomyces cerevisiae DDC1 gene belongs to the RAD17, MEC3 and RAD24 epistasis group which, together with RAD9, is proposed to act at the beginning of the DNA damage checkpoint pathway. Ddc1p is periodically phosphorylated during unperturbed cell cycle and hyperphosphorylated in response to DNA damage. We demonstrate that Ddc1p interacts physically in vivo with Mec3p, and this interaction requires Rad17p. We also show that phosphorylation of Ddc1p depends on the key checkpoint protein Mec1p and also on Rad24p, Rad17p and Mec3p. This suggests that Mec1p might act together with the Rad24 group of proteins at an early step of the DNA damage checkpoint response. On the other hand, Ddc1p phosphorylation is independent of Rad53p and Rad9p. Moreover, while Ddc1p is required for Rad53p phosphorylation, it does not play any major role in the phosphorylation of the anaphase inhibitor Pds1p, which requires RAD9 and MEC1. We suggest that Rad9p and Ddc1p might function in separated branches of the DNA damage checkpoint pathway, playing different roles in determining Mec1p activity and/or substrate specificity.     

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.     

Saccharomyces cerevisiae Rad9 acts as a Mec1 adaptor to allow Rad53 activation

BACKGROUND: The DNA damage checkpoint is a protein kinase-based signaling system that detects and signals physical alterations in DNA. Despite having identified many components of this signaling cascade, the exact mechanisms by which checkpoint kinases are activated after DNA damage, as well as the role of the checkpoint mediators, remain poorly understood. RESULTS: To elucidate the mechanisms that underlie the MEC1 and RAD9-dependent activation of Rad53, the Saccharomyces cerevisiae ortholog of Chk2, we mapped and characterized in vivo phosphorylation sites present on Rad53 after DNA damage by mass spectrometry. We find that Rad53 requires for its activation multisite phosphorylation on a number of typical and atypical Mec1 phosphorylation sites, thus confirming that Rad53 is a direct target of Mec1, the mammalian ATR homolog. Moreover, by using biochemical reconstitution experiments, we demonstrate that efficient and direct phosphorylation of Rad53 by Mec1 is only observed in the presence of purified Rad9, the archetypal checkpoint mediator. We find that the stimulatory activity of Rad9 requires a phospho- and FHA-dependent interaction with Rad53, which allows Rad53 to be recognized as a substrate for Mec1. CONCLUSIONS: Our results indicate that Rad9 acts as a bona fide signaling adaptor that enables Rad53 phosphorylation by Mec1. Given the high degree of conservation of checkpoint signaling in eukaryotes, we propose that one of the critical functions of checkpoint mediators such as MDC1, 53BP1, or Brca1 is to act as PIKK adaptors during the DNA damage response.     

LCD1: an essential gene involved in checkpoint control and regulation of the MEC1 signalling pathway in Saccharomyces cerevisiae

We identified YDR499W as a Saccharomyces cerevisiae open reading frame with homology to several checkpoint proteins, including S. cerevisiae Rfc5p and Schizosaccharomyces pombe Rad26. Disruption of YDR499W (termed LCD1) results in lethality that is rescued by increasing cellular deoxyribonucleotide levels. Cells lacking LCD1 are very sensitive to a range of DNA-damaging agents, including UV irradiation, and to the inhibition of DNA replication. LCD1 is necessary for the phosphorylation and activation of Rad53p in response to DNA damage or DNA replication blocks, and for Chk1p activation in response to DNA damage. LCD1 is also required for efficient DNA damage-induced phosphorylation of Rad9p and for the association of Rad9p with the FHA2 domain of Rad53p after DNA damage. In addition, cells lacking LCD1 are completely defective in the G(1)/S and G(2)/M DNA damage checkpoints. Finally, we reveal that endogenous Mec1p co-immunoprecipitates with Lcd1p both before and after treatment with DNA-damaging agents. These results indicate that Lcd1p is a pivotal checkpoint regulator, involved in both the essential and checkpoint functions of the Mec1p pathway.     

Cdc18 enforces long-term maintenance of the S phase checkpoint by anchoring the Rad3-Rad26 complex to chromatin

DNA replication is initiated by recruitment of Cdc18 to origins. During S phase, CDK-dependent destruction of Cdc18 occurs. We show that when DNA replication stalls, Cdc18 persists in a chromatin-bound complex including the checkpoint kinases Rad3 and Rad26. Rad26 directly binds Cdc18 and is required for Rad3 recruitment to chromatin. Depletion of Cdc18 when DNA replication is stalled leads to release of Rad3 and Rad26 from chromatin and entry into an aberrant mitosis even though replication intermediates can still be detected. These findings indicate that Cdc18 plays a pivotal role in checkpoint maintenance by anchoring the Rad3-Rad26 complex to chromatin. Cdc18 persistence during DNA-replication arrest requires the S phase checkpoint that inhibits the S phase CDK. We propose that S phase arrest activates the S phase checkpoint blocking mitosis onset and inhibiting Cdc18 degradation, and that the stabilized Cdc18, in turn, anchors Rad3 to chromatin to ensure long-term checkpoint maintenance.     

Telomere binding of checkpoint sensor and DNA repair proteins contributes to maintenance of functional fission yeast telomeres

Telomeres, the ends of linear chromosomes, are DNA double-strand ends that do not trigger a cell cycle arrest and yet require checkpoint and DNA repair proteins for maintenance. Genetic and biochemical studies in the fission yeast Schizosaccharomyces pombe were undertaken to understand how checkpoint and DNA repair proteins contribute to telomere maintenance. On the basis of telomere lengths of mutant combinations of various checkpoint-related proteins (Rad1, Rad3, Rad9, Rad17, Rad26, Hus1, Crb2, Chk1, Cds1), Tel1, a telomere-binding protein (Taz1), and DNA repair proteins (Ku70, Rad32), we conclude that Rad3/Rad26 and Tel1/Rad32 represent two pathways required to maintain telomeres and prevent chromosome circularization. Rad1/Rad9/Hus1/Rad17 and Ku70 are two additional epistasis groups, which act in the Rad3/Rad26 pathway. However, Rad3/Rad26 must have additional target(s), as cells lacking Tel1/Rad32, Rad1/Rad9/Hus1/Rad17, and Ku70 groups did not circularize chromosomes. Cells lacking Rad3/Rad26 and Tel1/Rad32 senesced faster than a telomerase trt1Delta mutant, suggesting that these pathways may contribute to telomere protection. Deletion of taz1 did not suppress chromosome circularization in cells lacking Rad3/Rad26 and Tel1/Rad32, also suggesting that two pathways protect telomeres. Chromatin immunoprecipitation analyses found that Rad3, Rad1, Rad9, Hus1, Rad17, Rad32, and Ku70 associate with telomeres. Thus, checkpoint sensor and DNA repair proteins contribute to telomere maintenance and protection through their association with telomeres.     

Rad17 phosphorylation is required for claspin recruitment and Chk1 activation in response to replication stress

The ATR-mediated checkpoint is not only critical for responding to genotoxic stress but also essential for cell proliferation. The RFC-related checkpoint protein Rad17, a phosphorylation substrate of ATR, is critical for ATR-mediated checkpoint signaling and cell survival. Here, we show that phosphorylation of Rad17 by ATR is important for genomic stability and restraint of S phase but is not essential for cell survival. The phosphomutant Rad17AA exhibits distinct defects in hydroxyurea- (HU) and ultraviolet- (UV) induced Chk1 activation, indicating that separate Rad17 functions are required differently in response to different types of replication interference. Although cells expressing Rad17AA can initiate Chk1 phosphorylation after HU treatment, they fail to sustain Chk1 phosphorylation after withdrawal of HU and are profoundly sensitive to HU. Importantly, we found that phosphorylated Rad17 interacts with Claspin and regulates its phosphorylation. These findings reveal a phosphorylation-dependent function of Rad17 in an ATR-Rad17-Claspin-Chk1-signaling cascade that responds to specific replication stress.     

Phosphorylation of human Rad9 is required for genotoxin-activated checkpoint signaling

Rad9, a key component of genotoxin-activated checkpoint signaling pathways, associates with Hus1 and Rad1 in a heterotrimeric complex (the 9-1-1 complex). Rad9 is inducibly and constitutively phosphorylated. However, the role of Rad9 phosphorylation is unknown. Here we identified nine phosphorylation sites, all of which lie in the carboxyl-terminal 119-amino acid Rad9 tail and examined the role of phosphorylation in genotoxin-triggered checkpoint activation. Rad9 mutants lacking a Ser-272 phosphorylation site, which is phosphorylated in response to genotoxins, had no effect on survival or checkpoint activation in Mrad9-/- mouse ES cells treated with hydroxyurea (HU), ionizing radiation (IR), or ultraviolet radiation (UV). In contrast, additional Rad9 tail phosphorylation sites were essential for Chk1 activation following HU, IR, and UV treatment. Consistent with a role for Chk1 in S-phase arrest, HU- and UV-induced S-phase arrest was abrogated in the Rad9 phosphorylation mutants. In contrast, however, Rad9 did not play a role in IR-induced S-phase arrest. Clonogenic assays revealed that cells expressing a Rad9 mutant lacking phosphorylation sites were as sensitive as Rad9-/- cells to UV and HU. Although Rad9 contributed to survival of IR-treated cells, the identified phosphorylation sites only minimally contributed to survival following IR treatment. Collectively, these results demonstrate that the Rad9 phospho-tail is a key participant in the Chk1 activation pathway and point to additional roles for Rad9 in cellular responses to IR.     

Role of a Complex Containing Rad17, Mec3, and Ddc1 in the Yeast DNA Damage Checkpoint Pathway

Genetic analysis has suggested that RAD17, RAD24, MEC3, and DDC1 play similar roles in the DNA damage checkpoint control in budding yeast. These genes are required for DNA damage-induced Rad53 phosphorylation and considered to function upstream of RAD53 in the DNA damage checkpoint pathway. Here we identify Mec3 as a protein that associates with Rad17 in a two-hybrid screen and demonstrate that Rad17 and Mec3 interact physically in vivo. The amino terminus of Rad17 is required for its interaction with Mec3, and the protein encoded by the rad17-1 allele, containing a missense mutation at the amino terminus, is defective for its interaction with Mec3 in vivo. Ddc1 interacts physically and cosediments with both Rad17 and Mec3, indicating that these three proteins form a complex. On the other hand, Rad24 is not found to associate with Rad17, Mec3, and Ddc1. DDC1 overexpression can partially suppress the phenotypes of the rad24Δ mutation: sensitivity to DNA damage, defect in the DNA damage checkpoint and decrease in DNA damage-induced phosphorylation of Rad53. Taken together, our results suggest that Rad17, Mec3, and Ddc1 form a complex which functions downstream of Rad24 in the DNA damage checkpoint pathway.     

Rad9 phosphorylation sites couple Rad53 to the Saccharomyces cerevisiae DNA damage checkpoint

Rad9 is required for the MEC1/TEL1-dependent activation of Saccharomyces cerevisiae DNA damage checkpoint pathways mediated by Rad53 and Chk1. DNA damage induces Rad9 phosphorylation, and Rad53 specifically associates with phosphorylated Rad9. We report here that multiple Mec1/Tel1 consensus [S/T]Q sites within Rad9 are phosphorylated in response to DNA damage. These Rad9 phosphorylation sites are selectively required for activation of the Rad53 branch of the checkpoint pathway. Consistent with the in vivo function in recruiting Rad53, Rad9 phosphopeptides are bound by Rad53 forkhead-associated (FHA) domains in vitro. These data suggest that functionally independent domains within Rad9 regulate Rad53 and Chk1, and support the model that FHA domain-mediated recognition of Rad9 phosphopeptides couples Rad53 to the DNA damage checkpoint pathway.     

A Tel1/MRX-dependent checkpoint inhibits the metaphase-to-anaphase transition after UV irradiation in the absence of Mec1

In Saccharomyces cerevisiae, Mec1/ATR plays a primary role in sensing and transducing checkpoint signals in response to different types of DNA lesions, while the role of the Tel1/ATM kinase in DNA damage checkpoints is not as well defined. We found that UV irradiation in G(1) in the absence of Mec1 activates a Tel1/MRX-dependent checkpoint, which specifically inhibits the metaphase-to-anaphase transition. Activation of this checkpoint leads to phosphorylation of the downstream checkpoint kinases Rad53 and Chk1, which are required for Tel1-dependent cell cycle arrest, and their adaptor Rad9. The spindle assembly checkpoint protein Mad2 also partially contributes to the G(2)/M arrest of UV-irradiated mec1Delta cells independently of Rad53 phosphorylation and activation. The inability of UV-irradiated mec1Delta cells to undergo anaphase can be relieved by eliminating the anaphase inhibitor Pds1, whose phosphorylation and stabilization in these cells depend on Tel1, suggesting that Pds1 persistence may be responsible for the inability to undergo anaphase. Moreover, while UV irradiation can trigger Mec1-dependent Rad53 phosphorylation and activation in G(1)- and G(2)-arrested cells, Tel1-dependent checkpoint activation requires entry into S phase independently of the cell cycle phase at which cells are UV irradiated, and it is decreased when single-stranded DNA signaling is affected by the rfa1-t11 allele. This indicates that UV-damaged DNA molecules need to undergo structural changes in order to activate the Tel1-dependent checkpoint. Active Clb-cyclin-dependent kinase 1 (CDK1) complexes also participate in triggering this checkpoint and are required to maintain both Mec1- and Tel1-dependent Rad53 phosphorylation, suggesting that they may provide critical phosphorylation events in the DNA damage checkpoint cascade.     

Cdc5 blocks in vivo rad53 activity,but not in situ activity (ISA)

DNA damage promotes the activation of a signal transduction cascade referred to as the DNA damage checkpoint. This pathway initiates with the Mec1/ATR kinase, which then phosphorylates the Rad53/Chk2 kinase. Mec1 phosphorylation of Rad53 is then thought to promote Rad53 autophosphorylation, ultimately leading to a fully active Rad53 molecule that can go on to phosphorylate substrates important for DNA damage resistance. In the absence of DNA repair, this checkpoint is eventually downregulated in a Cdc5-dependent process referred to as checkpoint adaptation. Recently, we showed that overexpression of Cdc5 leads to checkpoint inactivation and loss of the strong electrophoretic shift associated with Rad53 inactivation. Interestingly, this same overexpression did not strongly inhibit Rad53 autophosphorylation activity as measured by the in situ assay (ISA). The ISA involves incubating the re-natured Rad53 protein with γ ³²P labeled ATP after electrophoresis and western blotting. Using a newly identified Rad53 target, we show that despite strong ISA activity, Rad53 does not maintain phosphorylation of this substrate. We hypothesize that, during adaptation, Rad53 may be in a unique state in which it maintains some Mec1 phosphorylation but does not have the auto-phosphorylations required for full activity towards exogenous substrates.Key words: DNA damage, checkpoint, adaptation, CDC5, RAD53, ISA