Cds1 is the effector serine/threonine protein kinase of the DNA replication (intra-S phase) checkpoint of the fission yeast Schizosaccharomyces pombe, and is the ortholog of human CHK2 and budding-yeast Rad53 (CHEK2 subfamily). The 460-residue protein comprises an N-terminal SQ/TQ regulatory motif and a forkhead-associated (FHA) domain followed by a C-terminal protein kinase domain and a short C-terminal autoinhibitory tail. Cds1 is activated specifically in S phase when replication forks stall (for example upon nucleotide depletion by hydroxyurea, or when forks encounter DNA damage): the apical kinase Rad3 (ATR), working through the mediator Mrc1 (Claspin), phosphorylates Cds1 at Thr11, which primes FHA-dependent dimerization and autophosphorylation of the activation-loop residue Thr328, yielding the active kinase. Active Cds1 enforces the S/M checkpoint by phosphorylating Cdc25 (creating 14-3-3/Rad24 binding sites and excluding it from the nucleus) and by activating Wee1/Mik1, thereby keeping Cdc2 (CDK1) inhibited and blocking mitosis until replication is complete. Cds1 also phosphorylates targets that stabilize and protect stalled forks and govern the replication-stress response, including Dna2, Mus81, the DDK kinase Hsk1/Dfp1, Rad60, Mcm4, the MBF repressor Nrm1, and Rgf1. A Cds1-dependent replication checkpoint also operates during meiosis, where Cds1 plays an even more prominent role than in the mitotic cycle. Cds1 acts in the nucleus at chromatin/stalled forks. It is distinct in function from Chk1, the G2 DNA-damage checkpoint effector kinase.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0005737 cytoplasm | IBA GO_REF:0000033 | KEEP AS NON CORE | Summary: Phylogenetic (IBA) assignment of cytoplasmic localization. Cds1 is an abundant soluble kinase, but its functionally relevant site of action is the nucleus, at chromatin/stalled replication forks. A diffuse cytoplasmic pool is plausible but is not where the checkpoint function is executed. Reason: General compartment supported by phylogeny and large-scale localization, but not the site of the core checkpoint function (nucleus). Supporting Evidence: PMID:16823372 ORFeome cloning and global analysis of protein localization in the fission yeast Schizosaccharomyces pombe. |
| GO:0004674 protein serine/threonine kinase activity | IBA GO_REF:0000033 | ACCEPT | Summary: Core molecular function. Cds1 is a CHEK2-subfamily Ser/Thr protein kinase; the phylogenetic assignment is strongly corroborated by direct biochemical assays of Cds1 kinase activity on substrates such as Wee1, MBP, Hsk1 and Dna2. Reason: Phylogenetic inference matches abundant direct experimental evidence for protein serine/threonine kinase activity. Supporting Evidence: PMID:7723827 This gene, named cds1+ (checking DNA synthesis), encodes a typical protein kinase. Here we report that this protein kinase is a key component of the DNA replication-monitoring S/G2 checkpoint system. |
| GO:0005634 nucleus | IBA GO_REF:0000033 | ACCEPT | Summary: Core localization. Cds1 acts in the nucleus on chromatin at stalled replication forks; the phylogenetic assignment is corroborated by direct IDA evidence and by its chromatin recruitment during checkpoint activation. Reason: Nucleus is the site where the replication-checkpoint kinase function is executed; supported by phylogeny and direct experimental localization. Supporting Evidence: PMID:24478943 Chromatin recruitment of Cds1 is enhanced by Pcf1 overproduction but is attenuated by the Ξpcf1 mutation. |
| GO:0044773 mitotic DNA damage checkpoint signaling | IBA GO_REF:0000033 | MODIFY | Summary: This broad term encompasses G1, S and G2 DNA-damage checkpoints. For Cds1 the well-supported, specific role is the mitotic intra-S DNA damage checkpoint (slowing S phase when forks encounter damage). Cds1 is NOT the G2 DNA-damage checkpoint effector (that is Chk1). The annotation is best replaced by the precise intra-S child term. Reason: Term is too general/imprecise; Cds1 functions specifically in the intra-S DNA damage checkpoint, not the G2 DNA-damage checkpoint. Proposed replacements: mitotic intra-S DNA damage checkpoint signaling Supporting Evidence: PMID:10521402 Unlike the checkpoint Rad products, Cds1 is not required in the mitotic DNA damage checkpoint PMID:19037101 upon treatment with the alkylating agent methyl methane sulfonate, cds1Delta mutants display a complete checkpoint defect. |
| GO:0004672 protein kinase activity | IEA GO_REF:0000002 | MODIFY | Summary: InterPro-based electronic annotation of the parent term protein kinase activity. Correct but less specific than the experimentally supported protein serine/threonine kinase activity child term. Reason: Parent term is correct but too general; the specific Ser/Thr kinase term is directly supported by experiment. Proposed replacements: protein serine/threonine kinase activity Supporting Evidence: PMID:19357077 phosphorylation of a highly conserved threonine residue (Thr 328 ) in the activation loop is the only covalent modification required for kinase activation in vitro and in vivo. |
| GO:0004674 protein serine/threonine kinase activity | IEA GO_REF:0000003 | ACCEPT | Summary: EC 2.7.11.1-based electronic assignment of protein serine/threonine kinase activity, consistent with the experimentally demonstrated core function. Reason: Correct molecular function, redundantly supported by direct experimental evidence. Supporting Evidence: PMID:16618806 Cds1 kinase assays were performed in standard kinase buffer (20 mM Tris:HCl at pH 7.5, 5 mM MgCl 2 , 1 mM DTT, 75 mM KCl, 50 ΞΌM [Ξ³- 32 P]ATP) with GST-Wee1(1β152 amino acids) |
| GO:0005524 ATP binding | IEA GO_REF:0000002 | ACCEPT | Summary: Core molecular function. Cds1 has a canonical protein kinase fold with a glycine-rich ATP-binding loop (UniProt BINDING 173-181, 196); ATP binding is required for its autoactivation and for catalysis. Reason: ATP binding is an intrinsic property of the kinase domain, supported by sequence features and by the ATP requirement for autoactivation. Supporting Evidence: PMID:16618806 autoactivation of the Cds1 fusion construct requires both dimerization and the presence of ATP. |
| GO:0005634 nucleus | IEA GO_REF:0000117 | ACCEPT | Summary: ARBA machine-learning electronic annotation of nuclear localization, consistent with the IDA/IBA-supported nuclear site of action. Reason: Redundant with directly supported nuclear localization. Supporting Evidence: PMID:24478943 Chromatin recruitment of Cds1 is enhanced by Pcf1 overproduction but is attenuated by the Ξpcf1 mutation. |
| GO:0106310 protein serine kinase activity | IEA GO_REF:0000116 | ACCEPT | Summary: RHEA/Rhea-based electronic assignment of protein serine kinase activity. Cds1 phosphorylates serine residues on substrates (e.g. Cdc25 S99/S192/S359, Dna2 S220); this is a correct, more granular sibling of the Ser/Thr term. Reason: Consistent with experimentally demonstrated serine phosphorylation of substrates. Supporting Evidence: PMID:22682245 the Cds1(Chk2) effector kinase targets Dna2 on S220 to regulate, both in vivo and in vitro, Dna2 association with stalled replication forks in chromatin. |
| GO:0005515 protein binding | IPI PMID:18682565 DNA replication checkpoint promotes G1-S transcription by in... | MARK AS OVER ANNOTATED | Summary: Uninformative "protein binding" capturing the Cds1-Nrm1 interaction. Nrm1 is a substrate that Cds1 phosphorylates to relieve MBF repression; the biologically informative annotations are the kinase activity and checkpoint process terms. Reason: Bare protein binding is uninformative per curation guidance; the interaction reflects a kinase-substrate relationship already captured by MF and BP terms. Supporting Evidence: PMID:18682565 Phosphorylation of Nrm1 by the Cds1 (Chk2) checkpoint protein kinase, which is activated in response to DNA replication stress, promotes its dissociation from the MBF transcription factor. |
| GO:0004674 protein serine/threonine kinase activity | EXP PMID:11027263 Schizosaccharomyces pombe Hsk1p is a potential cds1p target ... | ACCEPT | Summary: Core molecular function, supported by direct in vitro kinase assay: Hsk1 (DDK catalytic subunit) is a direct substrate of purified Cds1. Reason: Direct experimental demonstration of Cds1 protein kinase activity. Supporting Evidence: PMID:11027263 We demonstrate that Hsk1p undergoes Cds1p-dependent phosphorylation in response to HU and that it is a direct substrate of purified Cds1p kinase in vitro. |
| GO:0004674 protein serine/threonine kinase activity | IDA PMID:12960401 Intra-G1 arrest in response to UV irradiation in fission yea... | ACCEPT | Summary: Core molecular function, supported by IDA: Cds1 immunoprecipitate kinase activity assayed on MBP. Reason: Direct assay of Cds1 kinase activity. Supporting Evidence: PMID:12960401 Immunoprecipitates of Cds1 were used to measure Cds1 kinase activity, with myelin basic protein (MBP) as substrate |
| GO:0033315 meiotic G2/MI DNA replication checkpoint signaling | IMP PMID:10521402 Meiotic DNA replication checkpoint control in fission yeast. | ACCEPT | Summary: Core biological process in meiosis. Cds1 is absolutely required for the meiotic DNA replication checkpoint that blocks meiosis I when replication is incomplete, playing an even more prominent role than in the mitotic cycle. Reason: Strong genetic (IMP) evidence that Cds1 enforces the meiotic replication checkpoint. Supporting Evidence: PMID:10521402 the Cds1 protein kinase are required for the DNA replication checkpoint during meiosis, with Cds1 playing a more prominent role than it does during mitosis. |
| GO:0004674 protein serine/threonine kinase activity | IDA PMID:16618806 Two-stage mechanism for activation of the DNA replication ch... | ACCEPT | Summary: Core molecular function, supported by direct in vitro kinase assays of purified Cds1 on GST-Wee1 and MBP. Reason: Direct biochemical demonstration of Cds1 kinase activity. Supporting Evidence: PMID:16618806 Cds1 kinase assays were performed in standard kinase buffer (20 mM Tris:HCl at pH 7.5, 5 mM MgCl 2 , 1 mM DTT, 75 mM KCl, 50 ΞΌM [Ξ³- 32 P]ATP) with GST-Wee1(1β152 amino acids) |
| GO:0004674 protein serine/threonine kinase activity | IDA PMID:28806726 Replication fork slowing and stalling are distinct, checkpoi... | ACCEPT | Summary: Core molecular function. This single-molecule combing study of the intra-S checkpoint used cds1 and supports Cds1 as the effector kinase of the checkpoint; kinase activity is the relevant molecular function. Reason: Consistent with Cds1's well-established role as the effector kinase. Supporting Evidence: PMID:28806726 the checkpoint slows replication by inhibiting origin firing, but not by decreasing fork rates. However, the checkpoint appears to facilitate replication of damaged templates, allowing forks to more quickly pass lesions. |
| GO:0031573 mitotic intra-S DNA damage checkpoint signaling | IMP PMID:28806726 Replication fork slowing and stalling are distinct, checkpoi... | ACCEPT | Summary: Core biological process. Cds1 is the effector of the intra-S checkpoint that responds to DNA damage during replication; this study dissects how the checkpoint inhibits origin firing and facilitates replication past lesions. Reason: Genetic evidence that Cds1 mediates the intra-S DNA damage checkpoint response. Supporting Evidence: PMID:28806726 This slowing is orchestrated by the intra-S checkpoint and involves inhibition of origin firing and reduction of replication fork speed. |
| GO:0004674 protein serine/threonine kinase activity | IDA PMID:9774107 Replication checkpoint requires phosphorylation of the phosp... | ACCEPT | Summary: Core molecular function. Cds1 phosphorylates Cdc25 on S99/S192/S359 to promote 14-3-3 binding, demonstrating substrate phosphorylation by the kinase. Reason: Direct demonstration of Cds1 phosphorylating a physiological substrate. Supporting Evidence: PMID:9774107 both kinases phosphorylate Cdc25 on the same sites, which include serine residues at positions 99, 192 and 359. Mutation of these residues reduces binding of 14-3-3 proteins to Cdc25 in vitro and disrupts the replication checkpoint in vivo. |
| GO:0004674 protein serine/threonine kinase activity | IDA PMID:21084840 The Mek1 phosphorylation cascade plays a role in meiotic rec... | ACCEPT | Summary: Protein serine/threonine kinase activity. This paper is primarily about Mek1, a Cds1/Rad53-related meiotic kinase; it reviews Cds1 activation as background but its biochemistry is on Mek1. Cds1 Ser/Thr kinase activity is nonetheless extensively documented elsewhere, so the annotation is correct though weakly supported by this particular reference. Reason: Correct core molecular function; note the experimental focus of this reference is the paralog Mek1, so support here is background-level, but the Ser/Thr kinase activity of Cds1 is well established overall. Supporting Evidence: PMID:21084840 Cds1 and its orthologs in other species ( S. cerevisiae Rad53 and mammalian CHK2) are characterized by an N-terminal Ser-Gln/Thr-Gln (SQ/TQ) cluster and forkhead-associated (FHA) domain and a Ser-Thr kinase domain. |
| GO:0004674 protein serine/threonine kinase activity | IDA PMID:24478458 The checkpoint-dependent nuclear accumulation of Rho1p excha... | ACCEPT | Summary: Core molecular function. Nuclear accumulation of the RhoGEF Rgf1 under replication stress depends on Cds1, which phosphorylates Cds1-consensus serines in Rgf1, illustrating substrate phosphorylation by the kinase. Reason: Supports Cds1 kinase activity acting on a downstream substrate. Supporting Evidence: PMID:24478458 Rgf1p nuclear accumulation during replication arrest depends on the 14-3-3 chaperone Rad24p and the DNA replication checkpoint kinase Cds1p. |
| GO:0005634 nucleus | IDA PMID:24478943 Pcf1, a large subunit of CAF-1, required for maintenance of ... | ACCEPT | Summary: Core localization, supported by IDA: Cds1 is recruited to chromatin (in the nucleus) where its checkpoint activity is maintained. Reason: Direct evidence that Cds1 acts at chromatin in the nucleus. Supporting Evidence: PMID:24478943 Chromatin recruitment of Cds1 is enhanced by Pcf1 overproduction but is attenuated by the Ξpcf1 mutation. |
| GO:0004674 protein serine/threonine kinase activity | IDA PMID:11313465 Threonine-11, phosphorylated by Rad3 and atm in vitro, is re... | ACCEPT | Summary: Core molecular function. This study maps Rad3-dependent priming of Cds1 at Thr11 required for activation of the kinase; Cds1 kinase activation upon HU treatment is directly assayed. Reason: Supports the regulated activation of Cds1 kinase activity. Supporting Evidence: PMID:11313465 Substitution of threonine-11 with alanine (T11A) abolished Cds1 activation that occurs when DNA replication is inhibited by hydroxyurea (HU) treatment. |
| GO:0033314 mitotic DNA replication checkpoint signaling | IMP PMID:11313465 Threonine-11, phosphorylated by Rad3 and atm in vitro, is re... | ACCEPT | Summary: Core biological process. Cds1 enforces the S-M (DNA replication) checkpoint that couples mitosis to completion of replication; the cds1-T11A activation mutant is unable to enforce this checkpoint and is HU-sensitive. Reason: Strong genetic evidence that Cds1 is required for the mitotic DNA replication checkpoint. Supporting Evidence: PMID:11313465 Cds1 enforces the S-M checkpoint that couples mitosis (M) to the completion of DNA synthesis (S). |
| GO:0005515 protein binding | IPI PMID:18180284 Minichromosome maintenance proteins interact with checkpoint... | MARK AS OVER ANNOTATED | Summary: Uninformative "protein binding" capturing the Cds1-Mcm4 interaction. Mcm4 binds and is phosphorylated by Cds1 during checkpoint activation; this is a kinase-substrate relationship already represented by the MF and BP terms. Reason: Bare protein binding is uninformative; the interaction is a kinase-substrate contact captured elsewhere. Supporting Evidence: PMID:18180284 Mcm4 binds the Cds1 kinase and undergoes Cds1-dependent phosphorylation. |
| GO:0033315 meiotic G2/MI DNA replication checkpoint signaling | IMP PMID:25492408 Meiotic nuclear movements in fission yeast are regulated by ... | ACCEPT | Summary: Core meiotic biological process. The Cds1-dependent replication checkpoint represses mei4 to delay meiotic progression (prolonged horsetail nuclear movement) when meiotic DNA replication is stalled. Reason: Genetic evidence (cds1 deletion rescues the prolongation) that Cds1 mediates the meiotic replication checkpoint. Supporting Evidence: PMID:25492408 this prolongation was caused by the Cds1-dependent replication checkpoint, which represses expression of the mei4(+) gene encoding a meiosis-specific transcription factor. |
| GO:0031573 mitotic intra-S DNA damage checkpoint signaling | IGI PMID:7723827 A kinase from fission yeast responsible for blocking mitosis... | ACCEPT | Summary: Core biological process. The founding study identified cds1 as a key component of the DNA replication-monitoring S/G2 checkpoint that blocks mitosis while DNA synthesis is in progress. Reason: Original genetic identification of Cds1 as a replication/intra-S checkpoint component. Supporting Evidence: PMID:7723827 this protein kinase is a key component of the DNA replication-monitoring S/G2 checkpoint system. |
| GO:0031573 mitotic intra-S DNA damage checkpoint signaling | IMP PMID:19037101 Mus81, Rhp51(Rad51), and Rqh1 form an epistatic pathway requ... | ACCEPT | Summary: Core biological process. cds1 deletion produces a complete S-phase DNA damage checkpoint defect (failure to slow replication after MMS), identifying downstream effectors Mus81 and Rqh1. Reason: Strong genetic evidence (complete checkpoint defect in cds1Ξ) for the intra-S DNA damage checkpoint. Supporting Evidence: PMID:19037101 upon treatment with the alkylating agent methyl methane sulfonate, cds1Delta mutants display a complete checkpoint defect. |
| GO:0004674 protein serine/threonine kinase activity | IDA PMID:19357077 Autoinhibition and autoactivation of the DNA replication che... | ACCEPT | Summary: Core molecular function. This study defines the autoactivation mechanism: trans-autophosphorylation of activation-loop Thr328 is the sole covalent modification required for Cds1 kinase activation in vitro and in vivo. Reason: Direct biochemical characterization of Cds1 kinase activation. Supporting Evidence: PMID:19357077 phosphorylation of a highly conserved threonine residue (Thr 328 ) in the activation loop is the only covalent modification required for kinase activation in vitro and in vivo. |
| GO:0004674 protein serine/threonine kinase activity | IDA PMID:22682245 The intra-S phase checkpoint targets Dna2 to prevent stalled... | ACCEPT | Summary: Core molecular function. Cds1 phosphorylates Dna2 on S220 both in vivo and in vitro to control Dna2 association with stalled forks and prevent fork reversal. Reason: Direct demonstration of Cds1 phosphorylating a physiological substrate. Supporting Evidence: PMID:22682245 the Cds1(Chk2) effector kinase targets Dna2 on S220 to regulate, both in vivo and in vitro, Dna2 association with stalled replication forks in chromatin. |
| GO:0005515 protein binding | IPI PMID:22682245 The intra-S phase checkpoint targets Dna2 to prevent stalled... | MARK AS OVER ANNOTATED | Summary: Uninformative "protein binding" capturing the Cds1-Dna2 interaction. Dna2 is a Cds1 substrate (phosphorylated on S220); this is better represented by the kinase activity and fork-protection process terms. Reason: Bare protein binding is uninformative; the interaction is a kinase-substrate contact captured elsewhere. Supporting Evidence: PMID:22682245 the Cds1(Chk2) effector kinase targets Dna2 on S220 to regulate, both in vivo and in vitro, Dna2 association with stalled replication forks in chromatin. |
| GO:0031573 mitotic intra-S DNA damage checkpoint signaling | IMP PMID:22682245 The intra-S phase checkpoint targets Dna2 to prevent stalled... | ACCEPT | Summary: Core biological process. Cds1, the intra-S checkpoint effector, phosphorylates Dna2 to stabilize stalled forks and prevent their reversal, a key fork-protection arm of the replication-stress response. Reason: Demonstrates Cds1-dependent fork protection as part of the intra-S checkpoint response. Supporting Evidence: PMID:22682245 We propose that Dna2 cleavage of regressed nascent strands prevents fork reversal and thus stabilizes stalled forks to maintain genome stability during replication stress. |
| GO:0005634 nucleus | HDA PMID:16823372 ORFeome cloning and global analysis of protein localization ... | ACCEPT | Summary: High-throughput GFP localization reporting nuclear signal, consistent with the nucleus being the site of Cds1 checkpoint function. Reason: Supports nuclear localization, corroborated by IDA evidence. Supporting Evidence: PMID:16823372 ORFeome cloning and global analysis of protein localization in the fission yeast Schizosaccharomyces pombe. |
| GO:0005829 cytosol | HDA PMID:16823372 ORFeome cloning and global analysis of protein localization ... | KEEP AS NON CORE | Summary: High-throughput GFP localization reporting cytosolic signal. Likely reflects the diffuse soluble pool of an abundant kinase rather than the functional site of action, which is the nucleus/chromatin. Reason: Bulk localization signal; not the site where the core checkpoint function is executed. Supporting Evidence: PMID:16823372 ORFeome cloning and global analysis of protein localization in the fission yeast Schizosaccharomyces pombe. |
| GO:0032153 cell division site | HDA PMID:16823372 ORFeome cloning and global analysis of protein localization ... | MARK AS OVER ANNOTATED | Summary: High-throughput GFP localization reporting cell-division-site signal. There is no functional evidence linking Cds1 to a role at the cell division site; this most likely reflects nonspecific or bulk signal in the global screen and is over-annotated. Reason: No experimental function at the division site; signal from a global screen not supported by targeted studies. Supporting Evidence: PMID:16823372 ORFeome cloning and global analysis of protein localization in the fission yeast Schizosaccharomyces pombe. |
| GO:0031573 mitotic intra-S DNA damage checkpoint signaling | IMP PMID:12032307 A single unbranched S-phase DNA damage and replication fork ... | ACCEPT | Summary: Core biological process. Genetic analysis shows the intra-S-phase slowing of replication in response to damage depends strongly on Cds1, defining a single unbranched S-phase damage/fork-blockage checkpoint pathway. Reason: Genetic dependence of the intra-S checkpoint on Cds1. Supporting Evidence: PMID:12032307 The slowing of S phase depends strongly on the six checkpoint-Rad proteins, on Cds1, and on Rad4/Cut5 |
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Download this section (compressed HTML)Q: Beyond the known substrates (Cdc25, Wee1/Mik1, Hsk1/Dfp1, Dna2, Mus81, Rad60, Mcm4, Nrm1, Rgf1), what is the complete in vivo Cds1 phosphoproteome at stalled forks, and which substrates mediate fork protection versus cell-cycle arrest?
Q: How is Cds1 inactivated and the checkpoint terminated after fork recovery, and what role do CAF-1/Pcf1 and histone H4 deacetylation play in resetting the kinase?
Experiment: Analog-sensitive cds1 (e.g. gatekeeper mutant) combined with quantitative phosphoproteomics in HU- and MMS-treated cells to define the direct, activation-dependent Cds1 substrate network and distinguish fork-protection from cell-cycle-arrest targets.
Experiment: Single-molecule DNA combing and fork-reversal assays (e.g. electron microscopy of replication intermediates) in cds1Ξ and Cds1 substrate-site mutants (dna2-S220A, mus81 phospho-mutants) to quantify the contribution of each substrate to stalled-fork stability.
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