Cdc18 is the fission-yeast ortholog of Cdc6, an AAA+ ATPase and essential pre-replicative complex (pre-RC) component required for DNA replication licensing. Recruited to ORC-bound replication origins in late G1/early S, it cooperates with Cdt1 to load the MCM2-7 replicative helicase onto origins, forming the pre-RC and licensing each origin for a single round of firing. Cdc18 is the rate-limiting initiator of S phase: its abundance is tightly cell-cycle controlled through MBF (Cdc10/Res1/Res2)-dependent transcription at START and through CDK (Cdc2)-mediated phosphorylation that targets it for SCF(Pop1/Pop2)-dependent ubiquitination and 26S proteasomal degradation. This regulation restricts replication to once per cycle, and Cdc18 overexpression or stabilization drives genome re-replication. Cdc18 additionally contributes to the S-phase (DNA replication) checkpoint: when replication forks stall it persists on chromatin and anchors the Rad3-Rad26 (ATR-ATRIP) checkpoint kinase complex, helping couple completion of S phase to the onset of mitosis. It localizes to the nucleus and is found at origins/chromatin as part of the pre-RC.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0005634 nucleus | IBA GO_REF:0000033 | ACCEPT | Summary: Cdc18 is a nuclear replication-licensing factor that acts at chromatin-bound origins. Nuclear localization is well established experimentally in S. pombe and by orthology, so this phylogenetic annotation is correct. Reason: Nuclear localization is supported both by IBA across the Cdc6/Cdc18 family and by direct experimental observation in fission yeast. Supporting Evidence: PMID:8521469 p65cdc18 is located in the nucleus when cdc18 is overexpressed. |
| GO:0006270 DNA replication initiation | IBA GO_REF:0000033 | ACCEPT | Summary: Cdc18 is a core initiator of DNA replication: it is required for replication and its regulated expression controls the onset of S phase. This is a primary, well-supported function and the phylogenetic call matches the conserved role of Cdc6/Cdc18 family members. Reason: Central, experimentally established function in fission yeast, conserved across the family, and the basis for the more specific mitotic DNA replication initiation annotation. Supporting Evidence: PMID:8521469 We propose that p65cdc18 acts at the initiation of DNA replication and plays a major role in controlling the onset of S phase. |
| GO:0003688 DNA replication origin binding | IBA GO_REF:0000033 | ACCEPT | Summary: Cdc18 is recruited to ORC-bound replication origins (e.g. ars1) where it nucleates pre-RC assembly. Origin association is conserved across the Cdc6/Cdc18 family, so this molecular function is appropriate. Reason: Cdc18 associates with origin DNA via ORC and is required for loading downstream factors at origins; supported by phylogenetic inference and fission-yeast biochemistry. Supporting Evidence: PMID:10747035 In budding and fission yeasts, these origins are bound throughout the cell cycle by the origin recognition complex (ORC) ( Diffley et al., 1994 ; Aparicio et al., 1997 ; Donovan et al., 1997 ; Tanaka et al., 1997 ; Lygerou and Nurse, 1999 ; Ogawa et al., 1999 ) but, early in the cell cycle, additional proteins bind at origins to form pre-replicative complexes (pre-RCs). |
| GO:0033314 mitotic DNA replication checkpoint signaling | IBA GO_REF:0000033 | ACCEPT | Summary: Cdc18 contributes to the S-phase/replication checkpoint. At stalled forks it persists on chromatin and anchors the Rad3-Rad26 (ATR-ATRIP) checkpoint kinase complex, which is needed for long-term checkpoint maintenance and for blocking mitosis until replication completes. Reason: Experimentally supported checkpoint role in fission yeast (Cdc18 anchors Rad3-Rad26), consistent with the phylogenetic annotation. Supporting Evidence: PMID:17531813 Cdc18 plays a pivotal role in checkpoint maintenance by anchoring the Rad3-Rad26 complex to chromatin. |
| GO:0005524 ATP binding | IEA GO_REF:0000002 | ACCEPT | Summary: Cdc18 is an AAA+ ATPase of the Cdc6/Cdc18 family with a Walker A/P-loop motif (residues 199-206). ATP binding is a conserved, expected feature of this family and underlies its helicase-loading function. Reason: InterPro-based ATP-binding inference is consistent with the AAA+ ATPase architecture and the annotated nucleotide-binding P-loop in UniProt. Supporting Evidence: GO_REF:0000002 Gene Ontology annotation through association of InterPro records with GO terms |
| GO:0006270 DNA replication initiation | IEA GO_REF:0000002 | ACCEPT | Summary: InterPro2GO annotation for DNA replication initiation, redundant with the experimentally and phylogenetically supported same term. The function is correct and core; this is a valid but lower-evidence duplicate. Reason: Correct core function; redundant with the IBA/IMP-supported DNA replication initiation annotations. Supporting Evidence: PMID:8521469 We propose that p65cdc18 acts at the initiation of DNA replication and plays a major role in controlling the onset of S phase. |
| GO:0006325 chromatin organization | IEA GO_REF:0000108 | MARK AS OVER ANNOTATED | Summary: This term is an automated inter-ontology inference from the chromatin-protein adaptor activity (GO:0140463) annotation. Cdc18's documented chromatin-related role is anchoring the Rad3-Rad26 checkpoint complex to chromatin, not remodeling or organizing chromatin structure. "Chromatin organization" overstates and mischaracterizes this activity. Reason: Logically inferred from the adaptor MF but not reflecting a genuine chromatin-organization role; the real function is checkpoint-complex anchoring, captured better by the adaptor and checkpoint terms. Supporting Evidence: PMID:17531813 Rad26 directly binds Cdc18 and is required for Rad3 recruitment to chromatin. |
| GO:0016887 ATP hydrolysis activity | IEA GO_REF:0000002 | ACCEPT | Summary: As an AAA+ ATPase of the Cdc6/Cdc18 family, Cdc18 has ATPase activity that contributes to MCM loading. ATP binding and hydrolysis are conserved, mechanistically important features of this family, so the InterPro-based inference is appropriate. Reason: Consistent with the AAA+ ATPase architecture; ATP hydrolysis is part of the helicase-loader mechanism, though the in vivo MCM-loading process is the more informative term. Supporting Evidence: GO_REF:0000002 Gene Ontology annotation through association of InterPro records with GO terms |
| GO:0051301 cell division | IEA GO_REF:0000002 | KEEP AS NON CORE | Summary: A generic, high-level process term derived from InterPro/keyword. While Cdc18 is essential for the cell-division cycle through its replication role, this term is uninformative relative to the specific replication-initiation, MCM-loading, and pre-RC assembly terms already present. Reason: Broadly true but generic; the specific replication-licensing terms capture the actual function far better. Supporting Evidence: GO_REF:0000002 Gene Ontology annotation through association of InterPro records with GO terms |
| GO:0005515 protein binding | IPI PMID:10209119 F-box/WD-repeat proteins pop1p and Sud1p/Pop2p form complexe... | MARK AS OVER ANNOTATED | Summary: This IntAct annotation records a physical interaction between Cdc18 and the F-box/WD-repeat protein Pop2 (Sud1), part of the SCF(Pop1/Pop2) ubiquitin ligase that targets Cdc18 for degradation. The interaction is real, but the bare "protein binding" term is uninformative about Cdc18's molecular function. Reason: Uninformative generic MF; the underlying interaction (Cdc18 recognized by the SCF(Pop1/Pop2) degradation machinery) is better captured at the process level (regulation of replication / proteolysis) rather than as a molecular function of Cdc18. Supporting Evidence: PMID:10209119 F-box/WD-repeat proteins pop1p and Sud1p/Pop2p form complexes that bind and direct the proteolysis of cdc18p. |
| GO:0005515 protein binding | IPI PMID:14970237 Requirement of the SCFPop1/Pop2 Ubiquitin Ligase for Degrada... | MARK AS OVER ANNOTATED | Summary: IntAct-curated Cdc18-Pop1 physical interaction associated with a study primarily on SCF(Pop1/Pop2)-mediated degradation of the S-phase cyclin Cig2. Cdc18 is an established Pop1 substrate, so the interaction is consistent, but bare "protein binding" is uninformative. Reason: Generic, uninformative MF; the biologically meaningful relationship is Cdc18 being targeted by the SCF(Pop1/Pop2) ligase, better expressed as regulation of Cdc18 stability. Supporting Evidence: PMID:14970237 Two F-box/WD proteins Pop1 and Pop2, homologues of budding yeast Cdc4 and human Fbw7, are responsible for Cig2 instability. |
| GO:0005515 protein binding | IPI PMID:9203581 Fission yeast WD-repeat protein pop1 regulates genome ploidy... | MARK AS OVER ANNOTATED | Summary: IntAct annotation capturing the in vivo Cdc18-Pop1 interaction, by which Pop1 recognizes Cdc18 for ubiquitin-proteasome degradation. The interaction is well established but the bare "protein binding" term conveys no functional specificity. Reason: Generic, uninformative MF; recognition of Cdc18 by Pop1 for degradation is better captured as a process controlling Cdc18 abundance and re-replication. Supporting Evidence: PMID:9203581 Pop1 binds Cdc18 in vivo. |
| GO:0005515 protein binding | IPI PMID:9653157 sud1(+) targets cyclin-dependent kinase-phosphorylated Cdc18... | MARK AS OVER ANNOTATED | Summary: IntAct annotation for the phosphorylation-dependent Cdc18-Sud1/Pop2 interaction that targets Cdc18 for degradation to limit replication to one round per cycle. Biologically important but the bare "protein binding" term is uninformative. Reason: Generic MF; the CDK-phosphorylation-dependent recognition of Cdc18 by Sud1/Pop2 is better represented at the level of regulated proteolysis and prevention of re-replication. Supporting Evidence: PMID:9653157 Sud1-Cdc18 binding requires prior phosphorylation of the Cdc18 polypeptide at CDK consensus sites. |
| GO:1902975 mitotic DNA replication initiation | IMP PMID:8521469 p65cdc18 plays a major role controlling the initiation of DN... | ACCEPT | Summary: Direct experimental (mutant phenotype) evidence in fission yeast: cdc18 is required for DNA replication and overexpression drives repeated rounds of DNA synthesis, demonstrating its role in initiating mitotic-cycle replication. This is a core function. Reason: Strong IMP evidence; cdc18 loss blocks replication and its deregulation re-fires origins, defining its initiation role in the mitotic cell cycle. Supporting Evidence: PMID:8521469 overexpressing cdc18 is able to bring about repeated rounds of DNA synthesis in the absence of mitosis and of continuing protein synthesis. |
| GO:0140463 chromatin-protein adaptor activity | IPI PMID:17531813 Cdc18 enforces long-term maintenance of the S phase checkpoi... | ACCEPT | Summary: Informative molecular function supported by direct interaction evidence: at stalled replication forks Cdc18 anchors the Rad3-Rad26 checkpoint kinase complex to chromatin, with Rad26 directly binding Cdc18 and being required for Rad3 chromatin recruitment. This adaptor role underpins long-term S-phase checkpoint maintenance. Reason: Well-supported, specific MF (with rad3 and rad26 partners) reflecting Cdc18's checkpoint anchoring activity on chromatin. Supporting Evidence: PMID:17531813 Rad26 directly binds Cdc18 and is required for Rad3 recruitment to chromatin. |
| GO:0140530 MCM complex loading | IMP PMID:10747035 Chromatin binding of the fission yeast replication factor mc... | ACCEPT | Summary: Core function. Chromatin binding of the MCM helicase subunit mcm4 requires both ORC (orc1) and cdc18, demonstrating that Cdc18 is required to load the MCM2-7 helicase onto origins to form the pre-RC. This is the defining licensing activity of Cdc6/Cdc18 family members. Reason: Direct mutant-phenotype evidence in fission yeast that Cdc18 is required for MCM (mcm4) chromatin loading. Supporting Evidence: PMID:10747035 Binding of mcm4 to chromatin requires orc1 and cdc18 (homologous to Cdc6 in budding yeast). |
| GO:0140530 MCM complex loading | IMP PMID:11532929 Expression of Cdc18/Cdc6 and Cdt1 during G2 phase induces in... | ACCEPT | Summary: Core function, independently supported: expression of Cdc18 (with Cdt1) during G2 induces initiation of DNA replication by recruiting the MCM helicase to chromatin and re-firing origins, confirming Cdc18's role in MCM loading. Consistent with the requirement of Cdc18 for mcm4 chromatin binding. Reason: Second independent IMP supporting Cdc18-dependent MCM loading; reinforces the licensing function. Supporting Evidence: PMID:10747035 Binding of mcm4 to chromatin requires orc1 and cdc18 (homologous to Cdc6 in budding yeast). |
| GO:0000785 chromatin | IDA PMID:17531813 Cdc18 enforces long-term maintenance of the S phase checkpoi... | ACCEPT | Summary: Direct evidence that Cdc18 is chromatin-associated: when replication stalls it persists in a chromatin-bound complex with Rad3 and Rad26. Consistent with its origin/pre-RC localization, this cellular-component annotation is well supported. Reason: IDA evidence that Cdc18 functions in a chromatin-bound complex; consistent with origin binding and pre-RC assembly. Supporting Evidence: PMID:17531813 when DNA replication stalls, Cdc18 persists in a chromatin-bound complex including the checkpoint kinases Rad3 and Rad26. |
| GO:0005634 nucleus | IDA PMID:15278909 The regulation of competence to replicate in meiosis by Cdc6... | ACCEPT | Summary: Direct evidence supports nuclear localization of Cdc18 in S. pombe. This paper examines Cdc6/Cdc18 control of replication competence including the fission-yeast nuclear context; nuclear localization is consistent with Cdc18 acting at chromosomal origins. Reason: Nuclear localization is the expected and experimentally supported site of action for this replication-licensing factor. Supporting Evidence: PMID:8521469 p65cdc18 is located in the nucleus when cdc18 is overexpressed. |
| GO:0005656 nuclear pre-replicative complex | ISO GO_REF:0000024 | ACCEPT | Summary: Cdc18 is a defining component of the pre-replicative complex: it assembles with ORC at origins and recruits the MCM helicase. Membership in the nuclear pre-RC, transferred by orthology, is fully consistent with its experimentally demonstrated licensing role. Reason: Cdc18/Cdc6 is a canonical pre-RC component; the ISO transfer is corroborated by direct fission-yeast MCM-loading evidence. Supporting Evidence: PMID:10747035 additional proteins bind at origins to form pre-replicative complexes (pre-RCs). Pre-RC formation establishes replication competence for the subsequent S phase |
| GO:0043596 nuclear replication fork | IC GO_REF:0000111 | KEEP AS NON CORE | Summary: Curator-inferred (from pre-RC membership) localization to the nuclear replication fork. Cdc18 acts before fork establishment (origin licensing) and during fork stalling (checkpoint anchoring), so a replication-fork association is plausible but indirect relative to its primary pre-RC/origin role. Reason: Plausible inferred localization, but Cdc18's core site of action is the origin/pre-RC; the fork association is secondary (e.g., stalled-fork checkpoint complex) rather than a defining location. Supporting Evidence: PMID:17531813 when DNA replication stalls, Cdc18 persists in a chromatin-bound complex including the checkpoint kinases Rad3 and Rad26. |
| GO:1902985 mitotic pre-replicative complex assembly | ISO GO_REF:0000024 | ACCEPT | Summary: Core function. Cdc18 is required for assembly of the pre-RC in the mitotic cell cycle: it is recruited to ORC-bound origins and loads the MCM helicase. The orthology-based annotation is strongly corroborated by direct fission-yeast evidence of Cdc18-dependent MCM loading. Reason: Pre-RC assembly is the central, experimentally supported role of Cdc18; the ISO transfer is accurate. Supporting Evidence: PMID:10747035 Binding of mcm4 to chromatin requires orc1 and cdc18 (homologous to Cdc6 in budding yeast). |
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Download this section (compressed HTML)Q: Does Cdc18 ATP binding/hydrolysis directly power MCM2-7 loading in fission yeast, and what are the in vitro biochemical requirements for Cdc18-Cdt1-mediated helicase loading onto S. pombe origins?
Suggested experts: Thomas J. Kelly, Stephen Kearsey
Q: How is the checkpoint-anchoring (Rad3-Rad26 adaptor) activity of Cdc18 mechanistically separated from its replication-licensing activity, and which Cdc18 domains/residues are required for each?
Suggested experts: Damien Hermand, Paul Nurse
Experiment: Reconstitute MCM2-7 loading in vitro with purified S. pombe ORC, Cdc18, Cdt1 and MCM2-7 on origin DNA, comparing wild-type Cdc18 with Walker A/B (ATP-binding/hydrolysis) mutants to test whether Cdc18 ATPase activity is required for productive helicase loading.
Hypothesis: Cdc18 ATP binding and hydrolysis are mechanistically required to load and/or activate the MCM2-7 helicase at origins.
Type: in vitro biochemical reconstitution
Experiment: Generate separation-of-function cdc18 alleles and test in vivo whether mutations that abolish Rad26/Rad3 chromatin anchoring (checkpoint maintenance) leave MCM loading and origin licensing intact, using chromatin-binding assays for MCM and Rad3 plus checkpoint and re-replication readouts.
Hypothesis: The checkpoint-anchoring function of Cdc18 is genetically and structurally separable from its pre-RC/MCM-loading function.
Type: genetic separation-of-function analysis
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