CDC21

UniProt ID: P06785
Organism: Saccharomyces cerevisiae S288C
Review Status: COMPLETE
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Gene Description

CDC21 (TMP1, CRT9) encodes thymidylate synthase (EC 2.1.1.45), a homodimeric enzyme that catalyses the reductive methylation of dUMP to dTMP with 5,10-methylene-tetrahydrofolate as the one-carbon donor and reductant, releasing 7,8-dihydrofolate. It is the only de novo source of thymidylate, which is phosphorylated to dTTP by Cdc8p and Ynk1p, and it is required for both nuclear and mitochondrial DNA synthesis. The cell-cycle mutant cdc21 is a temperature-sensitive allele of TMP1. Like tmp1 mutants it lacks thymidylate synthase activity and is a thymidylate auxotroph at the restrictive temperature. CDC21 transcription peaks just before S phase and depends on MluI cell-cycle box (MCB) elements and Swi6. The protein fractionates with nuclei and localises to the nuclear periphery. It is inhibited by FdUMP, the active form of 5-fluorouracil, and its dihydrofolate product is recycled to THF by Dfr1p.

Existing Annotations Review

GO Term Evidence Action Reason
GO:0004799 thymidylate synthase activity
IBA
GO_REF:0000033 		ACCEPT
Summary: IBA from the thymidylate synthase node PTN000167391 (CDC21/SGD:S000005600 is itself an experimental source). Core, universally conserved activity.
Supporting Evidence:
file:yeast/CDC21/CDC21-uniprot.txt
Reaction=dUMP + (6R)-5,10-methylene-5,6,7,8-tetrahydrofolate = 7,8-
GO:0004799 thymidylate synthase activity
IDA
PMID:6287238
Isolation of the thymidylate synthetase gene (TMP1) by compl... 		ACCEPT
Summary: Taylor et al. cloned TMP1/CDC21 by complementation and measured [3H]dUMP -> dTMP conversion: tmp1-6 extracts lacked activity which the plasmid restored; the yeast gene also complemented E. coli thyA. Core activity.
Supporting Evidence:
PMID:6287238
Lysates of pTL1 transformants of this strain, however, had thymidylate synthetase activity that was comparable to that of the wild-type strain.
PMID:6287238
Thus, yeast thymidylate synthetase is biologically active in Escherichia coli.
GO:0004799 thymidylate synthase activity
IEA
GO_REF:0000120 		ACCEPT
Summary: InterPro/RHEA/EC 2.1.1.45 mapping; correct.
Supporting Evidence:
file:yeast/CDC21/CDC21-uniprot.txt
Reaction=dUMP + (6R)-5,10-methylene-5,6,7,8-tetrahydrofolate = 7,8-
GO:0004799 thymidylate synthase activity
IMP
PMID:334734
Thymidine 5'-monophosphate-requiring mutants of Saccharomyce... 		ACCEPT
Summary: Bisson & Thorner showed tmp1 auxotrophs and cdc21 ts mutants lack thymidylate synthetase activity, and a ts allele produces thermolabile enzyme. Core activity.
Supporting Evidence:
PMID:334734
Extracts of a strain bearing the temperature-sensitive cell division cycle mutation cdc21 lacked detectable thymidylate synthetase activity under all conditions tested.
PMID:334734
non-conditional mutants auxotrophic for thymidine 5'-monophosphate (tmp1) lacked detectable thymidylate synthetase activity in cell-free extracts
GO:0004799 thymidylate synthase activity
RCA
GO_REF:0000123 		ACCEPT
Summary: YeastPathways RCA for EC 2.1.1.45 in ALL-CHORISMATE-PWY-1 (superpathway of chorismate metabolism); correct activity for Cdc21p.
Supporting Evidence:
file:yeast/CDC21/CDC21-uniprot.txt
Reaction=dUMP + (6R)-5,10-methylene-5,6,7,8-tetrahydrofolate = 7,8-
GO:0004799 thymidylate synthase activity
RCA
GO_REF:0000123 		ACCEPT
Summary: YeastPathways RCA for EC 2.1.1.45 in FOLSYN-PWY-1 (superpathway of tetrahydrofolate biosynthesis and salvage); correct activity for Cdc21p.
Supporting Evidence:
file:yeast/CDC21/CDC21-uniprot.txt
Reaction=dUMP + (6R)-5,10-methylene-5,6,7,8-tetrahydrofolate = 7,8-
GO:0004799 thymidylate synthase activity
RCA
GO_REF:0000123 		ACCEPT
Summary: YeastPathways RCA for EC 2.1.1.45 in PWY3O-697 (folate interconversions); correct activity for Cdc21p.
Supporting Evidence:
file:yeast/CDC21/CDC21-uniprot.txt
Reaction=dUMP + (6R)-5,10-methylene-5,6,7,8-tetrahydrofolate = 7,8-
GO:0004799 thymidylate synthase activity
RCA
GO_REF:0000123 		ACCEPT
Summary: YeastPathways RCA for EC 2.1.1.45 in YEAST-DE-NOVO-PYRMID-DNT (de novo biosynthesis of pyrimidine deoxyribonucleotides); correct activity for Cdc21p.
Supporting Evidence:
file:yeast/CDC21/CDC21-uniprot.txt
Reaction=dUMP + (6R)-5,10-methylene-5,6,7,8-tetrahydrofolate = 7,8-
GO:0005634 nucleus
IEA
GO_REF:0000044 		ACCEPT
Summary: UniProt SL-0191 Nucleus mapping, based on Poon & Storms who found TS associated with purified nuclei and at the nuclear periphery.
Supporting Evidence:
PMID:8132557
Subcellular fractionation experiments indicate thymidylate synthase is associated with purified nuclei.
GO:0005739 mitochondrion
IBA
GO_REF:0000033 		MARK AS OVER ANNOTATED
Summary: IBA from PTN002610699, seeded by human TYMS (mitochondrial pool) and plant/trypanosome bifunctional DHFR-TS. Yeast TS is needed for mtDNA synthesis, but that only requires dTTP; no mitochondrial localization of Cdc21p has been reported, and yeast data place it at the nuclear periphery.
Reason: No yeast evidence for a mitochondrial pool; mtDNA dependence does not imply mitochondrial location.
Propagation Review
Root cause: PROPAGATION BAD
Failure modes: COMPARTMENT OR COMPLEX MISMATCH LINEAGE OR TAXON MISMATCH
Sources checked:
UniProtKB:P04818 · human TYMS (mitochondrial pool reported) SUPPORTS SOURCE BUT NOT TARGET
AGI_LocusCode:AT4G34570 · Arabidopsis bifunctional DHFR-TS SUPPORTS SOURCE BUT NOT TARGET
Supporting Evidence:
file:yeast/CDC21/CDC21-uniprot.txt
Required for both nuclear and
PMID:8132557
immunofluorescence analysis suggests that thymidylate synthase is situated at the nuclear periphery
GO:0005829 cytosol
IBA
GO_REF:0000033 		MARK AS OVER ANNOTATED
Summary: IBA for cytosolic activity from PTN000167391, seeded by E. coli ThyA (a prokaryote has no nucleus). In yeast, TS fractionates with nuclei and is seen at the nuclear periphery, so a generic cytosol location is not supported for Cdc21p.
Reason: Yeast-specific localization data (nuclear periphery) contradict a default cytosolic location.
Propagation Review
Root cause: PROPAGATION BAD
Failure modes: COMPARTMENT OR COMPLEX MISMATCH LINEAGE OR TAXON MISMATCH
Sources checked:
UniProtKB:P0A884 · E. coli ThyA (prokaryote, no nucleus) SUPPORTS SOURCE BUT NOT TARGET
Supporting Evidence:
PMID:8132557
immunofluorescence analysis suggests that thymidylate synthase is situated at the nuclear periphery
GO:0005829 cytosol
RCA
GO_REF:0000123 		MARK AS OVER ANNOTATED
Summary: YeastPathways RCA cytosol assignment from ALL-CHORISMATE-PWY-1 (superpathway of chorismate metabolism), a pathway-level default rather than evidence for Cdc21p, which is nuclear-periphery associated in yeast.
Reason: Pathway default location, not supported by the yeast localization study.
Supporting Evidence:
PMID:8132557
immunofluorescence analysis suggests that thymidylate synthase is situated at the nuclear periphery
GO:0005829 cytosol
RCA
GO_REF:0000123 		MARK AS OVER ANNOTATED
Summary: YeastPathways RCA cytosol assignment from FOLSYN-PWY-1 (superpathway of tetrahydrofolate biosynthesis and salvage), a pathway-level default rather than evidence for Cdc21p, which is nuclear-periphery associated in yeast.
Reason: Pathway default location, not supported by the yeast localization study.
Supporting Evidence:
PMID:8132557
immunofluorescence analysis suggests that thymidylate synthase is situated at the nuclear periphery
GO:0005829 cytosol
RCA
GO_REF:0000123 		MARK AS OVER ANNOTATED
Summary: YeastPathways RCA cytosol assignment from PWY3O-697 (folate interconversions), a pathway-level default rather than evidence for Cdc21p, which is nuclear-periphery associated in yeast.
Reason: Pathway default location, not supported by the yeast localization study.
Supporting Evidence:
PMID:8132557
immunofluorescence analysis suggests that thymidylate synthase is situated at the nuclear periphery
GO:0005829 cytosol
RCA
GO_REF:0000123 		MARK AS OVER ANNOTATED
Summary: YeastPathways RCA cytosol assignment from YEAST-DE-NOVO-PYRMID-DNT (de novo biosynthesis of pyrimidine deoxyribonucleotides), a pathway-level default rather than evidence for Cdc21p, which is nuclear-periphery associated in yeast.
Reason: Pathway default location, not supported by the yeast localization study.
Supporting Evidence:
PMID:8132557
immunofluorescence analysis suggests that thymidylate synthase is situated at the nuclear periphery
GO:0006207 'de novo' pyrimidine nucleobase biosynthetic process
RCA
GO_REF:0000123 		MODIFY
Summary: RCA from the de novo pyrimidine deoxyribonucleotide pathway mapped to de novo pyrimidine nucleobase biosynthesis. TS methylates dUMP (a nucleotide) to dTMP; it does not build the pyrimidine ring or make a free nucleobase.
Reason: Wrong chemical class; the precise process is dTMP biosynthesis.
Proposed replacements: dTMP biosynthetic process
GO:0006231 dTMP biosynthetic process
IBA
GO_REF:0000033 		ACCEPT
Summary: IBA from the TS node; TS catalyses the only de novo route to dTMP. Core process.
Supporting Evidence:
file:yeast/CDC21/CDC21-uniprot.txt
Thymidylate synthase required for de novo biosynthesis of
GO:0006231 dTMP biosynthetic process
IEA
GO_REF:0000002 		ACCEPT
Summary: InterPro (IPR000398/IPR020940) mapping to dTMP biosynthesis; correct.
Supporting Evidence:
file:yeast/CDC21/CDC21-uniprot.txt
Reaction=dUMP + (6R)-5,10-methylene-5,6,7,8-tetrahydrofolate = 7,8-
GO:0006231 dTMP biosynthetic process
IMP
PMID:334734
Thymidine 5'-monophosphate-requiring mutants of Saccharomyce... 		ACCEPT
Summary: tmp1/cdc21 mutants lacking TS activity are dTMP auxotrophs, showing Cdc21p is required for and catalyses dTMP synthesis. Core process.
Supporting Evidence:
PMID:334734
non-conditional mutants auxotrophic for thymidine 5'-monophosphate (tmp1) lacked detectable thymidylate synthetase activity in cell-free extracts
GO:0006231 dTMP biosynthetic process
IMP
PMID:794696
Yeast cell-cycle mutant cdc21 is a temperature-sensitive thy... 		ACCEPT
Summary: Game showed that cdc21 and tmp1 are allelic and that the cdc21 mutant is a temperature-sensitive dTMP auxotroph. Core process.
Supporting Evidence:
PMID:794696
indicate that the CDC21 gene in yeast is the same as the TMP1 gene, whose mutant alleles confer an auxotrophic requirement for thymidine-5'-monophosphate (dTMP)
GO:0006235 dTTP biosynthetic process
IEA
GO_REF:0000041 		ACCEPT
Summary: UniPathway UPA00575 (dTTP biosynthesis) mapping; dTMP made by Cdc21p is phosphorylated by Cdc8p and Ynk1p to dTTP, so TS performs a step of dTTP biosynthesis.
Supporting Evidence:
file:yeast/CDC21/CDC21-uniprot.txt
PATHWAY: Pyrimidine metabolism; dTTP biosynthesis.
GO:0016741 transferase activity, transferring one-carbon groups
IEA
GO_REF:0000002 		MODIFY
Summary: InterPro IPR045097 (thymidylate synthase/dCMP hydroxymethylase) mapping to the generic one-carbon transferase term. Correct parent but uninformative.
Reason: Specific activity is established.
Proposed replacements: thymidylate synthase activity
Supporting Evidence:
file:yeast/CDC21/CDC21-uniprot.txt
Reaction=dUMP + (6R)-5,10-methylene-5,6,7,8-tetrahydrofolate = 7,8-
GO:0034399 nuclear periphery
IDA
PMID:8132557
Thymidylate synthase is localized to the nuclear periphery i... 		ACCEPT
Summary: Poon & Storms: subcellular fractionation and immunofluorescence place yeast thymidylate synthase at the nuclear periphery. This is the best-supported location for Cdc21p.
Supporting Evidence:
PMID:8132557
immunofluorescence analysis suggests that thymidylate synthase is situated at the nuclear periphery
PMID:8132557
Subcellular fractionation experiments indicate thymidylate synthase is associated with purified nuclei.
GO:0035999 folate cycle
RCA
GO_REF:0000123 		MARK AS OVER ANNOTATED
Summary: RCA from the THF superpathway. TS consumes methylene-THF and releases dihydrofolate, so it drains the C1 pool and creates the DHF that Dfr1p must recycle, but it does not transfer C1 units between THF molecules as the term defines.
Reason: TS is a consumer of THF-bound C1 units, not part of THF interconversion.
GO:0046417 chorismate metabolic process
RCA
GO_REF:0000123 		REMOVE
Summary: RCA from the superpathway of chorismate metabolism. Thymidylate synthase has no connection to chorismate chemistry; this is pure superpathway inflation.
Reason: No chorismate chemistry.
GO:0046655 folic acid metabolic process
RCA
GO_REF:0000123 		MARK AS OVER ANNOTATED
Summary: RCA from PWY3O-697 (folate interconversions). TS converts methylene-THF to DHF; it does not act on folic acid. Its role in folate chemistry is a side consequence of dTMP synthesis.
Reason: Folic acid is not a substrate; the meaningful process is dTMP biosynthesis.

Core Functions

Thymidylate synthase: reductive methylation of dUMP to dTMP using 5,10-methylene-THF as both C1 donor and reductant (yielding dihydrofolate); the sole de novo source of thymidylate for nuclear and mitochondrial DNA replication. The enzyme is associated with the nuclear periphery.

Molecular Function:
thymidylate synthase activity
Directly Involved In:
dTMP biosynthetic process
Cellular Locations:
nuclear periphery
Supporting Evidence:
  • file:yeast/CDC21/CDC21-uniprot.txt
    Reaction=dUMP + (6R)-5,10-methylene-5,6,7,8-tetrahydrofolate = 7,8-
  • PMID:6287238
    Lysates of pTL1 transformants of this strain, however, had thymidylate synthetase activity that was comparable to that of the wild-type strain.
  • PMID:334734
    Extracts of a strain bearing the temperature-sensitive cell division cycle mutation cdc21 lacked detectable thymidylate synthetase activity under all conditions tested.
  • PMID:794696
    indicate that the CDC21 gene in yeast is the same as the TMP1 gene, whose mutant alleles confer an auxotrophic requirement for thymidine-5'-monophosphate (dTMP)
  • PMID:8132557
    immunofluorescence analysis suggests that thymidylate synthase is situated at the nuclear periphery

References

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Suggested Questions for Experts

Q: How do mitochondria obtain dTTP for mtDNA replication when thymidylate synthase is nuclear-periphery associated?

Q: Is the nuclear-periphery localisation of Cdc21p cell-cycle regulated and does it reflect a dNTP-synthesis complex near replication sites?

Suggested Experiments

Experiment: Live-cell imaging of endogenously tagged Cdc21p through synchronized cell cycles with nuclear envelope markers, combined with proximity labelling (APEX2/BioID) to identify neighbours such as Cdc8p or RNR subunits.

Hypothesis: Cdc21p localisation to the nuclear periphery is S-phase enriched

Type: live-cell imaging / proximity proteomics

Deep Research

SGD

(CDC21-deep-research-sgd.md)
SGD gene description for CDC21 (yeast) SGD

SGD gene description for CDC21 (yeast)

Thymidylate synthase; required for de novo biosynthesis of pyrimidine deoxyribonucleotides; expression is induced at G1/S; human homolog TYMSOS can complement yeast cdc21 temperature-sensitive mutant at restrictive temperature

📚 Additional Documentation

Notes

(CDC21-notes.md)

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