NCU02539 encodes Mcm4, one of the six subunits of the Mcm2-7 complex, the conserved eukaryotic replicative DNA helicase. During late M and G1, Mcm2-7 is loaded onto replication origins by ORC, Cdc6 and Cdt1 as an inactive double hexamer around double-stranded DNA (origin licensing). In S phase it is activated with Cdc45 and GINS into the CMG helicase, which encircles the leading-strand template and unwinds DNA 3' to 5' at the replication fork, using ATP hydrolysed at the subunit interfaces of the AAA+ ring. Mcm4 contributes Walker A, Walker B and arginine-finger elements to these ATPase sites and helps the ring bind single-stranded DNA. The 1013-residue N. crassa protein has the N-terminal OB-fold region, the AAA+ ATPase domain with its lid, and the C-terminal winged-helix region typical of Mcm4, plus a longer disordered N-terminal tail than the yeast orthologs.
Existing Annotations Review
GO Term
Evidence
Action
Reason
GO:0000727 double-strand break repair via break-induced replication
IBA GO_REF:0000033
KEEP AS NON CORE
Summary: Mcm2-7, including Mcm4, is part of the replicative helicase that budding-yeast genetics shows is needed for break-induced replication.
Reason: Lydeard et al. showed that BIR in S. cerevisiae needs the CMG helicase (Cdc45, GINS, Mcm2-7) but not ORC or Cdc6. The unwinding that drives BIR DNA synthesis is the work of the same helicase, so the claim is helicase participation and not just necessity. PAINT placed this term at the Mcm4 subfamily node PTN000179968, with S. cerevisiae MCM4 as the seed. NCU02539 sits in that subfamily (PTHR11630:SF66). BIR is a repair use of the replicative helicase, secondary to genome replication, and has not been tested in Neurospora.
GO:0000727 double-strand break repair via break-induced replication
IEA GO_REF:0000107
KEEP AS NON CORE
Summary: Mcm2-7, including Mcm4, is part of the replicative helicase that budding-yeast genetics shows is needed for break-induced replication.
Reason: Lydeard et al. showed that BIR in S. cerevisiae needs the CMG helicase (Cdc45, GINS, Mcm2-7) but not ORC or Cdc6. The unwinding that drives BIR DNA synthesis is the work of the same helicase, so the claim is helicase participation and not just necessity. PAINT placed this term at the Mcm4 subfamily node PTN000179968, with S. cerevisiae MCM4 as the seed. NCU02539 sits in that subfamily (PTHR11630:SF66). BIR is a repair use of the replicative helicase, secondary to genome replication, and has not been tested in Neurospora.
Summary: Fission-yeast Mcm4 (cdc21) binds chromatin from anaphase to S phase in an ORC- and Cdc18-dependent way. The helicase acts on chromatinized DNA.
Reason: Kearsey et al. showed that S. pombe Mcm4 associates with chromatin in a licensing-dependent, cell-cycle-regulated way and is released as replication proceeds. Neurospora Mcm4 is a full-length member of the same subfamily, and the MCM complex is loaded onto and works on chromosomal DNA. Chromatin is where the protein acts.
Summary: DNA binding is correct but too general. The supported activity is ATP-dependent single-stranded DNA binding by the Mcm2-7/Mcm4-6-7 ring, which this gene already carries.
Reason: The InterPro2GO mapping gives generic DNA binding. Purified yeast MCM2-7 and Mcm467 complexes show ATP-dependent ssDNA binding. Mutation of mouse Mcm4 implicates this subunit in ssDNA binding by the complex. The more specific GO:0003697 is already on this gene (IBA and IEA), so generic DNA binding should be refined to it.
only the dimeric complex of the Mcm4/6/7 heterotrimer possessed single stranded DNA-dependent ATPase, ATP-dependent single stranded DNA binding, and 3' to 5' DNA helicase activities
Summary: DNA helicase activity is correct but too general. The MCM ring is a 3'-to-5' helicase that translocates on single-stranded DNA, and Mcm4 contributes to that activity as a subunit.
Reason: Purified S. cerevisiae Mcm2-7 is a helicase (Bochman and Schwacha 2008). S. pombe Mcm4/6/7 has 3'-to-5' DNA helicase activity (Lee and Hurwitz 2000). The Mcm4-6-7 ring binds the leading-strand template and unwinds by steric exclusion (Kaplan et al. 2003). The precise term GO:1990518 is already on this gene, and the activity belongs to the hexamer, so the contributes_to qualifier fits better than enables. NCU02539 keeps the Walker A, Walker B and SRF arginine-finger motifs of the yeast Mcm4 proteins.
only the dimeric complex of the Mcm4/6/7 heterotrimer possessed single stranded DNA-dependent ATPase, ATP-dependent single stranded DNA binding, and 3' to 5' DNA helicase activities
Summary: Mcm4 associates with chromatin once it is loaded as part of the MCM double hexamer. The underlying activity is topological encirclement of DNA, not a chromatin-reading binding activity.
Reason: The S. pombe experiments behind this transfer (Kearsey et al. 2000) show that Mcm4 associates with chromatin in a way that depends on ORC and Cdc18, which load the MCM ring. Mcm2-7 is loaded around DNA as a double hexamer that can slide along dsDNA (Remus et al. 2009). Chromatin binding is true at this level of description, but the informative activities are ssDNA binding and helicase activity, so the term is non-core.
Summary: Mcm2-7 is loaded onto origin DNA during licensing, but origin selection is done by ORC, Cdc6 and Cdt1. Loaded MCM double hexamers are not fixed to the origin sequence.
Reason: This term is transferred from S. cerevisiae MCM4 (IDA, including reconstituted pre-RC assembly on ARS1 DNA). Purified yeast proteins recruit ORC, Cdc6, Cdt1 and then Mcm2-7 in order onto ARS1 DNA, and ARS-derived ssDNA stimulates Mcm467 helicase activity. MCM does contact origin DNA. However, ORC defines the origin, and once loaded Mcm2-7 can slide passively along dsDNA. The term is kept as non-core and does not imply that Mcm4 recognises origin sequences.
Summary: Mcm4 contributes to the ATP-dependent single-stranded DNA binding of the MCM ring, the property that underlies its translocation on the leading-strand template.
Reason: Yeast MCM2-7 and Mcm467 both bind ssDNA in an ATP-dependent way, and S. pombe Mcm4/6/7 shows ATP-dependent ssDNA binding. Mutations in the mouse Mcm4 ATPase motifs implicate Mcm4 in ssDNA binding by the complex. PAINT placed this term at the MCM2-7 node PTN000179711, which includes the Mcm4 subfamily that contains NCU02539. The activity belongs to the ring, so contributes_to is the most accurate qualifier; the IBA row's enables reflects the PAINT default.
only the dimeric complex of the Mcm4/6/7 heterotrimer possessed single stranded DNA-dependent ATPase, ATP-dependent single stranded DNA binding, and 3' to 5' DNA helicase activities
Summary: Mcm4 contributes to the ATP-dependent single-stranded DNA binding of the MCM ring, the property that underlies its translocation on the leading-strand template.
Reason: Yeast MCM2-7 and Mcm467 both bind ssDNA in an ATP-dependent way, and S. pombe Mcm4/6/7 shows ATP-dependent ssDNA binding. Mutations in the mouse Mcm4 ATPase motifs implicate Mcm4 in ssDNA binding by the complex. PAINT placed this term at the MCM2-7 node PTN000179711, which includes the Mcm4 subfamily that contains NCU02539. The activity belongs to the ring, so contributes_to is the most accurate qualifier; the IBA row's enables reflects the PAINT default.
only the dimeric complex of the Mcm4/6/7 heterotrimer possessed single stranded DNA-dependent ATPase, ATP-dependent single stranded DNA binding, and 3' to 5' DNA helicase activities
Summary: Transfer of an in vitro observation that a fission-yeast Mcm4/6/7 preparation can bind RNA oligonucleotides. It cannot translocate on RNA, and no physiological RNA-binding role is known.
Reason: Shin and Kelman (2006) found that replicative helicases from all three domains bind both DNA and RNA in vitro, but translocate only on DNA, and only DNA stimulates their ATPase. RNA binding here is a side property of an ssDNA-translocating ring, not a function. No in vivo RNA-binding role of Mcm4 has been reported in any organism, and none has been studied in Neurospora.
Summary: NCU02539 keeps the Mcm4 Walker A motif GDPSTSKS (645-652) and the Walker B and SRF motifs. ATP binding and hydrolysis drive the MCM motor.
Reason: My alignment places the Walker A, Walker B and arginine-finger motifs of NCU02539 exactly where they sit in S. cerevisiae and S. pombe Mcm4. The UniProt MOTIF features at 662-669 and 726-729 are misplaced, which explains the PROSITE-ProRule caution, but the real motifs are intact. The Mcm2-7 ring is an AAA+ motor that uses ATP binding and hydrolysis to separate DNA strands.
The Mcm2-7 complex is the eukaryotic replicative helicase, a toroidal AAA(+) molecular motor that uses adenosine triphosphate (ATP) binding and hydrolysis to separate duplex DNA strands during replication.
Summary: Mcm4 acts in the nucleus as a subunit of the replicative helicase.
Reason: The Mcm2-7 helicase loads onto and unwinds nuclear chromosomal DNA. Fission-yeast Mcm4 is nuclear and binds chromatin. PAINT placed nucleus at PTN000179712, which covers the MCM2-7 subunits. NCU02539 is a full-length member of the Mcm4 subfamily.
The licensing of eukaryotic DNA replication origins, which ensures once-per-cell-cycle replication, involves the loading of six related minichromosome maintenance proteins (Mcm2-7) into prereplicative complexes (pre-RCs).
file:NEUCR/NCU02539/NCU02539-uniprot.txt
DR PANTHER; PTHR11630:SF66; DNA REPLICATION LICENSING FACTOR MCM4; 1.
Summary: Mcm4 acts in the nucleus as a subunit of the replicative helicase.
Reason: The Mcm2-7 helicase loads onto and unwinds nuclear chromosomal DNA. Fission-yeast Mcm4 is nuclear and binds chromatin. PAINT placed nucleus at PTN000179712, which covers the MCM2-7 subunits. NCU02539 is a full-length member of the Mcm4 subfamily.
The licensing of eukaryotic DNA replication origins, which ensures once-per-cell-cycle replication, involves the loading of six related minichromosome maintenance proteins (Mcm2-7) into prereplicative complexes (pre-RCs).
file:NEUCR/NCU02539/NCU02539-uniprot.txt
DR PANTHER; PTHR11630:SF66; DNA REPLICATION LICENSING FACTOR MCM4; 1.
Summary: Mcm2-7, including Mcm4, is the helicase core loaded into the pre-replicative complex at licensed origins.
Reason: Origin licensing loads Mcm2-7 into pre-RCs. In vitro reconstitution with purified budding-yeast ORC, Cdc6 and Cdt1-Mcm2-7 recruits these proteins in order onto ARS1. Mcm4 is a core part of the pre-RC in its loaded double-hexamer form.
The licensing of eukaryotic DNA replication origins, which ensures once-per-cell-cycle replication, involves the loading of six related minichromosome maintenance proteins (Mcm2-7) into prereplicative complexes (pre-RCs).
Summary: In budding yeast, CDK activity drives Mcm4 and other MCM subunits out of the nucleus outside G1. This is transferred to Neurospora by orthology.
Reason: Labib et al. (1999) and Nguyen et al. (2000) showed that S. cerevisiae Mcm2-7 is exported to the cytoplasm during S phase and excluded from the nucleus in G2/M, as a once-per-cycle control. A cytoplasmic pool is therefore documented for an ortholog. However, this is a regulatory storage state, not a site of function, and whether Neurospora regulates MCM nuclear export in this way is untested. Non-core.
Mcm2-4, 6 and 7 were concentrated in the nucleus in G1 phase, gradually exported to the cytoplasm during S phase, and excluded from the nucleus by G2 and M phase.
Summary: DNA replication is correct but too general. The supported process is nuclear genome replication: initiation and fork elongation as the replicative helicase.
Reason: The more specific terms GO:0033260, GO:0006271 and GO:1902975 are already on this gene. Generic DNA replication says less than these.
The licensing of eukaryotic DNA replication origins, which ensures once-per-cell-cycle replication, involves the loading of six related minichromosome maintenance proteins (Mcm2-7) into prereplicative complexes (pre-RCs).
file:NEUCR/NCU02539/NCU02539-uniprot.txt
DR PANTHER; PTHR11630:SF66; DNA REPLICATION LICENSING FACTOR MCM4; 1.
GO:0006267 pre-replicative complex assembly involved in nuclear cell cycle DNA replication
IEA GO_REF:0000107
ACCEPT
Summary: Mcm2-7 is loaded in an ATP-dependent step that forms the pre-RC. Mcm4 is a structural and ATPase-contributing part of the assembled complex.
Reason: Loading of Mcm2-7 onto origin DNA needs ORC, Cdc6, Cdt1 and ATP, and gives head-to-head double hexamers. The MCM ring is the core of the complex that is built, and its own ATPase sites take part in loading, so Mcm4 participates in pre-RC assembly rather than only being needed for it. Licensing is one of the central roles of the MCM complex.
The licensing of eukaryotic DNA replication origins, which ensures once-per-cell-cycle replication, involves the loading of six related minichromosome maintenance proteins (Mcm2-7) into prereplicative complexes (pre-RCs).
Summary: Mcm4 takes part in replication initiation as a subunit of the helicase that is licensed at origins and activated into CMG to melt and unwind origin DNA.
Reason: Loaded Mcm2-7 is the inactive core of the replicative helicase. At initiation it joins Cdc45 and GINS to form the CMG helicase that unwinds origin DNA. GO:0006270 is the right level for a gene that works in both mitotic and premeiotic S phase, so it is accepted alongside the more specific IBA term GO:1902975.
The licensing of eukaryotic DNA replication origins, which ensures once-per-cell-cycle replication, involves the loading of six related minichromosome maintenance proteins (Mcm2-7) into prereplicative complexes (pre-RCs).
The eukaryotic helicase is an 11-subunit CMG complex that encircles the leading strand during unwinding and consists of the Mcm2-7 motor subunit
GO:0006271 DNA strand elongation involved in DNA replication
IBA GO_REF:0000033
ACCEPT
Summary: The MCM ring, including Mcm4, is needed throughout fork progression as the motor of the CMG helicase.
Reason: Degron depletion of MCM proteins after initiation stops replication-fork progression in S. cerevisiae, which shows that MCM is the elongating fork helicase. Unwinding of template DNA is part of strand elongation. PAINT placed this term at the Mcm4 subfamily node PTN000179968, which covers NCU02539 (PTHR11630:SF66).
The eukaryotic helicase is an 11-subunit CMG complex that encircles the leading strand during unwinding and consists of the Mcm2-7 motor subunit
file:NEUCR/NCU02539/NCU02539-uniprot.txt
DR PANTHER; PTHR11630:SF66; DNA REPLICATION LICENSING FACTOR MCM4; 1.
GO:0006271 DNA strand elongation involved in DNA replication
IEA GO_REF:0000107
ACCEPT
Summary: The MCM ring, including Mcm4, is needed throughout fork progression as the motor of the CMG helicase.
Reason: Degron depletion of MCM proteins after initiation stops replication-fork progression in S. cerevisiae, which shows that MCM is the elongating fork helicase. Unwinding of template DNA is part of strand elongation. PAINT placed this term at the Mcm4 subfamily node PTN000179968, which covers NCU02539 (PTHR11630:SF66).
Summary: Premeiotic S phase uses the same Mcm2-7 replicative helicase as mitotic S phase. This is a valid but specialised context.
Reason: Mcm2-7 is the eukaryotic replicative helicase, and premeiotic DNA replication is nuclear genome replication before meiosis. The term is reasonable for a fungus with a sexual cycle, but no Neurospora experiment addresses it, and nuclear DNA replication already captures the core role.
The Mcm2-7 complex is the eukaryotic replicative helicase, a toroidal AAA(+) molecular motor that uses adenosine triphosphate (ATP) binding and hydrolysis to separate duplex DNA strands during replication.
file:NEUCR/NCU02539/NCU02539-uniprot.txt
DR PANTHER; PTHR11630:SF66; DNA REPLICATION LICENSING FACTOR MCM4; 1.
Summary: Transfer of an in vitro observation that the yeast Mcm4-6-7 subcomplex can drive Holliday-junction branch migration while translocating on duplex DNA.
Reason: Kaplan et al. (2003) showed that the isolated Mcm4,6,7 ring translocates along duplex DNA and so can push Holliday junctions. This is a mechanistic side effect of a subcomplex in vitro. The physiological helicase is CMG, built on the full Mcm2-7 ring, and no in vivo role of Mcm2-7 in four-way junction processing is known. The term overstates the biology for NCU02539.
Summary: Mcm4 contributes to ATP hydrolysis by the MCM ring. Its Walker A and B motifs are conserved, and active sites form at subunit interfaces.
Reason: MCM ATPase active sites are built from Walker motifs of one subunit and the arginine finger of the next. Single-subunit contribution is therefore the right description, matching the contributes_to qualifier. Purified S. pombe Mcm4/6/7 has ssDNA-dependent ATPase activity, and NCU02539 keeps all the catalytic motifs.
The Mcm2-7 complex is the eukaryotic replicative helicase, a toroidal AAA(+) molecular motor that uses adenosine triphosphate (ATP) binding and hydrolysis to separate duplex DNA strands during replication.
only the dimeric complex of the Mcm4/6/7 heterotrimer possessed single stranded DNA-dependent ATPase, ATP-dependent single stranded DNA binding, and 3' to 5' DNA helicase activities
Summary: Single-stranded DNA helicase activity is correct but less precise than supported. The MCM ring translocates 3' to 5' on the leading-strand template.
Reason: PAINT placed GO:0017116 at the MCM2-7 node PTN000179711, which covers the Mcm4 subfamily containing NCU02539, so the inheritance claim is sound. Biochemistry of the yeast complexes fixes the polarity: S. pombe Mcm4/6/7 has 3'-to-5' DNA helicase activity, and Mcm4,6,7 binds only the leading-strand template. GO:1990518 (single-stranded 3'-5' DNA helicase activity) is an is_a descendant of GO:0017116 and is already on this gene. Mcm4 contributes to the activity as a ring subunit.
Propagation Review
Root cause:TERM SCOPING PROBLEM
Failure modes:GRANULARITY MISMATCH
Sources checked:
PANTHER:PTN000179711SUPPORTS TRANSFER
MCM2-7 node seeded by yeast Mcm2-7 experimental helicase annotations; NCU02539 is inside this clade (PTHR11630:SF66). The transfer is sound; only the term granularity is refined to the 3'-5' child GO:1990518.
only the dimeric complex of the Mcm4/6/7 heterotrimer possessed single stranded DNA-dependent ATPase, ATP-dependent single stranded DNA binding, and 3' to 5' DNA helicase activities
Summary: Single-stranded DNA helicase activity is correct but less precise than supported. The MCM ring translocates 3' to 5' on the leading-strand template.
Reason: PAINT placed GO:0017116 at the MCM2-7 node PTN000179711, which covers the Mcm4 subfamily containing NCU02539, so the inheritance claim is sound. Biochemistry of the yeast complexes fixes the polarity: S. pombe Mcm4/6/7 has 3'-to-5' DNA helicase activity, and Mcm4,6,7 binds only the leading-strand template. GO:1990518 (single-stranded 3'-5' DNA helicase activity) is an is_a descendant of GO:0017116 and is already on this gene. Mcm4 contributes to the activity as a ring subunit.
only the dimeric complex of the Mcm4/6/7 heterotrimer possessed single stranded DNA-dependent ATPase, ATP-dependent single stranded DNA binding, and 3' to 5' DNA helicase activities
Summary: Transfer of the fission-yeast nuclear-structure term rDNA protrusion. Neurospora has no documented equivalent compartment for Mcm4, and the location adds nothing beyond chromatin.
Reason: rDNA protrusion is a term defined by the DAPI-visible rDNA arrays of S. pombe. MCM is loaded genome-wide at licensed origins, so rDNA repeats would carry MCM along with every other replicating region, and Mcm4 has no rDNA-specific role. Chromatin and nucleus capture the location. Carrying a fission-yeast-specific nuclear sub-structure over to Neurospora is an over-annotation.
The licensing of eukaryotic DNA replication origins, which ensures once-per-cell-cycle replication, involves the loading of six related minichromosome maintenance proteins (Mcm2-7) into prereplicative complexes (pre-RCs).
Summary: Loaded Mcm2-7 is joined by Cdc45 and other factors to form the preinitiation complex before origin firing.
Reason: Cdc45 binds chromatin after S-phase CDK activation, depending on Mcm2, and interacts physically with MCM proteins on chromatin. This defines the preinitiation complex built on the loaded MCM double hexamer. Mcm4, as a ring subunit, is part of this complex.
The licensing of eukaryotic DNA replication origins, which ensures once-per-cell-cycle replication, involves the loading of six related minichromosome maintenance proteins (Mcm2-7) into prereplicative complexes (pre-RCs).
Summary: Nuclear DNA replication is the core process of Mcm4, as a subunit of the licensing factor and replicative helicase.
Reason: Mcm2-7 is licensed at origins, activated into CMG and needed for fork progression. Together these make up nuclear DNA replication. NCU02539 is a full-length Mcm4-subfamily protein with intact motor motifs.
The licensing of eukaryotic DNA replication origins, which ensures once-per-cell-cycle replication, involves the loading of six related minichromosome maintenance proteins (Mcm2-7) into prereplicative complexes (pre-RCs).
Summary: Transfer of an in vitro observation that replicative helicases, including a eukaryotic MCM preparation, can displace RNA from a DNA strand while translocating on DNA.
Reason: Shin and Kelman (2006) showed RNA-DNA hybrid unwinding by replicative helicases while they translocate on ssDNA. This follows from the DNA-translocating mechanism and is not a separate function. The authors present a physiological role only as a possibility. The established activity is 3'-5' DNA helicase activity on DNA (GO:1990518). DNA/RNA hybrid helicase activity is an over-annotation unless an in vivo role is shown.
Summary: Mcm4 is a subunit of the heterohexameric Mcm2-7 (MCM) complex.
Reason: Mcm2-7 complexes containing Mcm4 have been purified and reconstituted from budding and fission yeast. PAINT placed MCM complex at the MCM2-7 node PTN000179711. NCU02539 is classified in the Mcm4 subfamily PTHR11630:SF66, and the InterPro MCM_4 signature identifies it as the Mcm4 subunit.
The licensing of eukaryotic DNA replication origins, which ensures once-per-cell-cycle replication, involves the loading of six related minichromosome maintenance proteins (Mcm2-7) into prereplicative complexes (pre-RCs).
file:NEUCR/NCU02539/NCU02539-uniprot.txt
DR PANTHER; PTHR11630:SF66; DNA REPLICATION LICENSING FACTOR MCM4; 1.
Summary: Mcm4 is a subunit of the heterohexameric Mcm2-7 (MCM) complex.
Reason: Mcm2-7 complexes containing Mcm4 have been purified and reconstituted from budding and fission yeast. PAINT placed MCM complex at the MCM2-7 node PTN000179711. NCU02539 is classified in the Mcm4 subfamily PTHR11630:SF66, and the InterPro MCM_4 signature identifies it as the Mcm4 subunit.
The licensing of eukaryotic DNA replication origins, which ensures once-per-cell-cycle replication, involves the loading of six related minichromosome maintenance proteins (Mcm2-7) into prereplicative complexes (pre-RCs).
file:NEUCR/NCU02539/NCU02539-uniprot.txt
DR PANTHER; PTHR11630:SF66; DNA REPLICATION LICENSING FACTOR MCM4; 1.
Summary: 3'-5' DNA helicase activity is correct, but the more specific single-stranded 3'-5' DNA helicase activity applies, since the ring translocates on ssDNA.
Reason: S. pombe Mcm4/6/7 has 3'-to-5' DNA helicase activity and translocates on the leading-strand template. GO:1990518 is an is_a descendant of GO:0043138 and is already on this gene. The contributes_to qualifier, reflecting that the activity belongs to the ring, should be kept.
only the dimeric complex of the Mcm4/6/7 heterotrimer possessed single stranded DNA-dependent ATPase, ATP-dependent single stranded DNA binding, and 3' to 5' DNA helicase activities
Summary: As the motor of the CMG helicase, Mcm4 travels with the nuclear replication fork.
Reason: CMG, made of the Mcm2-7 motor with Cdc45 and GINS, encircles the leading strand at the fork, and MCM function is needed throughout fork progression. Mcm4 is at the nuclear replication fork as part of the replisome.
Summary: Mcm4 is a subunit of the CMG (Cdc45-MCM-GINS) helicase, the active replicative helicase.
Reason: The eukaryotic helicase is the 11-subunit CMG complex, whose Mcm2-7 motor includes Mcm4. Cryo-EM of the budding-yeast replisome places CMG around the leading strand. The CMG complex is the active form in which NCU02539 does its core function.
Summary: Mcm4 forms the Mcm4/6/7 subcomplex, which has helicase activity in vitro. Whether this subcomplex has a separate in vivo role apart from Mcm2-7 is unclear.
Reason: S. pombe and S. cerevisiae Mcm4/6/7 complexes form stably and have helicase activity, and Lee and Hurwitz proposed Mcm4/6/7 as the catalytic core. Mcm4 is a real component. However, the physiological helicase is the full Mcm2-7 ring within CMG, so membership of the MCM core complex is kept as non-core.
only the dimeric complex of the Mcm4/6/7 heterotrimer possessed single stranded DNA-dependent ATPase, ATP-dependent single stranded DNA binding, and 3' to 5' DNA helicase activities
Summary: Mcm4 takes part in replication initiation in the mitotic cell cycle as part of the licensed and activated MCM helicase.
Reason: PAINT placed this term at the Mcm4 subfamily node PTN000179968, with S. pombe cdc21/Mcm4 as the seed. NCU02539 is in PTHR11630:SF66. Origin licensing and CMG activation are the MCM steps of initiation.
The licensing of eukaryotic DNA replication origins, which ensures once-per-cell-cycle replication, involves the loading of six related minichromosome maintenance proteins (Mcm2-7) into prereplicative complexes (pre-RCs).
During S phase, Cdc45p physically interacts with minichromosome maintenance (MCM) proteins on chromatin
file:NEUCR/NCU02539/NCU02539-uniprot.txt
DR PANTHER; PTHR11630:SF66; DNA REPLICATION LICENSING FACTOR MCM4; 1.
GO:1990518 single-stranded 3'-5' DNA helicase activity
IEA GO_REF:0000107
ACCEPT
Summary: Mcm4 contributes to the 3'-to-5' single-stranded DNA translocating helicase activity of the MCM ring. This is its core molecular function.
Reason: Purified Mcm2-7 is a helicase. S. pombe Mcm4/6/7 has ATP-dependent ssDNA binding and 3'-to-5' DNA helicase activity, and the Mcm4,6,7 ring unwinds by translocating on the leading-strand template. NCU02539 keeps the Mcm4 Walker A, Walker B and arginine-finger motifs. The contributes_to qualifier fits a subunit of a heterohexameric motor.
only the dimeric complex of the Mcm4/6/7 heterotrimer possessed single stranded DNA-dependent ATPase, ATP-dependent single stranded DNA binding, and 3' to 5' DNA helicase activities
The target Walker A motif GDPSTSKS at residues 645-652 is identical to that of both yeast Mcm4
Core Functions
Subunit of the Mcm2-7 ring that contributes to ATP-dependent 3'-5' single-stranded DNA helicase activity, as the motor of the CMG replicative helicase at nuclear replication forks.
only the dimeric complex of the Mcm4/6/7 heterotrimer possessed single stranded DNA-dependent ATPase, ATP-dependent single stranded DNA binding, and 3' to 5' DNA helicase activities
The licensing of eukaryotic DNA replication origins, which ensures once-per-cell-cycle replication, involves the loading of six related minichromosome maintenance proteins (Mcm2-7) into prereplicative complexes (pre-RCs).
Q: Does Neurospora regulate MCM nuclear export through the cell cycle, as budding yeast does, or are Mcm2-7 subunits nuclear throughout as in many other eukaryotes?
Q: Does the extended disordered N-terminal tail of Neurospora Mcm4 (residues 1-162) carry DDK/CDK phosphorylation sites that regulate CMG activation, as the Mcm4 N-terminal regulatory domain does in budding yeast?
Suggested Experiments
Experiment: Make a conditional (degron or temperature-sensitive) allele of NCU02539 and measure DNA content and origin firing, to confirm Mcm4 is needed for nuclear DNA replication in N. crassa.
Type: genetic depletion
External Prediction Reviews
These computational predictions are reviewed separately from the GOA annotation set used for this review. The assessments below are from this project and do not constitute official GO annotations or endorsement by GO/UniProt. They are not included in the existing annotation review above.
NCU02539 is the Neurospora Mcm4 subunit of the Mcm2-7 replicative helicase. The two process predictions repeat terms already in GOA and accepted by the gene review, so they are correct but not novel. The generic DNA-binding prediction is correct but less precise than the supported single-stranded DNA binding annotation.
Review rationale: NCU02539 carries the InterPro MCM_4 signature and is classified in the Mcm4 subfamily PTHR11630:SF66, the subfamily whose PAINT node gives it mitotic DNA replication initiation and DNA strand elongation. Budding-yeast experiments show that Mcm2-7 is loaded at origins during licensing and is needed for fork progression after initiation, which together make up nuclear DNA replication. GO:0033260 is already in GOA for this gene and the gene review accepts it as the level that covers both mitotic and premeiotic replication, so the prediction is correct but not novel.
PMID:19896182: "The licensing of eukaryotic DNA replication origins, which ensures once-per-cell-cycle replication, involves the loading of six related minichromosome maintenance proteins (Mcm2-7) into prereplicative complexes (pre-RCs)."
PMID:10834843: "depletion of MCMs after initiation irreversibly blocks the progression of replication forks in Saccharomyces cerevisiae"
Review rationale: Mcm4-subfamily membership places NCU02539 in the Mcm2-7 helicase. Mcm2-7 is loaded onto origins in the pre-replicative complex and becomes the motor of the CMG helicase that unwinds origin DNA, which is the Mcm2-7 contribution to initiation. GO:0006270 is already in GOA for this gene and is accepted in the gene review as a core process at the level that spans mitotic and premeiotic S phase, alongside the more specific IBA term GO:1902975. The prediction is correct but not novel. It concerns the Mcm4 subunit as part of the complex, not autonomous initiation activity.
PMID:19896182: "The licensing of eukaryotic DNA replication origins, which ensures once-per-cell-cycle replication, involves the loading of six related minichromosome maintenance proteins (Mcm2-7) into prereplicative complexes (pre-RCs)."
Review rationale: Purified yeast Mcm2-7 and Mcm4/6/7 complexes bind single-stranded DNA in an ATP-dependent way, and mutational analysis implicates Mcm4 in the single-stranded DNA binding of the complex. GO:0003697 single-stranded DNA binding, an is_a descendant of GO:0003677, is already annotated to this gene by PAINT (MCM2-7 node) and is accepted in the gene review, which refines the generic DNA-binding row to it. The generic prediction is correct but loses the substrate specificity, so it is less precise. The binding is a property of the assembled ring, not of an independent DNA-binding subunit.
PMID:17895243: "We demonstrate that both complexes are toroidal and possess a similar ATP-dependent single-stranded DNA (ssDNA) binding activity"
PMID:10567526: "the Mcm4 protein may play a role in the single-stranded DNA binding activity of the complex."
PMID:10770926: "only the dimeric complex of the Mcm4/6/7 heterotrimer possessed single stranded DNA-dependent ATPase, ATP-dependent single stranded DNA binding, and 3' to 5' DNA helicase activities"