Affinage

ORC5

Origin recognition complex subunit 5 · UniProt O43913

Length
435 aa
Mass
50.3 kDa
Annotated
2026-06-10
14 papers in source corpus 10 papers cited in narrative 11 extracted findings
Cross-family judge vs UniProt: Affinage preferred faithfulness: 5/5 claims corpus-supported (100%)

Mechanistic narrative

Synthesis pass · prose summary of the discoveries below

ORC5 is a core AAA+ ATPase subunit of the origin recognition complex (ORC), the multiprotein machine that marks replication origins, and was identified as a bona fide ORC subunit through co-purification with other ORC subunits in both human and Xenopus systems (PMID:9765232, PMID:9829972). Its structural integrity within the complex depends on ATP binding: mutation of the Walker A motif of yeast Orc5p disrupts its interaction with Orc4p—mediated by the N-terminal region of Orc5p and the C-terminal region of Orc4p—and triggers proteasomal degradation of the entire ORC, a defect rescued by Orc4p overproduction (PMID:14625297, PMID:17107343). Genetic dissection in budding and fission yeast reveals that ORC5 carries mechanistically separable functions in DNA replication initiation, transcriptional silencing, and, in fission yeast, loading of the cohesin subunit Rad21 for sister chromatid cohesion (PMID:9383052, PMID:18414064). In human cells, ORC5 contributes to origin licensing chromatin remodeling by associating with the histone H3 acetyltransferase GCN5 (KAT2A) to drive G1-phase chromatin decondensation and H3 acetylation at origins (PMID:26644179). Strikingly, CRISPR deletion of ORC5—alone or together with ORC2—in human cancer cell lines does not prevent normal MCM2-7 chromatin loading or origin firing, indicating the six-subunit ORC is dispensable for these steps in certain cellular contexts (PMID:32989049).

Mechanistic history

Synthesis pass · year-by-year structured walk · 9 steps
  1. 1998 High

    Establishing whether a vertebrate ORC5 homolog exists as a physical subunit of the origin recognition complex was the first step in defining its role; co-purification fixed it as a core ORC subunit conserved from yeast to humans.

    Evidence Co-immunoprecipitation and subcellular fractionation in human extracts, and single-step immunoaffinity purification with protein microsequencing in Xenopus

    PMID:9765232 PMID:9829972

    Open questions at the time
    • Stoichiometry and architecture of ORC5 within the assembled complex not resolved
    • Functional significance of the insoluble nuclear pool of human ORC5 unexplained
    • Role of the alternatively spliced HsORC5T isoform not functionally tested
  2. 1997 High

    Whether ORC5 served only in replication or had additional roles was unknown; allele dissection showed its replication and transcriptional silencing functions are genetically separable.

    Evidence Genetic complementation, allele analysis, and suppressor screening in S. cerevisiae

    PMID:9383052

    Open questions at the time
    • Molecular basis distinguishing the silencing and replication functions not defined
    • Does not establish which protein regions mediate each separable activity
  3. 2003 High

    The functional consequence of ATP binding by ORC5 was unclear; Walker A mutagenesis showed ATP binding is required for ORC stability and identified Orc4p as a specific functional partner.

    Evidence Walker A (K43E) mutagenesis, cell-cycle analysis, immunoblotting, and overexpression suppressor analysis in yeast

    PMID:14625297

    Open questions at the time
    • Whether ATP hydrolysis (versus binding) contributes was not separated
    • Mechanism linking ORC5 ATP status to whole-complex degradation not yet defined at this stage
  4. 2007 High

    How ORC5 ATP binding stabilized the complex was unresolved; the work showed ATP binding promotes the Orc5p-Orc4p interaction, whose loss causes proteasomal degradation of ORC.

    Evidence Yeast two-hybrid, co-immunoprecipitation, proteasome inhibitor treatment, and proteasome mutant strains

    PMID:17107343

    Open questions at the time
    • The ubiquitin ligase targeting destabilized ORC was not identified
    • Whether this regulatory degradation operates in metazoan cells untested
  5. 2008 High

    Whether ORC5 functions extend beyond replication into chromosome segregation was unknown; fission yeast alleles separated a replication-initiation function from a cohesin-loading function.

    Evidence Temperature-sensitive allele and execution-point analysis, FACS, checkpoint epistasis, and direct Rad21 cohesin loading assays in S. pombe

    PMID:18414064

    Open questions at the time
    • Molecular mechanism by which ORC5 promotes Rad21 loading not defined
    • Conservation of the cohesion function in metazoans untested
  6. 2015 Medium

    How ORC5 acts on chromatin at origins in human cells was unclear; tethering and interaction data linked it to GCN5-dependent chromatin decondensation and origin H3 acetylation.

    Evidence Chromatin tethering with live imaging, Orc5-GCN5 co-IP, and ChIP for H3 acetylation after Orc5 knockdown in human cells

    PMID:26644179

    Open questions at the time
    • Direct versus indirect nature of the Orc5-GCN5 interaction not resolved
    • Whether chromatin decondensation is required for origin licensing not established
  7. 2016 Medium

    Whether ORC5 serves as a docking point for replication-stress factors was unknown; mapping showed an N-terminal domain of the Rrm3 helicase binds Orc5 to control DNA synthesis under stress.

    Evidence Domain deletion mapping, binding/pulldown assays, genetic epistasis, and quantitative mass spectrometry in yeast

    PMID:27923055

    Open questions at the time
    • Structural basis of the Rrm3-Orc5 interaction not defined
    • Generality of ORC5 as a helicase-recruiting platform untested in other systems
  8. 2020 High

    Whether the six-subunit ORC is strictly required for MCM2-7 loading in human cells was assumed but untested; CRISPR knockout showed ORC5-null cancer cells still load MCM2-7 and fire origins normally.

    Evidence CRISPR-Cas9 deletion of ORC5 and ORC2, chromatin fractionation for MCM2-7, and DNA combing origin-firing assays in HCT116 cells

    PMID:32989049

    Open questions at the time
    • Whether residual undetected ORC subunits provide partial function not fully excluded
    • Whether ORC dispensability is specific to transformed/cancer cells unresolved
  9. 2024 Medium

    Whether origin site specification itself depends on ORC was open; genome-wide mapping in ORC5-deleted cells showed origins fire at the same sites with comparable excess MCM2-7 loading.

    Evidence Genome-wide origin mapping and MCM2-7 loading assays in CRISPR ORC5-deleted human cancer cell lines (preprint)

    PMID:bio_10.1101_2024.10.30.621095

    Open questions at the time
    • Single lab, preprint status not peer-reviewed
    • Alternative origin-licensing machinery operating without ORC not identified
    • Whether non-transformed cells behave the same untested

Open questions

Synthesis pass · forward-looking unresolved questions
  • The factor(s) that specify and license replication origins in the absence of a functional six-subunit ORC, and the reconciliation of ORC's essential role in yeast with its dispensability in human cancer cells, remain unresolved.
  • No alternative origin-licensing mechanism identified that bypasses ORC
  • Mechanism distinguishing ORC-dependent and ORC-independent cellular contexts unknown

Mechanism profile

Synthesis pass · controlled-vocabulary classification · explore literature graph →
Molecular activity
GO:0140657 ATP-dependent activity 3 GO:0003677 DNA binding 2
Localization
GO:0005694 chromosome 3 GO:0005634 nucleus 1
Pathway
R-HSA-69306 DNA Replication 3 R-HSA-1640170 Cell Cycle 2 R-HSA-4839726 Chromatin organization 1
Partners
GCN5ORC1ORC2ORC4RRM3
Complex memberships
origin recognition complex (ORC)

Evidence

Reading pass · 11 per-paper findings extracted from the source corpus
Year Finding Method Journal Conf PMIDs
1998 Human ORC5 (HsORC5p) is a 50-kDa protein that co-precipitates with HsORC2p and HsORC4p from cell extracts, establishing it as a bona fide subunit of the human origin recognition complex. The bulk of HsORC5p resides in an insoluble nuclear fraction, unlike HsORC1p, HsORC2p, and HsORC4p which are extractable in nuclear-soluble fractions. Co-immunoprecipitation from cell extracts; subcellular fractionation The Journal of biological chemistry Medium 9765232
1998 An alternatively spliced isoform (HsORC5T) from the ORC5 locus forms a complex with HsORC4p but not with HsORC2p, suggesting it may play a regulatory role in the assembly of different ORC subcomplexes. Co-immunoprecipitation from cell extracts The Journal of biological chemistry Low 9765232
1998 Immunoaffinity purification of Xenopus ORC using anti-Orc1p antibodies co-purified six polypeptides including Orc5p (~48 kDa), identifying Orc5p as a core subunit of the vertebrate ORC. Sequence comparison revealed that Orc5p is structurally related to Orc1p, Orc4p, and the replication initiation protein Cdc6p. Single-step immunoaffinity purification; microsequencing/protein sequencing; sequence homology analysis The Journal of biological chemistry High 9829972
1997 In Saccharomyces cerevisiae, ORC5 has separable functions in DNA replication initiation and transcriptional silencing: spontaneous revertants of orc5-1 were recovered that restored replication but not silencing, and other alleles were non-functional for replication but fully competent for silencing. Complementation between these two classes of alleles in the same cell established that the two functions are mechanistically separable. Genetic complementation; allele analysis; suppressor screen in yeast Genetics High 9383052
2003 Mutation of the Walker A motif (K43E) of yeast Orc5p causes temperature-sensitive growth and G2/M cell-cycle arrest. At non-permissive temperature, all ORC subunits are degraded. Overproduction of Orc4p, but not other ORC subunits, specifically suppresses this temperature sensitivity, indicating that Orc4p is specifically involved in the function of ATP binding to Orc5p or its role in DNA replication. Walker A motif mutagenesis in yeast; immunoblotting; overexpression suppressor analysis; cell-cycle analysis The Journal of biological chemistry High 14625297
2007 ATP binding to yeast Orc5p is required for efficient interaction with Orc4p; the orc5-A (Walker A) mutation diminishes the Orc5p–Orc4p interaction, leading to proteasomal degradation of the entire ORC. The interaction is mediated by the C-terminal region of Orc4p and the N-terminal region of Orc5p. Overproduction of Orc4p restores this interaction and suppresses ORC degradation. Yeast two-hybrid; co-immunoprecipitation; proteasome inhibitor treatment; proteasome mutant strains The Biochemical journal High 17107343
2008 In Schizosaccharomyces pombe, Orc5 has at least two genetically separable functions: temperature-sensitive allele orc5-H19 is defective in DNA replication initiation (with ORC function required before metaphase for next-cycle replication), while allele orc5-H37 completes DNA synthesis but arrests before M phase and shows premature sister chromatid separation due to a defect in loading of the cohesin component Rad21. Both mutants activate the rad3-chk1 checkpoint. Temperature-sensitive allele analysis; execution point analysis; FACS; checkpoint mutant epistasis; cohesin loading assay Cell cycle (Georgetown, Tex.) High 18414064
2015 Human Orc5, when ectopically tethered to a chromatin locus, induces large-scale chromatin decondensation predominantly during G1 phase. Orc5 associates with the histone H3 acetyltransferase GCN5 (KAT2A), and this association enhances Orc5's chromatin-opening function. Depletion of Orc5 reduces histone H3 acetylation at origins. Chromatin tethering assay (live imaging); co-immunoprecipitation (Orc5–GCN5); ChIP for H3 acetylation after Orc5 knockdown Journal of cell science Medium 26644179
2016 The yeast helicase Rrm3 associates with replication origins and controls DNA synthesis during replication stress via an N-terminal domain (residues 186–212) that is required for binding Orc5 of the ORC. This Orc5-binding domain-dependent function of Rrm3 is genetically separable from its ATPase/helicase activity in facilitating fork progression. Domain deletion mapping; pulldown/binding assay; genetic epistasis; quantitative mass spectrometry PLoS genetics Medium 27923055
2020 Human HCT116 colon cancer cells engineered via CRISPR-Cas9 to lack detectable ORC5 protein (with loss of 80% of the AAA+ ATPase domain including the Walker A motif) survive, show normal chromatin binding of MCM2-7, and initiate DNA replication from a similar number of origins as wild-type cells. Double-knockout cells lacking both ORC5 and ORC2 (which also destabilizes ORC1, ORC3, and ORC4) still recruit MCM2-7 normally and initiate replication, indicating that the six-subunit ORC is dispensable for MCM2-7 loading and origin firing in these cancer cells. CRISPR-Cas9 gene editing; chromatin fractionation for MCM2-7 binding; DNA combing/origin firing assay The Journal of biological chemistry High 32989049
2024 Mapping of replication origins in ORC5-deleted human cancer cell lines shows that specific origins are still used at mostly the same genomic sites as in wild-type cells, and excess MCM2-7 is still loaded at comparable rates in G1 phase, establishing that origin specification and excess MCM2-7 loading do not require the six-subunit ORC in human cancer cell lines. CRISPR-Cas9 ORC5 deletion; genome-wide origin mapping; MCM2-7 chromatin loading assays bioRxivpreprint Medium bio_10.1101_2024.10.30.621095

Source papers

Stage 0 corpus · 14 papers · ranked by NIH iCite citations
Year Title Journal Citations PMID
1998 The Orc4p and Orc5p subunits of the Xenopus and human origin recognition complex are related to Orc1p and Cdc6p. The Journal of biological chemistry 83 9829972
1998 ORC5L, a new member of the human origin recognition complex, is deleted in uterine leiomyomas and malignant myeloid diseases. The Journal of biological chemistry 65 9765232
1997 Separable functions of ORC5 in replication initiation and silencing in Saccharomyces cerevisiae. Genetics 64 9383052
2020 Juvenile hormone acts through FoxO to promote Cdc2 and Orc5 transcription for polyploidy-dependent vitellogenesis. Development (Cambridge, England) 37 32907849
1997 Isolation of human and fission yeast homologues of the budding yeast origin recognition complex subunit ORC5: human homologue (ORC5L) maps to 7q22. Genomics 37 9417919
2020 A human cancer cell line initiates DNA replication normally in the absence of ORC5 and ORC2 proteins. The Journal of biological chemistry 29 32989049
2016 A Novel Rrm3 Function in Restricting DNA Replication via an Orc5-Binding Domain Is Genetically Separable from Rrm3 Function as an ATPase/Helicase in Facilitating Fork Progression. PLoS genetics 17 27923055
2008 Schizosaccharomyces pombe Orc5 plays multiple roles in the maintenance of genome stability throughout the cell cycle. Cell cycle (Georgetown, Tex.) 15 18414064
2003 Analysis on origin recognition complex containing Orc5p with defective Walker A motif. The Journal of biological chemistry 15 14625297
2015 Orc5 induces large-scale chromatin decondensation in a GCN5-dependent manner. Journal of cell science 12 26644179
2021 PSMC2, ORC5 and KRTDAP are specific biomarkers for HPV-negative head and neck squamous cell carcinoma. Oncology letters 10 33732365
2001 Mutation analysis of the origin recognition complex subunit 5 (ORC5L) gene in adult patients with myeloid leukemias exhibiting deletions of chromosome band 7q22. Human genetics 10 11379876
1997 Sequence analysis of the 33 kb long region between ORC5 and SUI1 from the left arm of chromosome XIV from Saccharomyces cerevisiae. Yeast (Chichester, England) 8 9234673
2007 Mechanism for the degradation of origin recognition complex containing Orc5p with a defective Walker A motif and its suppression by over-production of Orc4p in yeast cells. The Biochemical journal 6 17107343

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