| 1996 |
Xenopus Orc2-related protein (XORC2) is required for chromosomal DNA replication: immunodepletion of XORC2 from Xenopus egg extracts abolishes replication of chromosomal DNA but not elongation synthesis on single-stranded DNA templates. XORC2 binds chromatin prior to DNA synthesis and prior to loading of replication licensing factors. |
Immunodepletion from Xenopus egg extracts; indirect immunofluorescence |
Nature |
High |
8552193
|
| 1996 |
Fission yeast Orp2 (ORC2 ortholog) physically interacts with Cdc2 kinase and with the replication activator Cdc18, and is required for DNA replication; cells lacking Orp2 undergo aberrant mitosis indicating Orp2 is involved in generating a checkpoint signal. |
Genetic interaction screen, co-immunoprecipitation, loss-of-function analysis in S. pombe |
Nature |
High |
8552194
|
| 2000 |
Drosophila ORC2 is required for normal replication timing: two alleles of Drosophila ORC2 disrupt the normal early-euchromatin/late-heterochromatin replication pattern, causing some euchromatic regions to replicate abnormally late, and resulting in defective mitotic chromosome condensation in those late-replicating regions. |
Genetic mutant analysis, BrdU incorporation timing assay, cytological examination of mitotic chromosomes in Drosophila larvae |
Current biology : CB |
High |
11137005
|
| 2004 |
Human ORC2 localizes to centrosomes throughout the entire cell cycle and to centromeres and heterochromatin in a cell-cycle-dependent manner. ORC2 is tightly bound to heterochromatin and HP1α/HP1β during G1 and early S phase, but during late S, G2, and M phases chromatin association is restricted to centromeres. Depletion of ORC2 by siRNA disrupts HP1 localization (without affecting H3K9 methylation), causes S-phase defects with reduced PCNA on chromatin (though MCM proteins remain), induces abnormal chromosome condensation, failed chromosome congression, and multiple centrosomes. |
siRNA depletion, immunofluorescence, cell fractionation, co-immunoprecipitation with HP1α and HP1β |
The EMBO journal |
High |
15215892
|
| 2006 |
Human ORC2 contains a single ORC assembly domain required in vivo for interaction with all other ORC subunits, and two nuclear localization signals (NLS) required for ORC accumulation in the nucleus. In the nucleus, ORC2 exists as ORC(2-5) or ORC(1-5) complexes; only ORC(1-5) is chromatin-bound, indicating Orc1 is required to load ORC(2-5) onto chromatin. Additionally, ORC2 suppresses expression of endogenous ORC2, indicating cells limit intracellular ORC2 levels. |
Stable expression of epitope-tagged domain-deletion mutants in HeLa cells, chromatin fractionation, co-immunoprecipitation |
The Journal of biological chemistry |
High |
16762929
|
| 2006 |
Co-expression of ORC2 with ORC1 prevents ORC1-induced apoptosis and restores uniform nuclear localization of ORC1. In the absence of ORC2 co-expression, unmodified ORC1 accumulates perinuclearly and rapidly induces p53-independent apoptosis. |
Transient expression of ORC1 ± ORC2 in cell lines, immunofluorescence, apoptosis assays |
Journal of cell science |
Medium |
16537645
|
| 2011 |
Polo-like kinase 1 (Plk1) phosphorylates ORC2 at Ser188 in vitro and in vivo. This phosphorylation is enhanced under DNA replication stress (UV, hydroxyurea, gemcitabine, aphidicolin). Cells expressing the unphosphorylatable S188A ORC2 mutant have defective DNA synthesis under stress, fail to maintain functional pre-replicative complex, and activate the intra-S-phase checkpoint. |
In vitro kinase assay, in vivo phosphorylation (mass spectrometry), site-directed mutagenesis (S188A), BrdU incorporation, pre-RC chromatin binding assays, checkpoint activation assays |
Molecular and cellular biology |
High |
21947279
|
| 2012 |
Cyclin-dependent kinase phosphorylates ORC2 at Thr-116 and Thr-226 during S phase, causing dissociation of ORC2, ORC3, ORC4, and ORC5 subunits from chromatin and replication origins. Phosphomimetic ORC2 shows defective binding to replication origins and chromatin; phosphodefective ORC2 persists in chromatin binding throughout the cell cycle. |
In vitro CDK phosphorylation assay, phosphomimetic and phosphodefective mutagenesis, chromatin fractionation, ChIP at replication origins |
The Journal of biological chemistry |
High |
22334659
|
| 2012 |
ORC2 protects the ORC-associated protein ORCA/LRWD1 from ubiquitin-mediated degradation. ORCA is polyubiquitinated via K48-linked chains by Cul4A-DDB1 E3 ligase at the WD40 repeat domain. ORC2 binds exclusively the non-ubiquitinated form of ORCA, and depletion of ORC2 leads to proteasome-mediated destabilization of ORCA. |
In vivo ubiquitination assay, co-immunoprecipitation, siRNA knockdown, proteasome inhibitor treatment |
Cell cycle (Georgetown, Tex.) |
Medium |
22935713
|
| 2012 |
Plk1 phosphorylation of ORC2 (at Ser188) maintains DNA replication under gemcitabine treatment; cells expressing a Plk1-unphosphorylatable ORC2 mutant are more sensitive to gemcitabine than wild-type ORC2-expressing cells. |
Phosphomimetic/phosphodefective ORC2 mutant expression in cancer cells, drug sensitivity assays, mouse xenograft model |
Molecular cancer therapeutics |
Medium |
23188630
|
| 2014 |
Protein phosphatase 1 (PP1) physically interacts with ORC2 via the consensus PP1-binding motif 119-KSVSF-123 on ORC2. PP1 dephosphorylates ORC2 at Thr116 and Thr226 in a cell-cycle-dependent manner (late M phase), which is required for re-binding of ORC2 and associated subunits to chromatin and replication origins. |
Co-immunoprecipitation of PP1 and ORC2, PP1 inhibitor treatment, overexpression and siRNA knockdown of PP1 isoforms, chromatin fractionation |
Biochemical and biophysical research communications / Biochemical and biophysical research communications |
Medium |
24732362 24792176
|
| 2016 |
ORC2 is SUMOylated by SUMO2 (but not SUMO1) at the G2/M phase of the cell cycle. SUMO2-modified ORC2 recruits the histone demethylase KDM5A to centromeres to convert H3K4me3 to H3K4me2, a permissive histone mark for α-satellite transcription. Loss of ORC2 SUMOylation (SUMO-less ORC2) reduces α-satellite transcription, impairs pericentric heterochromatin silencing, leads to heterochromatin DNA re-replication, activates DNA damage response, and causes polyploidy. |
In vivo SUMOylation assays, co-immunoprecipitation of KDM5A with SUMO2-ORC2, stable expression of SUMO-less ORC2 mutant, chromatin analysis, flow cytometry |
Cell reports |
High |
27052177
|
| 2016 |
Papillomavirus E2 protein binds ORC2; however, ORC2 is not detected at the viral origin. ORC2 depletion enhances PV replication and increases E1/E2 occupancy at the viral origin, indicating ORC2 suppresses E2 replicative function rather than promoting viral replication. Over-expression of HPV E2 decreases ORC2 occupation at mammalian replication origins. |
Co-immunoprecipitation of E2 and ORC2, siRNA depletion of ORC2, transient replication assay, ChIP at viral and cellular origins |
PLoS pathogens |
Medium |
27701460
|
| 2017 |
ORC2 SUMOylation is reversibly regulated: SUMO E3 ligase PIAS4 promotes ORC2 SUMOylation at G2/M, while de-SUMOylase SENP2 removes it. Depletion of PIAS4 or overexpression of SENP2 eliminates ORC2 SUMOylation, causes abnormal centromeric H3K4 methylation, and results in mitotic bypass and polyploidy; co-expression of ORC2-SUMO2 fusion protein reduces polyploid cell formation. |
PIAS4 siRNA depletion, SENP2 overexpression, ORC2-SUMO2 fusion rescue, flow cytometry, histone methylation analysis |
Oncotarget |
Medium |
29050267
|
| 2019 |
The ORC2 winged-helix domain (WHD) binds dsDNA through a flexible β-sheet hairpin anchor region with key residues R540, K548, and K549. NMR chemical shift perturbations reveal a unique dsDNA binding topology distinct from archaeal and yeast ORC WHDs; mutagenesis of these residues validates their importance for binding. |
Crystal structure determination, NMR backbone assignments and chemical shift perturbation, site-directed mutagenesis of DNA-binding residues, molecular dynamics simulation |
The FEBS journal |
High |
30963726
|
| 2020 |
Human HCT116 cancer cells can survive and maintain normal MCM2-7 chromatin loading and origin firing even when both ORC2 and ORC5 proteins are eliminated by CRISPR-Cas9 mutation, causing destabilization of ORC1, ORC3, and ORC4 as well. This demonstrates that in these selected cancer cells, the six-subunit ORC is not strictly required for MCM2-7 loading or origin specification. |
CRISPR-Cas9 gene editing, chromatin fractionation, origin firing assays, Western blot |
The Journal of biological chemistry |
Medium |
32989049
|
| 2024 |
The ORFIUS complex (BRD1, HBO1, BRCA1, BARD1) promotes ORC2 localization at replication origins. Depletion of BRD1 and/or HBO1 reduces origin firing and reduces the number of nuclei with ORC2 foci. BRCA1 regulates BRD1, HBO1, and ORC2 localization at origins; in BRCA1-mutant HGSC cells, ORC2 remains at origins and is unresponsive to replication stress signals. |
siRNA/CRISPR depletion of complex components, immunofluorescence for ORC2 foci, origin firing assays, ChIP |
NAR cancer |
Medium |
38288445
|
| 2025 |
DNA-bound ORC2 (independent of the full six-subunit ORC) compacts chromatin and attracts repressive histone marks at focal genomic sites, while also activating chromatin and protecting genes from repressive marks at other sites. ORC2 also prevents CTCF acquisition at focal sites to regulate chromatin loops and indirectly affects epigenetics. Individual ORC subunits bind thousands of sites without co-occupancy of other subunits. |
Multi-omics analysis (ChIP-seq, ATAC-seq, Hi-C) in ORC-mutant cancer cell lines (ORC1, ORC2, ORC5 CRISPR knockouts) |
Cell reports |
Medium |
40504688
|
| 2002 |
Elevated cyclin A-dependent kinase activity in Xenopus egg extract prevents XORC2 from binding to chromatin from permeable erythrocyte nuclei; kinase inhibition reverses this effect. However, inhibiting nuclear-accumulated kinase activity within intact erythrocyte nuclei does not facilitate XORC2 binding to chromatin, suggesting additional mechanisms prevent ORC association within intact terminally differentiated nuclei. |
Xenopus egg extract replication system, permeable and intact erythrocyte nuclei, cyclin A-CDK titration, kinase inhibitor treatment |
Experimental cell research |
Medium |
11900493
|
| 2011 |
Sp1 transcription factor participates in recruiting ORC2 to the chicken lysozyme GAS41 replication origin; knockdown of Sp1 by RNA interference reduces specific ORC2 binding to this origin, which maps to a region containing multiple Sp1/Sp3-binding sites coinciding with DNase I hypersensitive sites. |
Chromatin immunoprecipitation (ChIP), quantitative real-time PCR, siRNA knockdown of Sp1 |
DNA and cell biology |
Low |
21879882
|