Architecture of the human origin recognition complex

All the human homologs of the six subunits of Saccharomyces cerevisiae origin recognition complex have been reported so far. However, not much has been reported on the nature and the characteristics of the human origin recognition complex. In an attempt to purify recombinant human ORC from insect cells infected with baculoviruses expressing HsORC subunits, we found that human ORC2, -3, -4, and -5 form a core complex. HsORC1 and HsORC6 subunits did not enter into this core complex, suggesting that the interaction of these two subunits with the core ORC2-5 complex is extremely labile. We found that the C-terminal region of ORC2 interacts directly with the N-terminal region of ORC3. The C-terminal region of ORC3 was, however, necessary to bring ORC4 and ORC5 into the core complex. A fragment containing the N-terminal 200 residues of ORC3 (ORC3N) competitively inhibited the ORC2-ORC3 interaction. Overexpression of this fragment in U2OS cells blocked the cells in G(1), providing the first evidence that a mammalian ORC subunit is important for the G(1)-S transition in mammalian cells.     

Assembly of the human origin recognition complex

The six-subunit origin recognition complex (ORC) was originally identified in the yeast Saccharomyces cerevisiae. Yeast ORC binds specifically to origins of replication and serves as a platform for the assembly of additional initiation factors, such as Cdc6 and the Mcm proteins. Human homologues of all six ORC subunits have been identified by sequence similarity to their yeast counterparts, but little is known about the biochemical characteristics of human ORC (HsORC). We have extracted HsORC from HeLa cell chromatin and probed its subunit composition using specific antibodies. The endogenous HsORC, identified in these experiments, contained homologues of Orc1-Orc5 but lacked a putative homologue of Orc6. By expressing HsORC subunits in insect cells using the baculovirus system, we were able to identify a complex containing all six subunits. To explore the subunit-subunit interactions that are required for the assembly of HsORC, we carried out extensive co-immunoprecipitation experiments with recombinant ORC subunits expressed in different combinations. These studies revealed the following binary interactions: HsOrc2-HsOrc3, HsOrc2-HsOrc4, HsOrc3-HsOrc4, HsOrc2-HsOrc6, and HsOrc3-HsOrc6. HsOrc5 did not form stable binary complexes with any other HsORC subunit but interacted with sub-complexes containing any two of subunits HsOrc2, HsOrc3, or HsOrc4. Complex formation by HsOrc1 required the presence of HsOrc2, HsOrc3, HsOrc4, and HsOrc5 subunits. These results suggest that the subunits HsOrc2, HsOrc3, and HsOrc4 form a core upon which the ordered assembly of HsOrc5 and HsOrc1 takes place. The characterization of HsORC should facilitate the identification of human origins of DNA replication.     

The origin recognition complex in human diseases

ORC (origin recognition complex) serves as the initiator for the assembly of the pre-RC (pre-replication complex) and the subsequent DNA replication. Together with many of its non-replication functions, ORC is a pivotal regulator of various cellular processes. Notably, a number of reports connect ORC to numerous human diseases, including MGS (Meier–Gorlin syndrome), EBV (Epstein–Barr virus)-infected diseases, American trypanosomiasis and African trypanosomiasis. However, much of the underlying molecular mechanism remains unclear. In those genetic diseases, mutations in ORC alter its function and lead to the dysregulated phenotypes; whereas in some pathogen-induced symptoms, host ORC and archaeal-like ORC are exploited by these organisms to maintain their own genomes. In this review, I provide detailed examples of ORC-related human diseases, and summarize the current findings on how ORC is involved and/or dysregulated. I further discuss how these discoveries can be generalized as model systems, which can then be applied to elucidating other related diseases and revealing potential targets for developing effective therapies.     

RNA-dependent recruitment of the origin recognition complex

The origin recognition complex (ORC) has an important function in determining the initiation sites of DNA replication. In higher eukaryotes, ORC lacks sequence-specific DNA binding, and the mechanisms of ORC recruitment and origin determination are poorly understood. ORC is recruited with high efficiency to the Epstein-Barr virus origin of plasmid replication (OriP) through a complex mechanism involving interactions with the virus-encoded EBNA1 protein. We present evidence that ORC recruitment to OriP and DNA replication function depends on RGG-like motifs, referred to as LR1 and LR2, in the EBNA1 amino-terminal domain. Moreover, we show that LR1 and LR2 recruitment of ORC is RNA dependent. HMGA1a, which can functionally substitute for LR1 and LR2 domain, can also recruit ORC in an RNA-dependent manner. EBNA1 and HMGA1a RGG motifs bound to structured G-rich RNA, as did ORC1 peptides, which interact with EBNA1. RNase A treatment of cellular chromatin released a fraction of the total ORC, suggesting that ORC association with chromatin, and possibly cellular origins, is stabilized by RNA. We propose that structural RNA molecules mediate ORC recruitment at some cellular and viral origins, similar to OriP.     

An exonic splicing silencer represses spliceosome assembly after ATP-dependent exon recognition

Precursor messenger RNA splicing is catalyzed by the spliceosome, a macromolecular complex that assembles in a stepwise process. The spliceosome's dynamic nature suggests the potential for regulation at numerous points along the assembly pathway; however, thus far, naturally occurring regulation of splicing has only been found to influence a small subset of spliceosomal intermediates. Here we report that the exonic splicing silencer (ESS1) that represses splicing of PTPRC (encoding CD45) exon 4 does not function by the typical mechanism of inhibiting binding of U1 or U2 small nuclear ribonucleoproteins (snRNPs) to the splice sites. Instead, a U1-, U2- and ATP-dependent complex forms across exon 4 that is required for inhibiting progression to the U4-U6-U5 tri-snRNP-containing B complex. Such inhibition represents a new mechanism for splicing regulation and suggests that regulation can probably occur at many of the transitions along the spliceosome assembly pathway.     

The origin recognition complex marks a replication origin in the human TOP1 gene promoter

The locations of the origin recognition complex (ORC) in mammalian genomes have been elusive. We have therefore analyzed the DNA sequences associated with human ORC via in vivo cross-linking and chromatin immunoprecipitation. Antibodies specific for hOrc2 protein precipitate chromatin fragments that also contain other ORC proteins, suggesting that the proteins form multisubunit complexes on chromatin in vivo. A binding region for ORC was identified at the CpG island upstream of the human TOP1 gene. Nascent strand abundance assays show that the ORC binding region coincides with an origin of bidirectional replication. The TOP1 gene includes two well characterized matrix attachment regions. The matrix attachment region elements analyzed contain no ORC and constitute no sites for replication initiation. In initial attempts to use the chromatin immunoprecipitation technique for the identification of additional ORC sites in the human genome, we isolated a sequence close to another actively transcribed gene (TOM1) and an alphoid satellite sequence that underlies centromeric heterochromatin. Nascent strand abundance assays gave no indication that the heterochromatin sequence serves as a replication initiation site, suggesting that an ORC on this site may perform functions other than replication initiation.     

The many faces of the origin recognition complex

The hetero-hexameric origin recognition complex (ORC) is well known for its separable roles in DNA replication and heterochromatin assembly. However, ORC and its individual subunits have been implicated in diverse cellular activities in both the nucleus and the cytoplasm. Some of ORC's implied functions, such as cell cycle checkpoint control and mitotic chromosome assembly, may be indirectly related to its roles in replication control and/or heterochromatin assembly. Other suggested roles in ribosomal biogenesis and in centrosome and kinetochore function are based on localization/interaction data and are as yet inconclusive. However, recent findings directly link ORC to sister chromatin cohesion, cytokinesis and neural dendritic branching.     

The origin recognition complex protein family

Origin recognition complex (ORC) proteins were first discovered as a six-subunit assemblage in budding yeast that promotes the initiation of DNA replication. Orc1-5 appear to be present in all eukaryotes, and include both AAA+ and winged-helix motifs. A sixth protein, Orc6, shows no structural similarity to the other ORC proteins, and is poorly conserved between budding yeast and most other eukaryotic species. The replication factor Cdc6 has extensive sequence similarity with Orc1 and phylogenetic analysis suggests the genes that encode them may be paralogs. ORC proteins have also been found in the archaea, and the bacterial DnaA replication protein has ORC-like functional domains. In budding yeast, Orc1-6 are bound to origins of DNA replication throughout the cell cycle. Following association with Cdc6 in G1 phase, the sequential hydrolysis of Cdc6 - then ORC-bound ATP loads the Mcm2-7 helicase complex onto DNA. Localization of ORC subunits to the kinetochore and centrosome during mitosis and to the cleavage furrow during cytokinesis has been observed in metazoan cells and, along with phenotypes observed following knockdown with short interfering RNAs, point to additional roles at these cell-cycle stages. In addition, ORC proteins function in epigenetic gene silencing through interactions with heterochromatin factors such as Sir1 in budding yeast and HP1 in higher eukaryotes. Current avenues of research have identified roles for ORC proteins in the development of neuronal and muscle tissue, and are probing their relationship to genome integrity.     

Genetic analysis of human Orc2 reveals specific domains that are required in vivo for assembly and nuclear localization of the origin recognition complex

Eukaryotic DNA replication begins with the binding of a six subunit origin recognition complex (ORC) to DNA. To study the assembly and function of mammalian ORC proteins in their native environment, HeLa cells were constructed that constitutively expressed an epitope-tagged, recombinant human Orc2 subunit that had been genetically altered. Analysis of these cell lines revealed that Orc2 contains a single ORC assembly domain that is required in vivo for interaction with all other ORC subunits, as well as two nuclear localization signals (NLSs) that are required for ORC accumulation in the nucleus. The recombinant Orc2 existed in the nucleus either as an ORC-(2-5) or ORC-(1-5) complex; no other combinations of ORC subunits were detected. Moreover, only ORC-(1-5) was bound to the chromatin fraction, suggesting that Orc1 is required in vivo to load ORC-(2-5) onto chromatin. Surprisingly, recombinant Orc2 suppressed expression of endogenous Orc2, revealing that mammalian cells limit the intracellular level of Orc2, and thereby limit the amount of ORC-(2-5) in the nucleus. Because this suppression required only the ORC assembly and NLS domains, these domains appear to constitute the functional domain of Orc2.     

Assembly of the human origin recognition complex occurs through independent nuclear localization of its components

Initiation of eukaryotic genome duplication begins when a six-subunit origin recognition complex (ORC) binds to DNA. However, the mechanism by which this occurs in vivo and the roles played by individual subunits appear to differ significantly among organisms. Previous studies identified a soluble human ORC(2-5) complex in the nucleus, an ORC(1-5) complex bound to chromatin, and an Orc6 protein that binds weakly, if at all, to other ORC subunits. Here we show that stable ORC(1-6) complexes also can be purified from human cell extracts and that Orc6 and Orc1 each contain a single nuclear localization signal that is essential for nuclear localization but not for ORC assembly. The Orc6 nuclear localization signal, which is essential for Orc6 function, is facilitated by phosphorylation at its cyclin-dependent kinase consensus site and by association with Kpna6/1, nuclear transport proteins that did not co-purify with other ORC subunits. These and other results support a model in which Orc6, Orc1, and ORC(2-5) are transported independently to the nucleus where they can either assemble into ORC(1-6) or function individually.     

ADP-binding to origin recognition complex of Saccharomyces cerevisiae

The origin recognition complex (ORC), a possible initiator of chromosomal DNA replication in eukaryotes, binds to ATP through its subunits Orc1p and Orc5p. Orc1p possesses ATPase activity. As for DnaA, the Escherichia coli initiator, the ATP-DnaA complex is active but the ADP-DnaA complex is inactive for DNA replication and, therefore, the ATPase activity of DnaA inactivates the ATP-DnaA complex to suppress the re-initiation of chromosomal DNA replication. We investigated ADP-binding to ORC by a filter-binding assay. The K(d) values for ADP-binding to wild-type ORC and to ORC-1A (ORC containing Orc1p with a defective Walker A motif) were less than 10nM, showing that Orc5p can bind to ADP with a high affinity, similar to ATP. ORC-5A (ORC containing Orc5p with a defective Walker A motif) did not bind to ADP, suggesting that the ADP-Orc1p complex is too unstable to be detected by the filter-binding assay. ADP dissociated more rapidly than ATP from wild-type ORC and ORC-1A. Origin DNA fragments did not stimulate ADP-binding to any type of ORC. In the presence of ADP, ORC could not bind to origin DNA in a sequence-specific manner. Thus, in eukaryotes, the ADP-ORC complex may be unable to initiate chromosomal DNA replication, and in this it resembles the ADP-DnaA complex in prokaryotes. However, overall control may be different. In eukaryotes, the ADP-ORC complex is unstable, suggesting that the ADP-ORC complex might rapidly become an ATP-ORC complex; whereas in prokaryotes, ADP remains bound to DnaA, keeping DnaA inactive, and preventing re-initiation for some periods.     

Association of human origin recognition complex 1 with chromatin DNA and nuclease-resistant nuclear structures

An origin recognition complex (ORC) consisting of six polypeptides has been identified as a DNA replication origin-binding factor in Saccharomyces cerevisiae. Homologues of ORC subunits have been discovered among eukaryotes, and we have prepared monoclonal antibodies against a human homologue of ORC1 (hORC1) to study its localization in human cells. It was thus found to associate with nuclei throughout the cell cycle and to be resistant to nonionic detergent treatment, in contrast to MCM proteins, which are other replication factors, the association of which with nuclei is clearly dependent on the phase of the cell cycle. A characteristic feature of hORC1 is dissociation by NaCl in a narrow concentration range around 0.25 M, suggesting interaction with some specific partner(s) in nuclei. Nuclease treatment experiments and UV cross-linking experiments further indicated interaction with both nuclease-resistant nuclear structures and chromatin DNA. Although its DNA binding was unaffected, some variation in the cell cycle was apparent, the association with nuclear structures being less stable in the M phase. Interestingly, the less stable association occurred concomitantly with hyperphosphorylation of hORC1, suggesting that this hyperphosphorylation may be involved in M phase changes.     

Expression and characterization of human malaria parasite Plasmodium falciparum origin recognition complex subunit 1

In eukaryotes, the origin recognition complex (ORC) is essential for the initiation of DNA replication. The largest subunit of this complex (ORC1) has a regulatory role in origin activation. Here we report the cloning and functional characterization of Plasmodium falciparum ORC1 homolog. Using immunofluorescence and immunoelectron microscopy, we show here that PfORC1 is expressed in the nucleus during the late trophozoite and schizont stages where maximum amount of DNA replication takes place. Homology modelling of the carboxy terminal region of PfORC1 (781-1033) using Saccharomyces pombe Cdc6/Cdc18 homolog as a template reveals the presence of a similar AAA+ type nucleotide-binding fold. This region shows ATPase activity in vitro that is important for the origin activity. To our knowledge, this is the first evidence of an individual ORC subunit that shows ATPase activity. These observations strongly suggest that PfORC1 might be involved in DNA replication initiation during the blood stage of the parasitic life cycle.     

Chromatin association of human origin recognition complex, cdc6, and minichromosome maintenance proteins during the cell cycle: assembly of prereplication complexes in late mitosis

Evidence obtained from studies with yeast and Xenopus indicate that the initiation of DNA replication is a multistep process. The origin recognition complex (ORC), Cdc6p, and minichromosome maintenance (MCM) proteins are required for establishing prereplication complexes, upon which initiation is triggered by the activation of cyclin-dependent kinases and the Dbf4p-dependent kinase Cdc7p. The identification of human homologues of these replication proteins allows investigation of S-phase regulation in mammalian cells. Using centrifugal elutriation of several human cell lines, we demonstrate that whereas human Orc2 (hOrc2p) and hMcm proteins are present throughout the cell cycle, hCdc6p levels vary, being very low in early G(1) and accumulating until cells enter mitosis. hCdc6p can be polyubiquitinated in vivo, and it is stabilized by proteasome inhibitors. Similar to the case for hOrc2p, a significant fraction of hCdc6p is present on chromatin throughout the cell cycle, whereas hMcm proteins alternate between soluble and chromatin-bound forms. Loading of hMcm proteins onto chromatin occurs in late mitosis concomitant with the destruction of cyclin B, indicating that the mitotic kinase activity inhibits prereplication complex formation in human cells.     

Nucleotide-dependent conformational changes in the DnaA-like core of the origin recognition complex

Structural details of initiator proteins for DNA replication have provided clues to the molecular events in this process. EM reconstructions of the Drosophila melanogaster origin recognition complex (ORC) reveal nucleotide-dependent conformational changes in the core of the complex. All five AAA+ domains in ORC contain a conserved structural element that, in DnaA, promotes formation of a right-handed helix, indicating that helical AAA+ substructures may be a feature of all initiators. A DnaA helical pentamer can be docked into ORC, and the location of Orc5 uniquely positions this core. The results suggest that ATP-dependent conformational changes observed in ORC derive from reorientation of the AAA+ domains. By analogy to the DNA-wrapping activity of DnaA, we posit that ORC together with Cdc6 prepares origin DNA for helicase loading through mechanisms related to the established pathway of prokaryotes.     

Origin recognition complex binding to a metazoan replication origin

The initiation of DNA replication in eukaryotic cells at the onset of S phase requires the origin recognition complex (ORC) [1]. This six-subunit complex, first isolated in Saccharomyces cerevisiae [2], is evolutionarily conserved [1]. ORC participates in the formation of the prereplicative complex [3], which is necessary to establish replication competence. The ORC-DNA interaction is well established for autonomously replicating sequence (ARS) elements in yeast in which the ARS consensus sequence [4] (ACS) constitutes part of the ORC binding site [2, 5]. Little is known about the ORC-DNA interaction in metazoa. For the Drosophila chorion locus, it has been suggested that ORC binding is dispersed [6]. We have analyzed the amplification origin (ori) II/9A of the fly, Sciara coprophila. We identified a distinct 80-base pair (bp) ORC binding site and mapped the replication start site located adjacent to it. The binding of ORC to this 80-bp core region is ATP dependent and is necessary to establish further interaction with an additional 65-bp of DNA. This is the first time that both the ORC binding site and the replication start site have been identified in a metazoan amplification origin. Thus, our findings extend the paradigm from yeast ARS1 to multicellular eukaryotes, implicating ORC as a determinant of the position of replication initiation.     

Kinetics of ATP binding to the origin recognition complex of Saccharomyces cerevisiae

Origin recognition complex (ORC), a candidate initiator of chromosomal DNA replication in eukaryotes, binds specifically to ATP through two of its subunits (Orc1p and Orc5p). In this study, we investigated the kinetics of ATP binding to ORC by a filter binding assay. The Kd values for the ATP of wild-type ORC and ORC-1A (mutant ORC containing Orc1p with a defective Walker A motif) were less than 10 nm, suggesting that the affinity of Orc5p for ATP is very high. On the other hand, the Kd values for the ATP of ORC-5A (mutant ORC containing Orc5p with a defective Walker A motif) was much higher (about 1.5 microm), suggesting that the affinity of Orc1p for ATP is relatively low in the absence of origin DNA. ATP dissociated more rapidly from its complex with ORC-5A than from its complex with ORC-1A, suggesting that the ATP-Orc5p complex is more stable than ATP-Orc1p complex. Origin DNA fragments decreased the Kd value of ORC-5A for ATP and stabilized the complex of ATP with ORC-5A. Wild-type ORC, ORC-1A, and ORC-5A required different concentrations of ATP for specific binding to origin DNA. All of these results imply that ATP binding to Orc5p, ATP binding to Orc1p, and origin DNA binding to ORC are co-operatively regulated, which may be important for the initiation of DNA replication.     

Acidic phospholipids with unsaturated fatty acids inhibit the binding of origin recognition complex to origin DNA

Origin Recognition Complex (ORC) is a candidate initiator of chromosomal DNA replication in eukaryotes. We recently reported that cardiolipin inhibits the interaction of Origin Recognition Complex ORC with origin DNA, as is the case of DnaA, the initiator of chromosomal DNA replication in prokaryotes. We report here that another acidic phospholipid, phosphatidylglycerol (PG), also inhibits the interaction. Synthetic PG with only unsaturated fatty acids inhibits ORC-binding to origin DNA more strongly than PG with only saturated fatty acids. On the other hand, phosphatidylcholine (neutral phospholipid) does not affect the ORC-origin interaction, regardless of the presence of saturated or unsaturated fatty acids. These results suggest that an acidic moiety and unsaturated fatty acids are important factors for the inhibitory effect of phospholipids on ORC binding to origin DNA, as is the case for DnaA. The inhibitory effect of cardiolipin on ORC binding to origin DNA was more apparent at 30 degrees C than at 4 degrees C. Furthermore, chlorpromazine restored the ORC-origin interaction in the presence of cardiolipin. Since the presence of unsaturated fatty acids, low incubation temperatures, and the addition of chlorpromazine all decrease membrane fluidity, these results suggest that membrane fluidity is important for the inhibitory effect of acidic phospholipids on ORC-binding to origin DNA, as is the case for DnaA.     

ATP-dependent membrane assembly of F-actin facilitates membrane fusion

We recently established an in vitro assay that monitors the fusion between latex-bead phagosomes and endocytic organelles in the presence of J774 macrophage cytosol (). Here, we show that different reagents affecting the actin cytoskeleton can either inhibit or stimulate this fusion process. Because the membranes of purified phagosomes can assemble F-actin de novo from pure actin with ATP (), we focused here on the ability of membranes to nucleate actin in the presence of J774 cytosolic extracts. For this, we used F-actin sedimentation, pyrene actin assays, and torsional rheometry, a biophysical approach that could provide kinetic information on actin polymerization and gel formation. We make two major conclusions. First, under our standard in vitro conditions (4 mg/ml cytosol and 1 mM ATP), the presence of membranes actively catalyzed the assembly of cytosolic F-actin, which assembled into highly viscoelastic gels. A model is discussed that links these results to how the actin may facilitate fusion. Second, cytosolic actin paradoxically polymerized more under ATP depletion than under high-ATP conditions, even in the absence of membranes; we discuss these data in the context of the well described, large increases in F-actin seen in many cells during ischemia.