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.     

Dynamic association of ORCA with prereplicative complex components regulates DNA replication initiation

In eukaryotes, initiation of DNA replication requires the assembly of a multiprotein prereplicative complex (pre-RC) at the origins. We recently reported that a WD repeat-containing protein, origin recognition complex (ORC)-associated (ORCA/LRWD1), plays a crucial role in stabilizing ORC to chromatin. Here, we find that ORCA is required for the G(1)-to-S-phase transition in human cells. In addition to binding to ORC, ORCA associates with Cdt1 and its inhibitor, geminin. Single-molecule pulldown experiments demonstrate that each molecule of ORCA can bind to one molecule of ORC, one molecule of Cdt1, and two molecules of geminin. Further, ORCA directly interacts with the N terminus of Orc2, and the stability of ORCA is dependent on its association with Orc2. ORCA associates with Orc2 throughout the cell cycle, with Cdt1 during mitosis and G(1), and with geminin in post-G(1) cells. Overexpression of geminin results in the loss of interaction between ORCA and Cdt1, suggesting that increased levels of geminin in post-G(1) cells titrate Cdt1 away from ORCA. We propose that the dynamic association of ORCA with pre-RC components modulates the assembly of its interacting partners on chromatin and facilitates DNA replication initiation.     

Dephosphorylation of Orc2 by protein phosphatase 1 promotes the binding of the origin recognition complex to chromatin

Phosphorylation of Orc2, one of the six subunits of the origin recognition complex (ORC), by cyclin A/CDK2 during S phase leads to the dissociation of Orc2, Orc3, Orc4, and Orc5 subunits (Orc2–5) from human chromatin and replication origins. Dephosphorylation of the phosphorylated Orc2 by protein phosphatase 1 (PP1) is accompanied by the binding of the dissociated subunits to chromatin. Here we show that PP1 physically interacts with Orc2. The binding of PP1 to Orc2 and the dephosphorylation of Orc2 by PP1 occurred in a cell cycle-dependent manner through an interaction with 119-KSVSF-123, which is the consensus motif for the binding of PP1, of Orc2. The dephosphorylation of Orc2 by PP1 is required for the binding of Orc2 to chromatin. These results support that PP1 dephosphorylates Orc2 to promote the binding of ORC to chromatin and replication origins for the subsequent round of the cell cycle.     

Role for Cdk1 (Cdc2)/cyclin A in preventing the mammalian origin recognition complex's largest subunit (Orc1) from binding to chromatin during mitosis

The eukaryotic origin recognition complex (ORC) selects the genomic sites where prereplication complexes are assembled and DNA replication begins. In proliferating mammalian cells, ORC activity appears to be regulated by reducing the affinity of the Orc1 subunit for chromatin during S phase and then preventing reformation of a stable ORC-chromatin complex until mitosis is completed and a nuclear membrane is assembled. Here we show that part of the mechanism by which this is accomplished is the selective association of Orc1 with Cdk1 (Cdc2)/cyclin A during the G(2)/M phase of cell division. This association accounted for the appearance in M-phase cells of hyperphosphorylated Orc1 that was subsequently dephosphorylated during the M-to-G(1) transition. Moreover, inhibition of Cdk activity in metaphase cells resulted in rapid binding of Orc1 to chromatin. However, chromatin binding was not mediated through increased affinity of Orc1 for Orc2, suggesting that additional events are involved in the assembly of functional ORC-chromatin sites. These results reveal that the same cyclin-dependent protein kinase that initiates mitosis in mammalian cells also concomitantly inhibits assembly of functional ORC-chromatin sites.     

Human Mcm proteins at a replication origin during the G1 to S phase transition

Previous work with yeast cells and with Xenopus egg extracts had shown that eukaryotic pre-replication complexes assemble on chromatin in a step-wise manner whereby specific loading factors promote the recruitment of essential Mcm proteins at pre-bound origin recognition complexes (ORC with proteins Orc1p–Orc6p). While the order of assembly—Mcm binding follows ORC binding—seems to be conserved in cycling mammalian cells in culture, it has not been determined whether mammalian Mcm proteins associate with ORC-bearing chromatin sites. We have used a chromatin immunoprecipitation approach to investigate the site of Mcm binding in a genomic region that has previously been shown to contain an ORC-binding site and an origin of replication. Using chromatin from HeLa cells in G₁ phase, antibodies against Orc2p as well as antibodies against Mcm proteins specifically immunoprecipitate chromatin enriched for a DNA region that includes a replication origin. However, with chromatin from cells in S phase, only Orc2p-specific antibodies immunoprecipitate the origin-containing DNA region while Mcm-specific antibodies immunoprecipitate chromatin with DNA from all parts of the genomic region investigated. Thus, human Mcm proteins first assemble at or adjacent to bound ORC and move to other sites during genome replication.     

ATP-dependent assembly of the human origin recognition complex

The origin recognition complex (ORC) was initially discovered in budding yeast extracts as a protein complex that binds with high affinity to autonomously replicating sequences in an ATP-dependent manner. We have cloned and expressed the human homologs of the ORC subunits as recombinant proteins. In contrast to other eukaryotic initiators examined thus far, assembly of human ORC in vitro is dependent on ATP binding. Mutations in the ATP-binding sites of Orc4 or Orc5 impair complex assembly, whereas Orc1 ATP binding is not required. Immunofluorescence staining of human cells with anti-Orc3 antibodies demonstrate cell cycle-dependent association with a nuclear structure. Immunoprecipitation experiments show that ORC disassembles as cells progress through S phase. The Orc6 protein binds directly to the Orc3 subunit and interacts as part of ORC in vivo. These data suggest that the assembly and disassembly of ORC in human cells is uniquely regulated and may contribute to restricting DNA replication to once in every cell division cycle.     

Role of the Orc6 protein in origin recognition complex-dependent DNA binding and replication in Drosophila melanogaster

The six-subunit origin recognition complex (ORC) is a DNA replication initiator protein in eukaryotes that defines the localization of the origins of replication. We report here that the smallest Drosophila ORC subunit, Orc6, is a DNA binding protein that is necessary for the DNA binding and DNA replication functions of ORC. Orc6 binds DNA fragments containing Drosophila origins of DNA replication and prefers poly(dA) sequences. We have defined the core replication domain of the Orc6 protein which does not include the C-terminal domain. Further analysis of the core replication domain identified amino acids that are important for DNA binding by Orc6. Alterations of these amino acids render reconstituted Drosophila ORC inactive in DNA binding and DNA replication. We show that mutant Orc6 proteins do not associate with chromosomes in vivo and have dominant negative effects in Drosophila tissue culture cells. Our studies provide a molecular analysis for the functional requirement of Orc6 in replicative functions of ORC in Drosophila and suggest that Orc6 may contribute to the sequence preferences of ORC in targeting to the origins.     

An essential role for Orc6 in DNA replication through maintenance of pre-replicative complexes

The heterohexameric origin recognition complex (ORC) acts as a scaffold for the G(1) phase assembly of pre-replicative complexes (pre-RC). Only the Orc1-5 subunits appear to be required for origin binding in budding yeast, yet Orc6 is an essential protein for cell proliferation. Imaging of Orc6-YFP in live cells revealed a punctate pattern consistent with the organization of replication origins into subnuclear foci. Orc6 was not detected at the site of division between mother and daughter cells, in contrast to observations for metazoans, and is not required for mitosis or cytokinesis. An essential role for Orc6 in DNA replication was identified by depleting it at specific cell cycle stages. Interestingly, Orc6 was required for entry into S phase after pre-RC formation, in contrast to previous models suggesting ORC is dispensable at this point in the cell cycle. When Orc6 was depleted in late G(1), Mcm2 and Mcm10 were displaced from chromatin, cells failed to progress through S phase, and DNA combing analysis following bromodeoxyuridine incorporation revealed that the efficiency of replication origin firing was severely compromised.     

Interaction between ORC and Cdt1p of Saccharomyces cerevisiae

Origin recognition complex (ORC), a six-protein complex, is the most likely initiator of chromosomal DNA replication in eukaryotes. Throughout the cell cycle, ORC binds to chromatin at origins of DNA replication and functions as a 'landing pad' for the binding of other proteins, including Cdt1p, to form a prereplicative complex. In this study, we used yeast two-hybrid analysis to examine the interaction between Cdt1p and every ORC subunit. We observed potent interaction with Orc6p, and weaker interaction with Orc2p and Orc5p. Coimmunoprecipitation assay confirmed that Cdt1p interacted with Orc6p, as well as with Orc1p and Orc2p. We mapped the C-terminal region, and a middle region of Orc6p (amino acids residues 394-435, and 121-175, respectively), as important for interaction with Cdt1p. Cdt1p was purified to examine its direct interaction with ORC, and its effect on the activity of ORC. Glutathione-S-transferase pull-down analysis revealed that Cdt1p binds directly to ORC. Cdt1p neither bound to origin DNA and ATP nor affected ORC-binding to origin DNA and ATP. These results suggest that interaction of Cdt1p with ORC is involved in the formation of the prereplicative complex, rather than in regulation of the activity of ORC.     

Cyclin A-dependent kinase activity affects chromatin binding of ORC, Cdc6, and MCM in egg extracts of Xenopus laevis.

The initiation of DNA replication in eukaryotes requires the loading of the origin recognition complex (ORC), Cdc6, and minichromosome maintenance (MCM) proteins onto chromatin to form the preinitiation complex. In Xenopus egg extract, the proteins Orc1, Orc2, Cdc6, and Mcm4 are underphosphorylated in interphase and hyperphosphorylated in metaphase extract. We find that chromatin binding of ORC, Cdc6, and MCM proteins does not require cyclin-dependent kinase activities. High cyclin A-dependent kinase activity inhibits the binding and promotes the release of Xenopus ORC, Cdc6, and MCM from sperm chromatin, but has no effect on chromatin binding of control proteins. Cyclin A together with ORC, Cdc6 and MCM proteins is bound to sperm chromatin in DNA replicating pseudonuclei. In contrast, high cyclin E/cdk2 was not detected on chromatin, but was found soluble in the nucleoplasm. High cyclin E kinase activity allows the binding of Xenopus ORC and Cdc6, but not MCM, to sperm chromatin, even though the kinase does not phosphorylate MCM directly. We conclude that chromatin-bound cyclin A kinase controls DNA replication by protein phosphorylation and chromatin release of Cdc6 and MCM, whereas soluble cyclin E kinase prevents rereplication during the cell cycle by the inhibition of premature MCM chromatin association.     

An episomal mammalian replicon: sequence-independent binding of the origin recognition complex

An extrachromosomally replicating plasmid was used to investigate the specificity by which the origin recognition complex (ORC) interacts with DNA sequences in mammalian cells in vivo. We first showed that the plasmid pEPI-1 replicates semiconservatively in a once-per-cell-cycle manner and is stably transmitted over many cell generations in culture without selection. Chromatin immunoprecipitations and quantitative polymerase chain reaction analysis revealed that, in G1-phase cells, Orc1 and Orc2, as well as Mcm3, another component of the prereplication complex, are bound to multiple sites on the plasmid. These binding sites are functional because they show the S-phase-dependent dissociation of Orc1 and Mcm3 known to be characteristic for prereplication complexes in mammalian cells. In addition, we identified replicative nascent strands and showed that they correspond to many plasmid DNA regions. This work has implications for current models of replication origins in mammalian systems. It indicates that specific DNA sequences are not required for the chromatin binding of ORC in vivo. The conclusion is that epigenetic mechanisms determine the sites where mammalian DNA replication is initiated.     

Phylogenetic Conservation and Homology Modeling Help Reveal a Novel Domain within the Budding Yeast Heterochromatin Protein Sir1

The yeast Sir1 protein's ability to bind and silence the cryptic mating-type locus HMRa requires a protein-protein interaction between Sir1 and the origin recognition complex (ORC). A domain within the C-terminal half of Sir1, the Sir1 ORC interaction region (Sir1OIR), and the conserved bromo-adjacent homology (BAH) domain within Orc1, the largest subunit of ORC, mediate this interaction. The structure of the Sir1OIR-Orc1BAH complex is known. Sir1OIR and Orc1BAH interacted with a high affinity in vitro, but the Sir1OIR did not inhibit Sir1-dependent silencing when overproduced in vivo, suggesting that other regions of Sir1 helped it bind HMRa. Comparisons of diverged Sir1 proteins revealed two highly conserved regions, N1 and N2, within Sir1's poorly characterized N-terminal half. An N-terminal portion of Sir1 (residues 27 to 149 [Sir1^27-149]) is similar in sequence to the Sir1OIR; homology modeling predicted a structure for Sir1^27-149 in which N1 formed a submodule similar to the known Orc1BAH-interacting surface on Sir1. Consistent with these findings, two-hybrid assays indicated that the Sir1 N terminus could interact with BAH domains. Amino acid substitutions within or near N1 or N2 reduced full-length Sir1's ability to bind and silence HMRa and to interact with Orc1BAH in a two-hybrid assay. Purified recombinant Sir1 formed a large protease-resistant structure within which the Sir1OIR domain was protected, and Orc1BAH bound Sir1OIR more efficiently than full-length Sir1 in vitro. Thus, the Sir1 N terminus exhibited both positive and negative roles in the formation of a Sir1-ORC silencing complex. This functional duality might contribute to Sir1's selectivity for silencer-bound ORCs in vivo.     

Human Orc2 localizes to centrosomes, centromeres and heterochromatin during chromosome inheritance

The initiation of DNA replication in S phase requires the prior assembly of an origin recognition complex (ORC)-dependent pre-replicative complex on chromatin during G1 phase of the cell division cycle. In human cells, the Orc2 subunit localized to the nucleus as expected, but it also localized to centrosomes throughout the entire cell cycle. Furthermore, Orc2 was tightly bound to heterochromatin and heterochromatin protein 1alpha (HP1alpha) and HP1beta in G1 and early S phase, but during late S, G2 and M phases tight chromatin association was restricted to centromeres. Depletion of Orc2 by siRNA caused multiple phenotypes. A population of cells showed an S-phase defect with little proliferating cell nuclear antigen (PCNA) on chromatin, although MCM proteins remained. Orc2 depletion also disrupted HP1 localization, but not histone-H3-lysine-9 methylation at prominent heterochromatic foci. Another subset of Orc2-depleted cells containing replicated DNA arrested with abnormally condensed chromosomes, failed chromosome congression and multiple centrosomes. These results implicate Orc2 protein in chromosome duplication, chromosome structure and centrosome copy number control, suggesting that it coordinates all stages of the chromosome inheritance cycle.     

Identification of a Binding Region for Human Origin Recognition Complex Proteins 1 and 2 That Coincides with an Origin of DNA Replication

We investigated the binding regions of components of the origin recognition complex (ORC) in the human genome. For this purpose, we performed chromatin immunoprecipitation assays with antibodies against human Orc1 and Orc2 proteins. We identified a binding region for human Orc proteins 1 and 2 in a <1-kbp segment between two divergently transcribed human genes. The region is characterized by CpG tracts and a central sequence rich in AT base pairs. Both, Orc1 and Orc2 proteins are found at the intergenic region in the G(1) phase, but S-phase chromatin contains only Orc2 protein, supporting the notion that Orc1p dissociates from its binding site in the S phase. Sequences corresponding to the intergenic region are highly abundant in a fraction of nascent DNA strands, strongly suggesting that this region not only harbors the binding sites for Orc1 protein and Orc2 protein but also serves as an origin of bidirectional DNA replication.     

A WD-repeat protein stabilizes ORC binding to chromatin

Origin recognition complex (ORC) plays critical roles in the initiation of DNA replication and cell-cycle progression. In metazoans, ORC associates with origin DNA during G1 and with heterochromatin in postreplicated cells. However, what regulates the binding of ORC to chromatin is not understood. We have identified a highly conserved, leucine-rich repeats and WD40 repeat domain-containing protein 1 (LRWD1) or ORC-associated (ORCA) in human cells that interacts with ORC and modulates chromatin association of ORC. ORCA colocalizes with ORC and shows similar cell-cycle dynamics. We demonstrate that ORCA efficiently recruits ORC to chromatin. Depletion of ORCA in human primary cells and embryonic stem cells results in loss of ORC association to chromatin, concomitant reduction of MCM binding, and a subsequent accumulation in G1 phase. Our results suggest ORCA-mediated association of ORC to chromatin is critical to initiate preRC assembly in G1 and chromatin organization in post-G1 cells.