The BAH domain facilitates the ability of human Orc1 protein to activate replication origins in vivo

Selection of initiation sites for DNA replication in eukaryotes is determined by the interaction between the origin recognition complex (ORC) and genomic DNA. In mammalian cells, this interaction appears to be regulated by Orc1, the only ORC subunit that contains a bromo-adjacent homology (BAH) domain. Since BAH domains mediate protein-protein interactions, the human Orc1 BAH domain was mutated, and the mutant proteins expressed in human cells to determine their affects on ORC function. The BAH domain was not required for nuclear localization of Orc1, association of Orc1 with other ORC subunits, or selective degradation of Orc1 during S-phase. It did, however, facilitate reassociation of Orc1 with chromosomes during the M to G1-phase transition, and it was required for binding Orc1 to the Epstein-Barr virus oriP and stimulating oriP-dependent plasmid DNA replication. Moreover, the BAH domain affected Orc1's ability to promote binding of Orc2 to chromatin as cells exit mitosis. Thus, the BAH domain in human Orc1 facilitates its ability to activate replication origins in vivo by promoting association of ORC with chromatin.     

Selective instability of Orc1 protein accounts for the absence of functional origin recognition complexes during the M-G(1) transition in mammals

To investigate the events leading to initiation of DNA replication in mammalian chromosomes, the time when hamster origin recognition complexes (ORCs) became functional was related to the time when Orc1, Orc2 and Mcm3 proteins became stably bound to hamster chromatin. Functional ORCs, defined as those able to initiate DNA replication, were absent during mitosis and early G(1) phase, and reappeared as cells progressed through G(1) phase. Immunoblotting analysis revealed that hamster Orc1 and Orc2 proteins were present in nuclei at equivalent concentrations throughout the cell cycle, but only Orc2 was stably bound to chromatin. Orc1 and Mcm3 were easily eluted from chromatin during mitosis and early G(1) phase, but became stably bound during mid-G(1) phase, concomitant with the appearance of a functional pre-replication complex at a hamster replication origin. Since hamster Orc proteins are closely related to their human and mouse homologs, the unexpected behavior of hamster Orc1 provides a novel mechanism in mammals for delaying assembly of pre-replication complexes until mitosis is complete and a nuclear structure has formed.     

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.     

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.     

Mammalian Orc1 Protein Is Selectively Released from Chromatin and Ubiquitinated during the S-to-M Transition in the Cell Division Cycle

Previous studies have shown that changes in the affinity of the hamster Orc1 protein for chromatin during the M-to-G(1) transition correlate with the activity of hamster origin recognition complexes (ORCs) and the appearance of prereplication complexes at specific sites. Here we show that Orc1 is selectively released from chromatin as cells enter S phase, converted into a mono- or diubiquitinated form, and then deubiquitinated and re-bound to chromatin during the M-to-G(1) transition. Orc1 is degraded by the 26S proteasome only when released into the cytosol, and peptide additions to Orc1 make it hypersensitive to polyubiquitination. In contrast, Orc2 remains tightly bound to chromatin throughout the cell cycle and is not a substrate for ubiquitination. Since the concentration of Orc1 remains constant throughout the cell cycle, and its half-life in vivo is the same as that of Orc2, ubiquitination of non-chromatin-bound Orc1 presumably facilitates the inactivation of ORCs by sequestering Orc1 during S phase. Thus, in contrast to yeast (Saccharomyces cerevisiae and Schizosaccharomyces pombe), mammalian ORC activity appears to be regulated during each cell cycle through selective dissociation and reassociation of Orc1 from chromatin-bound ORCs.     

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.     

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.     

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.     

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.     

Protein phosphatase 1 dephosphorylates Orc2

Phosphorylation of Thr¹¹⁶ and Thr²²⁶ on 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. The phosphorylated Orc2 becomes dephosphorylated in the late M phase of the cell cycle. Here we show that protein phosphatase 1 (PP1) dephosphorylates Orc2. Dephosphorylation of Orc2 was accompanied by associating the dissociated Orc subunits with chromatin. Inhibitors of PP1 preferentially inhibited the dephosphorylation of Orc2. The overexpression of the α, β and γ PP1 isoforms decreased the amount of phosphorylated Orc2, and the depletion of these isoforms by RNA interference increased the amount of phosphorylated Orc2. These results suggest that PP1 dephosphorylates Orc2 to promote the binding of ORC to chromatin.     

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.     

Interactions and subcellular distribution of DNA replication initiation proteins in eukaryotic cells

For initiation of eukaryotic DNA replication the origin recognition complex (ORC) associates with chromatin sites and constitutes a landing pad allowing Cdc6, Cdt1 and MCM proteins to accomplish the pre-replication complex (pre-RC). In S phase, the putative MCM helicase is assumed to move away from the ORC to trigger DNA unwinding. By using the fluorescence-based assays bioluminescence resonance energy transfer (BRET) and bimolecular fluorescence complementation (BiFC) we show in live mammalian cells that one key interaction in pre-RC assembly, the interaction between Orc2 and Orc3, is not restricted to the nucleus but also occurs in the cytoplasm. BRET assays also revealed a direct interaction between Orc2 and nuclear localization signal (NLS)-depleted Orc3. Further, we assessed the subcellular distribution of Orc2 and Orc3 in relation to MCM proteins Mcm3 and Mcm6 as well as to a key protein involved in elongation of DNA replication, proliferating nuclear cell antigen (PCNA). Our findings illustrate the spatial complexity of the elaborated process of DNA replication as well as that the BRET and BiFC techniques are novel tools that could contribute to our understanding of the processes at the very beginning of the duplication of the genome.     

Protein kinase A is required for chromosomal DNA replication.

Passage through mitosis resets cells for a new round of chromosomal DNA replication [1]. In late mitosis, the pre-replication complex - which includes the origin recognition complex (ORC), Cdc6 and the minichromosome maintenance (MCM) proteins - binds chromatin as a pre-requisite for DNA replication. S-phase-promoting cyclin-dependent kinases (Cdks) and the kinase Dbf4-Cdc7 then act to initiate replication. Before the onset of replication Cdc6 dissociates from chromatin. S-phase and M-phase Cdks block the formation of a new pre-replication complex, preventing DNA over-replication during the S, G2 and M phases of the cell cycle [1]. The nuclear membrane also contributes to limit genome replication to once per cell cycle [2]. Thus, at the end of M phase, nuclear membrane breakdown and the collapse of Cdk activity reset cells for a new round of chromosomal replication. We showed previously that protein kinase A (PKA) activity oscillates during the cell cycle in Xenopus egg extracts, peaking in late mitosis. The oscillations are induced by the M-phase-promoting Cdk [3] [4]. Here, we found that PKA oscillation was required for the following phase of DNA replication. PKA activity was needed from mitosis exit to the formation of the nuclear envelope. PKA was not required for the assembly of ORC2, Cdc6 and MCM3 onto chromatin. Inhibition of PKA activity, however, blocked the release of Cdc6 from chromatin and subsequent DNA replication. These data suggest that PKA activation in late M phase is required for the following S phase.     

Recruitment of Xenopus Scc2 and cohesin to chromatin requires the pre-replication complex

Cohesin is a multi-subunit, ring-shaped protein complex that holds sister chromatids together from the time of their synthesis in S phase until they are segregated in anaphase. In yeast, the loading of cohesin onto chromosomes requires the Scc2 protein. In vertebrates, cohesins first bind to chromosomes as cells exit mitosis, but the mechanism is unknown. Concurrent with cohesin binding, pre-replication complexes (pre-RCs) are assembled at origins of DNA replication through the sequential loading of the initiation factors ORC, Cdc6, Cdt1 and MCM2-7 (the 'licensing' reaction). In S phase, the protein kinase Cdk2 activates pre-RCs, causing origin unwinding and DNA replication. Here, we use Xenopus egg extracts to show that the recruitment of cohesins to chromosomes requires fully licensed chromatin and is dependent on ORC, Cdc6, Cdt1 and MCM2-7, but is independent of Cdk2. We further show that Xenopus Scc2 is required for cohesin loading and that binding of XScc2 to chromatin is MCM2-7 dependent. Our results define a novel pre-RC-dependent pathway for cohesin recruitment to chromosomes in a vertebrate model system.     

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.