The ORC1 cycle in human cells: II. Dynamic changes in the human ORC complex during the cell cycle

The origin recognition complex (ORC) plays a central role in regulating the initiation of DNA replication in eukaryotes. The level of the ORC1 subunit oscillates throughout the cell cycle, defining an ORC1 cycle. ORC1 accumulates in G1 and is degraded in S phase, although other ORC subunits (ORCs 2-5) remain at almost constant levels. The behavior of ORC components in human cell nuclei with respect to the ORC1 cycle demonstrates that ORCs 2-5 form a complex that is present throughout the cell cycle and that associates with ORC1 when it accumulates in G1 nuclei. ORCs 2-5 are found in both nuclease-insoluble and -soluble fractions. The appearance of nuclease-insoluble ORCs 2-5 parallels the increase in the level of ORC1 associating with nuclease-insoluble, non-chromatin nuclear structures. Thus, ORCs 2-5 are temporally recruited to nuclease-insoluble structures by formation of the ORC1-5 complex. An artificial reduction in the level of ORC1 in human cells by RNA interference results in a shift of ORC2 to the nuclease-soluble fraction, and the association of MCM proteins with chromatin fractions is also blocked by this treatment. These results indicate that ORC1 regulates the status of the ORC complex in human nuclei by tethering ORCs 2-5 to nuclear structures. This dynamic shift is further required for the loading of MCM proteins onto chromatin. Thus, the pre-replication complex in human cells may be regulated by the temporal accumulation of ORC1 in G1 nuclei.     

Metazoan origin selection: origin recognition complex chromatin binding is regulated by CDC6 recruitment and ATP hydrolysis

Using a plasmid competition assay, we have measured the stability of origin recognition complex (ORC) associated with sperm chromatin under physiological conditions. Under conditions in which pre-RCs are formed, both ORC and CDC6 dissociate from sperm chromatin with a relatively fast t(1/2) of 15 min. ORC dissociation from chromatin is regulated through the recruitment of CDC6 and MCM proteins as well as ATP hydrolysis. The t(1/2) for ORC alone in the absence of Cdc6 is 40 min and increases 8-fold to >2 h when Cdc6 is present. Strikingly, the presence of a non-hydrolyzable ATP derivative, ATPgammaS, not only increases both ORC and CDC6 t(1/2) but also inhibits the loading of MCM. The very stable association of ORC and Cdc6 with chromatin in this sequence-independent replication system suggests that origin selection in metazoans cannot be strictly dependent on the interaction of ORCs with specific DNA binding sequences.     

The roles of the MCM, ORC, and Cdc6 proteins in determining the replication competence of chromatin in quiescent cells

Most eukaryotic cell types can withdraw from proliferative cell cycles and remain quiescent for extended periods. Intact nuclei isolated from quiescent murine NIH3T3 cells fail to replicate in vitro when incubated in Xenopus egg extracts, although intact nuclei from proliferating cells replicate well. Permeabilization of the nuclear envelope rescues the ability of quiescent nuclei to replicate in the extract. We show that origin replication complex (ORC), minichromosome maintenance (MCM), and Cdc6 proteins are all present in early quiescent cells. Immunodepletion of Cdc6 or the MCM complex from Xenopus egg extract inhibits replication of permeable, quiescent, but not proliferating, NIH3T3 nuclei. Immunoblotting results demonstrate that mouse homologues of Mcm2, Mcm5, and Cdc6 are displaced from chromatin in quiescent cells. However, this absence of chromatin-bound Cdc6 and MCM proteins from quiescent cells appears not to be due to the absence of ORC subunits as murine homologues of Orc1 and Orc2 remain chromatin-bound in quiescent cells. Surprisingly, intact quiescent nuclei fail to bind exogenously added XCdc6 or to replicate in Xenopus egg extracts immunodepleted of ORC, even though G1- or S-phase nuclei still replicate in these extracts. Our results identify Cdc6 and the MCM complex as essential replication components absent from quiescent chromatin due to nonfunctional chromatin-bound ORC proteins. These results can explain why quiescent mammalian nuclei are unable to replicate in vivo and in Xenopus egg extracts.     

Interaction and assembly of murine pre-replicative complex proteins in yeast and mouse cells

Eukaryotic cells coordinate chromosome duplication by the assembly of protein complexes at origins of DNA replication by sequential binding of member proteins of the origin recognition complex (ORC), CDC6, and minichromosome maintenance (MCM) proteins. These pre-replicative complexes (pre-RCs) are activated by cyclin-dependent kinases and DBF4/CDC7 kinase. Here, we carried out a comprehensive yeast two-hybrid screen to establish sequential interactions between two individual proteins of the mouse pre-RC that are probably required for the initiation of DNA replication. The studies revealed multiple interactions among ORC subunits and MCM proteins as well as interactions between individual ORC and MCM proteins. In particular CDC6 was found to bind strongly to ORC1 and ORC2, and to MCM7 proteins. DBF4 interacts with the subunits of ORC as well as with MCM proteins. It was also demonstrated that CDC7 binds to different ORC and MCM proteins. CDC45 interacts with ORC1 and ORC6, and weakly with MCM3, -6, and -7. The three subunits of the single-stranded DNA binding protein RPA show interactions with various ORC subunits as well as with several MCM proteins. The data obtained by yeast two-hybrid analysis were paradigmatically confirmed in synchronized murine FM3A cells by immunoprecipitation of the interacting partners. Some of the interactions were found to be cell-cycle-dependent; however, most of them were cell-cycle-independent. Altogether, 90 protein-protein interactions were detected in this study, 52 of them were found for the first time in any eukaryotic pre-RC. These data may help to understand the complex interplay of the components of the mouse pre-RC and should allow us to refine its structural architecture as well as its assembly in real time.     

Cell cycle regulation of human CDC6 protein. Intracellular localization, interaction with the human mcm complex, and CDC2 kinase-mediated hyperphosphorylation.

The binding of mammalian MCM complexes to chromatin is cell cycle-regulated and under CDC2 kinase negative control. Here, we investigated the properties of mammalian CDC6 protein, a candidate regulator of MCM. The levels of CDC6 were relatively constant during the HeLa cell cycle. In asynchronous cells, CDC6 was mainly detected in the nuclei with immunostaining, but some CDC6 was not extractable with nonionic detergent. In contrast to the chromatin-bound MCM, this fraction of CDC6 was resistant to DNase I treatment, suggesting that it binds to the detergent- and nuclease-resistant nuclear structure. In S phase cells, CDC6 became detectable in the cytoplasm with immunostaining; however, the level of the bound CDC6 was unchanged. In G(2)/M phase cells, the level of the bound CDC6 was still maintained, which was hyperphosphorylated by CDC2 kinase. These data suggest that some CDC6 protein is associated with the specific nuclear structure throughout the cell cycle and that major binding sites on chromatin differ between MCM and CDC6. However, co-immunoprecipitation assays with chemical cross-linking indicated that a small part of the chromatin-bound MCM is present close to the bound CDC6.     

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.     

Mcm2, but not RPA, is a component of the mammalian early G1-phase prereplication complex.

Previous experiments in Xenopus egg extracts identified what appeared to be two independently assembled prereplication complexes (pre-RCs) for DNA replication: the stepwise assembly of ORC, Cdc6, and Mcm onto chromatin, and the FFA-1-mediated recruitment of RPA into foci on chromatin. We have investigated whether both of these pre-RCs can be detected in Chinese hamster ovary (CHO) cells. Early- and late-replicating chromosomal domains were pulse-labeled with halogenated nucleotides and prelabeled cells were synchronized at various times during the following G1-phase. The recruitment of Mcm2 and RPA to these domains was examined in relation to the formation of a nuclear envelope, specification of the dihydrofolate reductase (DHFR) replication origin and entry into S-phase. Mcm2 was loaded gradually and cumulatively onto both early- and late-replicating chromatin from late telophase throughout G1-phase. During S-phase, detectable Mcm2 was rapidly excluded from PCNA-containing active replication forks. By contrast, detergent-resistant RPA foci were undetectable until the onset of S-phase, when RPA joined only the earliest-firing replicons. During S-phase, RPA was present with PCNA specifically at active replication forks. Together, our data are consistent with a role for Mcm proteins, but not RPA, in the formation of mammalian pre-RCs during early G1-phase.     

Noc3p,a bHLH protein,plays an integral role in the initiation of DNA replication in budding yeast

Initiation of eukaryotic DNA replication requires many proteins that interact with one another and with replicators. Using a yeast genetic screen, we have identified Noc3p (nucleolar complex-associated protein) as a novel replication-initiation protein. Noc3p interacts with MCM proteins and ORC and binds to chromatin and replicators throughout the cell cycle. It functions as a critical link between ORC and other initiation proteins to effect chromatin association of Cdc6p and MCM proteins for the establishment and maintenance of prereplication complexes. Noc3p is highly conserved in eukaryotes and is the first identified bHLH (basic helix-loop-helix) protein required for replication initiation. As Noc3p is also required for pre-rRNA processing, Noc3p is a multifunctional protein that plays essential roles in two vital cellular processes.     

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.     

Selective interactions of human kin17 and RPA proteins with chromatin and the nuclear matrix in a DNA damage- and cell cycle-regulated manner

Several proteins involved in DNA synthesis are part of the so-called 'replication factories' that are anchored on non-chromatin nuclear structures. We report here that human kin17, a nuclear stress-activated protein, associates with both chromatin and non-chromatin nuclear structures in a cell cycle- and DNA damage-dependent manner. After L-mimosine block and withdrawal we observed that kin17 protein was recruited in the nucleus during re-entry and progression through S phase. These results are consistent with a role of kin17 protein in DNA replication. About 50% of the total amount of kin17 protein was detected on nuclear structures and could not be released by detergents. Furthermore, the amount of kin17 protein greatly increased in both G(1)/S and S phase-arrested cells in fractions containing proteins anchored to nuclear structures. The detection of kin17 protein showed for the first time its preferential assembly within non-chromatin nuclear structures in G(1)/S and S phase-arrested cells, while the association with these structures was found to be less stable in the G(2)/M phase, as judged by fractionation of human cells and immunostaining. In asynchronous growing cells, kin17 protein interacted with both chromatin DNA and non-chromatin nuclear structures, while in S phase-arrested cells it interacted mostly with non-chromatin nuclear structures, as judged by DNase I treatment and in vivo UV cross-linking. In the presence of DNA damage in S phase cells, the distribution of kin17 protein became mainly associated with chromosomal DNA, as judged by limited formaldehyde cross-linking of living cells. The physical interaction of kin17 protein with components of the nuclear matrix was confirmed and visualized by indirect immunofluorescence and immunoelectron microscopy. Our results indicate that, during S phase, a fraction of the human kin17 protein preferentially associates with the nuclear matrix, a fundamentally non-chromatin higher order nuclear structure, and to chromatin DNA in the presence of DNA damage.     

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.     

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.     

Dynamics of DNA binding of replication initiation proteins during de novo formation of pre-replicative complexes in Xenopus egg extracts

We investigated the dynamics of DNA binding of replication initiation proteins during formation of the pre-replicative complex (pre-RC) on plasmids in Xenopus egg extracts. The pre-RC was efficiently formed on plasmids at 23 degrees C, with one or a few origin recognition complex (ORC) molecules and approximately 10-20 mini-chromosome maintenance 2 (MCM2) molecules loaded onto each plasmid. Although geminin inhibited MCM loading, MCM interacted weakly but stoichiometrically with the plasmid in an ORC-dependent manner, even in the presence of geminin (with approximately 10 MCM2 molecules per plasmid). Interestingly, DNA binding of ORC, CDC6, and CDT1 was significantly stabilized in the presence of geminin, under which conditions approximately 10-20 molecules each of ORC and CDC6 were bound. Moreover, a similarly stable ORC-CDC6-CDT1 complex rapidly formed on DNA at lower temperature (0 degrees C) without geminin, with approximately 10-20 molecules each of ORC and CDC6 bound to the plasmid, but almost no binding of MCM. However, upon shifting the temperature to 23 degrees C, most ORC, CDC6, and CDT1 molecules were displaced from the DNA, leaving about one ORC molecule on the plasmid, whereas approximately 10 MCM2 molecules were loaded onto each plasmid. Furthermore, it was possible to load MCM onto DNA when the isolated ORC-CDC6-CDT1-DNA complex was mixed with purified MCM proteins. These results suggest that an ORC-CDC6-CDT1 complex pre-formed on DNA is directly involved in MCM loading and imply that each DNA-bound ORC molecule loads only one or a few MCM2-7 complexes during metazoan pre-RC formation.     

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.     

Localization of proteins bound to a replication origin of human DNA along the cell cycle

The proteins bound in vivo at the human lamin B2 DNA replication origin and their precise sites of binding were investigated along the cell cycle utilizing two novel procedures based on immunoprecipitation following UV irradiation with a pulsed laser light source. In G(1), the pre-replicative complex contains CDC6, MCM3, ORC1 and ORC2 proteins; of these, the post-replicative complex in S phase contains only ORC2; in M phase none of them are bound. The precise nucleotide of binding was identified for the two ORC and the CDC6 proteins near the start sites for leading-strand synthesis; the transition from the pre- to the post-replicative complex is accompanied by a 17 bp displacement of the ORC2 protein towards the start site.     

A human cancer cell line initiates DNA replication normally in the absence of ORC5 and ORC2 proteins

The origin recognition complex (ORC), composed of six subunits, ORC1–6, binds to origins of replication as a ring-shaped heterohexameric ATPase that is believed to be essential to recruit and load MCM2–7, the minichromosome maintenance protein complex, around DNA and initiate DNA replication. We previously reported the creation of viable cancer cell lines that lacked detectable ORC1 or ORC2 protein without a reduction in the number of origins firing. Here, using CRISPR-Cas9–mediated mutations, we report that human HCT116 colon cancer cells also survive when ORC5 protein expression is abolished via a mutation in the initiator ATG of the ORC5 gene. Even if an internal methionine is used to produce an undetectable, N terminally deleted ORC5, the protein would lack 80% of the AAA+ ATPase domain, including the Walker A motif. The ORC5-depleted cells show normal chromatin binding of MCM2–7 and initiate replication from a similar number of origins as WT cells. In addition, we introduced a second mutation in ORC2 in the ORC5 mutant cells, rendering both ORC5 and ORC2 proteins undetectable in the same cells and destabilizing the ORC1, ORC3, and ORC4 proteins. Yet the double mutant cells grow, recruit MCM2–7 normally to chromatin, and initiate DNA replication with normal number of origins. Thus, in these selected cancer cells, either a crippled ORC lacking ORC2 and ORC5 and present at minimal levels on the chromatin can recruit and load enough MCM2–7 to initiate DNA replication, or human cell lines can sometimes recruit MCM2–7 to origins independent of ORC.     

Pre-replication complex proteins assemble at regions of low nucleosome occupancy within the Chinese hamster dihydrofolate reductase initiation zone

Genome-scale mapping of pre-replication complex proteins has not been reported in mammalian cells. Poor enrichment of these proteins at specific sites may be due to dispersed binding, poor epitope availability or cell cycle stage-specific binding. Here, we have mapped sites of biotin-tagged ORC and MCM protein binding in G1-synchronized populations of Chinese hamster cells harboring amplified copies of the dihydrofolate reductase (DHFR) locus, using avidin-affinity purification of biotinylated chromatin followed by high-density microarray analysis across the DHFR locus. We have identified several sites of significant enrichment for both complexes distributed throughout the previously identified initiation zone. Analysis of the frequency of initiations across stretched DNA fibers from the DHFR locus confirmed a broad zone of de-localized initiation activity surrounding the sites of ORC and MCM enrichment. Mapping positions of mononucleosomal DNA empirically and computing nucleosome-positioning information in silico revealed that ORC and MCM map to regions of low measured and predicted nucleosome occupancy. Our results demonstrate that specific sites of ORC and MCM enrichment can be detected within a mammalian initiation zone, and suggest that initiation zones may be regions of generally low nucleosome occupancy where flexible nucleosome positioning permits flexible pre-RC assembly sites.     

Site-specific loading of an MCM protein complex in a DNA replication initiation zone upstream of the c-MYC gene in the HeLa cell cycle

The MCM proteins participate in an orderly association, beginning with the origin recognition complex, that culminates in the initiation of chromosomal DNA replication. Among these, MCM proteins 4, 6, and 7 constitute a subcomplex that reportedly possesses DNA helicase activity. Little is known about DNA sequences initially bound by these MCM proteins or about their cell cycle distribution in the chromatin. We have determined the locations of certain MCM and associated proteins by chromatin immunoprecipitation (ChIP) in a zone of initiation of DNA replication upstream of the c-MYC gene in the HeLa cell cycle. MCM7 and its clamp-loading partner Cdc6 are highly specifically colocalized by ChIP and re-ChIP in G(1) and early S on a 198-bp segment located near the center of the initiation zone. ChIP and Re-ChIP colocalizes MCM7 and ORC1 to the same segment specifically in late G(1). MCM proteins 6 and 7 can be coimmunoprecipitated throughout the cell cycle, whereas MCM4 is reduced in the complex in late S and G(2), reappearing upon mitosis. MCM7 is not visualized by immunohistochemistry on metaphase chromosomes. MCM7 is recruited to multiple sites in chromatin in S and G(2), at which time it is not detected with ORC1. The rate of dissemination is surprisingly slow and is unlikely to be simply attributed to progression with replication forks. Results indicate sequence-specific loading of MCM proteins onto DNA in late G(1) followed by a recruitment to multiple sites in chromatin subsequent to replication.     

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.