Activation of a human chromosomal replication origin by protein tethering

The specification of mammalian chromosomal replication origins is incompletely understood. To analyze the assembly and activation of prereplicative complexes (pre-RCs), we tested the effects of tethered binding of chromatin acetyltransferases and replication proteins on chromosomal c-myc origin deletion mutants containing a GAL4-binding cassette. GAL4^DBD (DNA binding domain) fusions with Orc2, Cdt1, E2F1 or HBO1 coordinated the recruitment of the Mcm7 helicase subunit, the DNA unwinding element (DUE)-binding protein DUE-B and the minichromosome maintenance (MCM) helicase activator Cdc45 to the replicator, and restored origin activity. In contrast, replication protein binding and origin activity were not stimulated by fusion protein binding in the absence of flanking c-myc DNA. Substitution of the GAL4-binding site for the c-myc replicator DUE allowed Orc2 and Mcm7 binding, but eliminated origin activity, indicating that the DUE is essential for pre-RC activation. Additionally, tethering of DUE-B was not sufficient to recruit Cdc45 or activate pre-RCs formed in the absence of a DUE. These results show directly in a chromosomal background that chromatin acetylation, Orc2 or Cdt1 suffice to recruit all downstream replication initiation activities to a prospective origin, and that chromosomal origin activity requires singular DNA sequences.     

Protein Phosphatase 2A and Cdc7 Kinase Regulate the DNA Unwinding Element-binding Protein in Replication Initiation

The DNA unwinding element (DUE)-binding protein (DUE-B) binds to replication origins coordinately with the minichromosome maintenance (MCM) helicase and the helicase activator Cdc45 in vivo, and loads Cdc45 onto chromatin in Xenopus egg extracts. Human DUE-B also retains the aminoacyl-tRNA proofreading function of its shorter orthologs in lower organisms. Here we report that phosphorylation of the DUE-B unstructured C-terminal domain unique to higher organisms regulates DUE-B intermolecular binding. Gel filtration analyses show that unphosphorylated DUE-B forms multiple high molecular weight (HMW) complexes. Several aminoacyl-tRNA synthetases and Mcm2–7 proteins were identified by mass spectrometry of the HMW complexes. Aminoacyl-tRNA synthetase binding is RNase A sensitive, whereas interaction with Mcm2–7 is nuclease resistant. Unphosphorylated DUE-B HMW complex formation is decreased by PP2A inhibition or direct DUE-B phosphorylation, and increased by inhibition of Cdc7. These results indicate that the state of DUE-B phosphorylation is maintained by the equilibrium between Cdc7-dependent phosphorylation and PP2A-dependent dephosphorylation, each previously shown to regulate replication initiation. Alanine mutation of the DUE-B C-terminal phosphorylation target sites increases MCM binding but blocks Cdc45 loading in vivo and inhibits cell division. In egg extracts alanine mutation of the DUE-B C-terminal phosphorylation sites blocks Cdc45 loading and inhibits DNA replication. The effects of DUE-B C-terminal phosphorylation reveal a novel S phase kinase regulatory mechanism for Cdc45 loading and MCM helicase activation.     

Fission yeast Cdc23/Mcm10 functions after pre-replicative complex formation to promote Cdc45 chromatin binding

Using a cytological assay to monitor the successive chromatin association of replication proteins leading to replication initiation, we have investigated the function of fission yeast Cdc23/Mcm10 in DNA replication. Inactivation of Cdc23 before replication initiation using tight degron mutations has no effect on Mcm2 chromatin association, and thus pre-replicative complex (pre-RC) formation, although Cdc45 chromatin binding is blocked. Inactivating Cdc23 during an S phase block after Cdc45 has bound causes a small reduction in Cdc45 chromatin binding, and replication does not terminate in the absence of Mcm10 function. These observations show that Cdc23/Mcm10 function is conserved between fission yeast and Xenopus, where in vitro analysis has indicated a similar requirement for Cdc45 binding, but apparently not compared with Saccharomyces cerevisiae, where Mcm10 is needed for Mcm2 chromatin binding. However, unlike the situation in Xenopus, where Mcm10 chromatin binding is dependent on Mcm2-7, we show that the fission yeast protein is bound to chromatin throughout the cell cycle in growing cells, and only displaced from chromatin during quiescence. On return to growth, Cdc23 chromatin binding is rapidly reestablished independently from pre-RC formation, suggesting that chromatin association of Cdc23 provides a link between proliferation and competence to execute DNA replication.     

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.     

Drosophila MCM10 interacts with members of the prereplication complex and is required for proper chromosome condensation

Mcm10 is required for the initiation of DNA replication in Saccharomyces cerevisiae. We have cloned MCM10 from Drosophila melanogaster and show that it complements a ScMCM10 null mutant. Moreover, Mcm10 interacts with key members of the prereplication complex: Mcm2, Dup (Cdt1), and Orc2. Interactions were also detected between Mcm10 and itself, Cdc45, and Hp1. RNAi depletion of Orc2 and Mcm10 in KC cells results in loss of DNA content. Furthermore, depletion of Mcm10, Cdc45, Mcm2, Mcm5, and Orc2, respectively, results in aberrant chromosome condensation. The condensation defects observed resemble previously published reports for Orc2, Orc5, and Mcm4 mutants. Our results strengthen and extend the argument that the processes of chromatin condensation and DNA replication are linked.     

The c-myc DNA-unwinding element-binding protein modulates the assembly of DNA replication complexes in vitro

The presence of DNA-unwinding elements (DUEs) at eukaryotic replicators has raised the question of whether these elements contribute to origin activity by their intrinsic helical instability, as protein-binding sites, or both. We used the human c-myc DUE as bait in a yeast one-hybrid screen and identified a DUE-binding protein, designated DUE-B, with a predicted mass of 23.4 kDa. Based on homology to yeast proteins, DUE-B was previously classified as an aminoacyl-tRNA synthetase; however, the human protein is approximately 60 amino acids longer than its orthologs in yeast and worms and is primarily nuclear. In vivo, chromatin-bound DUE-B localized to the c-myc DUE region. DUE-B levels were constant during the cell cycle, although the protein was preferentially phosphorylated in cells arrested early in S phase. Inhibition of DUE-B protein expression slowed HeLa cell cycle progression from G1 to S phase and induced cell death. DUE-B extracted from HeLa cells or expressed from baculovirus migrated as a dimer during gel filtration and co-purified with ATPase activity. In contrast to endogenous DUE-B, baculovirus-expressed DUE-B efficiently formed high molecular mass complexes in Xenopus egg and HeLa extracts. In Xenopus extracts, baculovirus-expressed DUE-B inhibited chromatin replication and replication protein A loading in the presence of endogenous DUE-B, suggesting that differential covalent modification of these proteins can alter their effect on replication. Recombinant DUE-B expressed in HeLa cells restored replication activity to egg extracts immunodepleted with anti-DUE-B antibody, suggesting that DUE-B plays an important role in replication in vivo.     

Regulation of replication licensing by acetyltransferase Hbo1

The initiation of DNA replication is tightly regulated in eukaryotic cells to ensure that the genome is precisely duplicated once and only once per cell cycle. This is accomplished by controlling the assembly of a prereplicative complex (pre-RC) which involves the sequential binding to replication origins of the origin recognition complex (ORC), Cdc6/Cdc18, Cdt1, and the minichromosome maintenance complex (Mcm2-Mcm7, or Mcm2-7). Several mechanisms of pre-RC regulation are known, including ATP utilization, cyclin-dependent kinase levels, protein turnover, and Cdt1 binding by geminin. Histone acetylation may also affect the initiation of DNA replication, but at present neither the enzymes nor the steps involved are known. Here, we show that Hbo1, a member of the MYST histone acetyltransferase family, is a previously unrecognized positive regulatory factor for pre-RC assembly. When Hbo1 expression was inhibited in human cells, Mcm2-7 failed to associate with chromatin even though ORC and Cdc6 loading was normal. When Xenopus egg extracts were immunodepleted of Xenopus Hbo1 (XHbo1), chromatin binding of Mcm2-7 was lost, and DNA replication was abolished. The binding of Mcm2-7 to chromatin in XHbo1-depleted extracts could be restored by the addition of recombinant Cdt1.     

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.     

The Cdc45·Mcm2-7·GINS protein complex in trypanosomes regulates DNA replication and interacts with two Orc1-like proteins in the origin recognition complex

Accurate DNA replication requires a complex interplay of many regulatory proteins at replication origins. The CMG (Cdc45·Mcm2-7·GINS) complex, which is composed of Cdc45, Mcm2-7, and the GINS (Go-Ichi-Ni-San) complex consisting of Sld5 and Psf1 to Psf3, is recruited by Cdc6 and Cdt1 onto origins bound by the heterohexameric origin recognition complex (ORC) and functions as a replicative helicase. Trypanosoma brucei, an early branched microbial eukaryote, appears to express an archaea-like ORC consisting of a single Orc1/Cdc6-like protein. However, unlike archaea, trypanosomes possess components of the eukaryote-like CMG complex, but whether they form an active helicase complex, associate with the ORC, and regulate DNA replication remains unknown. Here, we demonstrated that the CMG complex is formed in vivo in trypanosomes and that Mcm2-7 helicase activity is activated by the association with Cdc45 and the GINS complex in vitro. Mcm2-7 and GINS proteins are confined to the nucleus throughout the cell cycle, whereas Cdc45 is exported out of the nucleus after DNA replication, indicating that nuclear exclusion of Cdc45 constitutes one mechanism for preventing DNA re-replication in trypanosomes. With the exception of Mcm4, Mcm6, and Psf1, knockdown of individual CMG genes inhibits DNA replication and cell proliferation. Finally, we identified a novel Orc1-like protein, Orc1b, as an additional component of the ORC and showed that both Orc1b and Orc1/Cdc6 associate with Mcm2-7 via interactions with Mcm3. All together, we identified the Cdc45·Mcm2-7·GINS complex as the replicative helicase that interacts with two Orc1-like proteins in the unusual origin recognition complex in trypanosomes.     

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.     

Deregulated Cdc6 inhibits DNA replication and suppresses Cdc7-mediated phosphorylation of Mcm2–7 complex

Mcm2–7 is recruited to eukaryotic origins of DNA replication by origin recognition complex, Cdc6 and Cdt1 thereby licensing the origins. Cdc6 is essential for origin licensing during DNA replication and is readily destabilized from chromatin after Mcm2–7 loading. Here, we show that after origin licensing, deregulation of Cdc6 suppresses DNA replication in Xenopus egg extracts without the involvement of ATM/ATR-dependent checkpoint pathways. DNA replication is arrested specifically after chromatin binding of Cdc7, but before Cdk2-dependent pathways and deregulating Cdc6 after this step does not impair activation of origin firing or elongation. Detailed analyses revealed that Cdc6 deregulation leads to strong suppression of Cdc7-mediated hyperphosphorylation of Mcm4 and subsequent chromatin loading of Cdc45, Sld5 and DNA polymerase α. Mcm2 phosphorylation is also repressed although to a lesser extent. Remarkably, Cdc6 itself does not directly inhibit Cdc7 kinase activity towards Mcm2–4–6–7 in purified systems, rather modulates Mcm2–7 phosphorylation on chromatin context. Taken together, we propose that Cdc6 on chromatin acts as a modulator of Cdc7-mediated phosphorylation of Mcm2–7, and thus destabilization of Cdc6 from chromatin after licensing is a key event ensuring proper transition to the initiation of DNA replication.     

Biochemical analysis of components of the pre-replication complex of Archaeoglobus fulgidus

The eukaryotic pre-replication complex is assembled at replication origins in a reaction called licensing. Licensing involves the interactions of a variety of proteins including the origin recognition complex (ORC), Cdc6 and the Mcm2-7 helicase, homologues of which are also found in archaea. The euryarchaeote Archaeoglobus fulgidus encodes two genes with homology to Orc/Cdc6 and a single Mcm homologue. The A.fulgidus Mcm protein and one Orc/Cdc6 homologue have been purified and investigated in vitro. The Mcm protein is an ATP-dependent, hexameric helicase that can unwind between 200 and 400 bp of duplex DNA. Deletion of 112 amino acids from the N-terminus of A.f Mcm produced a protein, which was still capable of forming a hexamer, was competent in DNA binding and was able to unwind at least 1 kb of duplex DNA. The purified Orc/Cdc6 homologue was also able to bind DNA. Both Mcm and Orc/Cdc6 show a preference for specific DNA structures, namely molecules containing a single stranded bubble that mimics early replication intermediates. Nuclease protection showed that the binding sites for Mcm and Orc/Cdc6 overlap. The Orc/Cdc6 protein bound more tightly to these substrates and was able to displace pre-bound Mcm hexamer.     

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.     

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.     

Sld3, which interacts with Cdc45 (Sld4), functions for chromosomal DNA replication in Saccharomyces cerevisiae

Cdc45, which binds to the minichromosomal maintenance (Mcm) proteins, has a pivotal role in the initiation and elongation steps of chromosomal DNA replication in eukaryotes. Here we show that throughout the cell cycle in Saccharomyces cerevisiae, Cdc45 forms a complex with a novel factor, Sld3. Consistently, Sld3 and Cdc45 associate simultaneously with replication origins in the chromatin immunoprecipitation assay: both proteins associate with early-firing origins in G(1) phase and with late-firing origins in late S phase. Moreover, the origin associations of Sld3 and Cdc45 are mutually dependent. The temperature-sensitive sld3 mutation confers a defect in DNA replication at the restrictive temperature and reduces an interaction not only between Sld3 and Cdc45, but also between Cdc45 and Mcm2. These results suggest that the Sld3-Cdc45 complex associates with replication origins through Mcm proteins. At the restrictive temperature in sld3-5 cells, replication factor A, a single-strand DNA binding protein, does not associate with origins. Therefore, the origin association of Sld3-Cdc45 complex is prerequisite for origin unwinding in the initiation of DNA replication.     

Licensing for DNA replication requires a strict sequential assembly of Cdc6 and Cdt1 onto chromatin in Xenopus egg extracts

Replication origins are licensed for a single initiation event by the loading of Mcm2-7 proteins during late mitosis and G1. Sequential associations of origin recognition complex, Cdc6 and Mcm2-7 are essential for completion of the licensing. Although Cdt1 also binds to the chromatin when the licensing reaction takes place, whether the binding is a requirement for Cdt1 to function is unclear. To analyze the relevance of the chromatin association of Cdt1, we carried out chromatin transfer experiments using either immunodepleted Xenopus egg extracts or purified proteins. Licensing assay and immunoblotting analyses indicated that Cdt1 could only license DNA replication and load Mcm2-7 onto DNA when it binds to chromatin that has already associated with Cdc6. These results provide evidence supporting that Cdc6 and Cdt1 must bind to chromatin in a strict order for DNA licensing to occur.     

Clb/Cdc28 kinases promote nuclear export of the replication initiator proteins Mcm2-7

BACKGROUND: In the budding yeast Saccharomyces cerevisiae, the cyclin-dependent kinases of the Clb/Cdc28 family restrict the initiation of DNA replication to once per cell cycle by preventing the re-assembly of pre-replicative complexes (pre-RCs) at replication origins that have already initiated replication. This assembly involves the Cdc6-dependent loading of six minichromosome maintenance (Mcm) proteins, Mcm2-7, onto origins. How Clb/Cdc28 kinases prevent pre-RC assembly is not understood. RESULTS: In living cells, the Mcm proteins were found to colocalize in a cell-cycle-regulated manner. Mcm2-4, 6 and 7 were concentrated in the nucleus in G1 phase, gradually exported to the cytoplasm during S phase, and excluded from the nucleus by G2 and M phase. Tagging any single Mcm protein with the SV40 nuclear localization signal made all Mcm proteins constitutively nuclear. In the absence of functional Cdc6, Clb/Cdc28 kinases were necessary and sufficient for efficient net nuclear export of a fusion protein between Mcm7 and the green fluorescent protein (Mcm7-GFP), whereas inactivation of these kinases at the end of mitosis coincided with the net nuclear import of Mcm7-GFP. In contrast, in the presence of functional Cdc6, which loads Mcm proteins onto chromatin, S-phase progression as well as Clb/Cdc28 kinases was required for Mcm-GFP export. CONCLUSIONS: We propose that Clb/Cdc28 kinases prevent pre-RC reassembly in part by promoting the net nuclear export of Mcm proteins. We further propose that Mcm proteins become refractory to this regulation when they load onto chromatin and must be dislodged by DNA replication before they can be exported. Such an arrangement could ensure that Mcm proteins complete their replication function before they are removed from the nucleus.     

MCM2-7 form double hexamers at licensed origins in Xenopus egg extract

In late mitosis and G1, Mcm2-7 are assembled onto replication origins to license them for initiation in the upcoming S phase. After initiation, Mcm2-7 provide helicase activity to unwind DNA at the replication fork. Here we examine the structure of Mcm2-7 on chromatin in Xenopus egg extracts. We show that prior to replication initiation, Mcm2-7 is present at licensed replication origins in a complex with a molecular mass close to double that of the Mcm2-7 hexamer. This complex has approximately stoichiometric quantities of the 6 Mcm2-7 proteins and we conclude that it consists of a double heterohexamer. This provides a configuration potentially capable of initiating a pair of bidirectional replication forks in S phase. We also show that after initiation, Mcm2-7 associate with Cdc45 and GINS to form a relatively stable CMG (Cdc45-MCM-GINS) complex. The CMG proteins also associate less strongly with other replication proteins, consistent with the idea that a single CMG complex forms the core of the replisome.