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PubMed1995

Stepwise assembly of initiation complexes at budding yeast replication origins during the cell cycle.

DNA replication is a pivotal event in the cell cycle and, as a consequence, is tightly controlled in eukaryotic cells. The initiation of DNA replication is dependent upon the completion of mitosis and upon the commitment to complete the cell cycle made during G(1). Characterisation of the protein factors required for initiating DNA replication is essential to understand how the cell cycle is regulated. Recent results indicate that initiation complexes assemble in multiple stages during the cell cycle. First, origins are bound by the multisubunit origin recognition complex (ORC) which is essential for DNA replication in vivo. ORC, present at little more than one complete complex per replication origin, binds to origins immediately after initiation in the previous cell cycle. ORC binding occurs by the recognition of a bipartite sequence that includes the essential ARS consensus sequence (ACS) and the functionally important B1 element adjacent to the ACS. A novel pre-replicative complex (pre-RC) assembles at origins at the end of mitosis in actively cycling cells and remains at origins until DNA replication initiates. Finally, Dbf4, which is periodically synthesised at the end of G(1), interacts with replication origins. Dbf4-origin interaction requires an intact ACS strongly suggesting that interaction occurs through ORC. Dbf4 interacts with and is required for the activation of the Cdc7 protein kinase and together, Dbf4 and Cdc7 are required for the G(1)-S transition. Separate regions of Dbf4 are required for Cdc7- and origin-interaction suggesting that Dbf4 may act to recruit Cdc7 to replication origins where phosphorylation of some key component may cause origin firing.

Base Sequence↗
PubMed2000

Xenopus cdc7 function is dependent on licensing but not on XORC, XCdc6, or CDK activity and is required for XCdc45 loading.

The assembly and disassembly of protein complexes at replication origins play a crucial role in the regulation of chromosomal DNA replication. The sequential binding of the origin recognition complex (ORC), Cdc6, and the minichromosome maintenance (MCM/P1) proteins produces a licensed replication origin. Before the initiation of replication can occur, each licensed origin must be acted upon by S phase-inducing CDKs and the Cdc7 protein kinase. In the present report we describe the role of Xenopus Cdc7 (XCdc7) in DNA replication using cell-free extracts of Xenopus eggs. We show that XCdc7 binds to chromatin during G(1) and S phase. XCdc7 associates with chromatin only once origins have been licensed, but this association does not require the continued presence of XORC or XCdc6 once they have fulfilled their essential role in licensing. Moreover, XCdc7 is required for the subsequent CDK-dependent loading of XCdc45 but is not required for the destabilization of origins that occurs once licensing is complete. Finally, we show that CDK activity is not necessary for XCdc7 to associate with chromatin, induce MCM/P1 phosphorylation, or perform its essential replicative function. From these results we suggest a simple model for the assembly of functional initiation complexes in the Xenopus system.

Animals↗
PubMed2003

Radiation-mediated proteolysis of CDT1 by CUL4-ROC1 and CSN complexes constitutes a new checkpoint.

Genomic integrity is maintained by checkpoints that guard against undesired replication after DNA damage. Here, we show that CDT1, a licensing factor of the pre-replication complex (preRC), is rapidly proteolysed after UV- or gamma-irradiation. The preRC assembles on replication origins at the end of mitosis and during G1 to license DNA for replication in S phase. Once the origin recognition complex (ORC) binds to origins, CDC6 and CDT1 associate with ORC and promote loading of the MCM2-7 proteins onto chromatin, generating the preRC. We show that radiation-mediated CDT1 proteolysis is independent of ATM and CHK2 and can occur in G1-phase cells. Loss of the COP9-signalosome (CSN) or CUL4-ROC1 complexes completely suppresses CDT1 proteolysis. CDT1 is specifically polyubiquitinated by CUL4 complexes and the interaction between CDT1 and CUL4 is regulated in part by gamma-irradiation. Our study reveals an evolutionarily conserved and uncharacterized G1 checkpoint that induces CDT1 proteolysis by the CUL4-ROC1 ubiquitin E3 ligase and CSN complexes in response to DNA damage.

Cell Cycle↗
PubMed2006

Sequential ATP hydrolysis by Cdc6 and ORC directs loading of the Mcm2-7 helicase.

Loading of the Mcm2-7 DNA replicative helicase onto origin-proximal DNA is a critical and tightly regulated event during the initiation of eukaryotic DNA replication. The resulting protein-DNA assembly is called the prereplicative complex (pre-RC), and its formation requires the origin recognition complex (ORC), Cdc6, Cdt1, and ATP. ATP hydrolysis by ORC is required for multiple rounds of Mcm2-7 loading. Here, we investigate the role of ATP hydrolysis by Cdc6 during pre-RC assembly. We find that Cdc6 is an ORC- and origin DNA-dependent ATPase that functions at a step preceding ATP hydrolysis by ORC. Inhibiting Cdc6 ATP hydrolysis stabilizes Cdt1 on origin DNA and prevents Mcm2-7 loading. In contrast, the initial association of Mcm2-7 with the other pre-RC components does not require ATP hydrolysis by Cdc6. Importantly, these coordinated yet distinct functions of ORC and Cdc6 ensure the correct temporal and spatial regulation of pre-RC formation.

Adenosine Triphosphate↗
PubMed1996

Regulatory roles of the nuclear envelope.

Roles of the nuclear envelope are considered in the regulation of nuclear protein import, ribonucleoprotein export, and coupling of DNA replication to the cell cycle. First, evidence is discussed that indicates that neutral and acidic amino acids can be important in nuclear localization signals as well as the widely acknowledged basic amino acids. Second, the recognition of nuclear localization signals by their receptor "importin" is discussed, focusing on the different roles of the two subunits of importin. Third, a role for the alpha subunit of importin in RNP export is considered together with the question of how the direction of traffic through nuclear pores is determined. The final part of this article considers evidence that the nuclear membrane prevents reinitiation of DNA replication in Xenopus eggs, by excluding a "licensing factor" that is essential for DNA replication. Replication licensing in Xenopus appears to involve several proteins including the MCM (minichromosome maintenance) complex and ORC, the origin recognition complex, which must bind before the MCM complex can bind to chromatin.

Amino Acid Sequence↗
PubMed1996

Expression of the HsOrc1 gene, a human ORC1 homolog, is regulated by cell proliferation via the E2F transcription factor.

The initiation of DNA replication in Saccharomyces cerevisiae requires the action of a multisubunit complex of six proteins known as the origin recognition complex (ORC). The identification of higher eukaryotic homologs of several ORC components suggests a universal role for this complex in DNA replication. We now demonstrate that the expression of one of these homologs is regulated by cell proliferation. Expression of the human Orc1 gene (HsOrc1) is low in quiescent cells, and it is then dramatically induced upon stimulation of cell growth. In contrast, expression of the HsOrc2 gene does not appear to be similarly regulated. We have isolated the promoter that regulates HsOrc1 transcription, and we show that the promoter confers cell growth-dependent expression. We also demonstrate that the cell growth control is largely the consequence of E2F-dependent negative transcription control in quiescent cells. Activation of HsOrc1 transcription following growth stimulation requires G1 cyclin-dependent kinase activity, and forced E2F1 expression can bypass this requirement. These results thus provide a direct link between the initiation of DNA replication and the cell growth regulatory pathway involving G1 cyclin-dependent kinases, the Rb tumor suppressor, and E2F.

Base Sequence↗
PubMed1998

Cell cycle dependent topological changes of chromosomal replication origins in Saccharomyces cerevisiae.

BACKGROUND: The ORC (Origin Recognition Complex) of Saccharomyces cerevisiae is a protein complex for the initiation of replication which interacts with a cis-element, ACS (ARS Consensus Sequence), essential for DNA replication. The protein-DNA complex detected by the DNase I genomic footprinting method has been shown to vary depending on cell cycle progression. Further studies on topological changes of replication origin in vivo caused by ORC association are crucial for an understanding of chromosomal DNA replication in S. cerevisiae. RESULTS: Topological changes in the replication origins of the S. cerevisiae chromosome were studied by an in vivo UV photofootprinting method which is capable of detecting the change in the flexibility of DNA caused by protein binding. The footprinting method detected the inhibition and enhancement of UV-induced pyrimidine dimer formation in A and B1 elements of a chromosomal origin, ARS1, depending on the activity of native ORC subunits. Furthermore, footprint patterns were reproduced in vitro with purified ORC. The inhibition regarding the A element was stronger during the S to late M phase than that during the progression through the G1 phase. Functional CDC6 and MCM5 were required for maintaining the weaker inhibition state in G1-arrested cells. CONCLUSION: The application of in vivo UV photofootprinting in studies of topological changes of S. cerevisiae replication origins revealed the presence of two modes of topological ORC-ACS interaction. The weaker footprint in the G1 phase represents a specific topology of ACS, resulting from an alteration of the ORC-ACS interaction aided by CDC6 and MCM5, and this topological change may make the replication origin competent for initiating DNA replication.

Base Sequence↗
PubMed2003

Complex protein-DNA dynamics at the latent origin of DNA replication of Epstein-Barr virus.

The sequential binding of the origin recognition complex (ORC), Cdc6p and the minichromosome maintenance proteins (MCM2-7) mediates replication competence at eukaryotic origins of DNA replication. The latent origin of Epstein-Barr virus, oriP, is a viral origin known to recruit ORC. OriP also binds EBNA1, a virally encoded protein that lacks any activity predicted to be required for replication initiation. Here, we used chromatin immunoprecipitation and chromatin binding to compare the cell-cycle-dependent binding of pre-RC components and EBNA1 to oriP and to global cellular chromatin. Prereplicative-complex components such as the Mcm2p-Mcm7p proteins and HsOrc1p are regulated in a cell-cycle-dependent fashion, whereas other HsOrc subunits and EBNA1 remain constantly bound. In addition, HsOrc1p becomes sensitive to the 26S proteasome after release from DNA during S phase. These results show that the complex protein-DNA dynamics at the viral oriP are synchronized with the cell division cycle. Chromatin-binding and chromatin-immunoprecipitation experiments on G0 arrested cells indicated that the ORC core complex (ORC2-5) and EBNA1 remain bound to chromatin and oriP. HsOrc6p and the MCM2-7 complex are released in resting cells. HsOrc1p is partly liberated from chromatin. Our data suggest that origins remain marked in resting cells by the ORC core complex to ensure a rapid and regulated reentry into the cell cycle. These findings indicate that HsOrc is a dynamic complex and that its DNA binding activity is regulated differently in the various stages of the cell cycle.

Animals↗
PubMed2004

Modular structure of the human lamin B2 replicator.

The cis-acting elements necessary for the activity of DNA replication origins in metazoan cells are still poorly understood. Here we report a thorough characterization of the DNA sequence requirements of the origin associated with the human lamin B2 gene. A 1.2-kb DNA segment, comprising the start site of DNA replication and located within a large protein-bound region, as well as a CpG island, displays origin activity when moved to different ectopic positions. Genomic footprinting analysis of both the endogenous and the ectopic origins indicates that the large protein complex is assembled in both cases around the replication start site. Replacement of this footprinted region with an unrelated sequence, maintaining the CpG island intact, abolishes origin activity and the interaction with hORC2, a subunit of the origin recognition complex. Conversely, the replacement of 17 bp within the protected region reduces the extension of the protection without affecting the interaction with hORC2. This substitution does not abolish the origin activity but makes it more sensitive to the integration site. Finally, the nearby CpG island positively affects the efficiency of initiation. This analysis reveals the modular structure of the lamin B2 origin and supports the idea that sequence elements close to the replication start site play an important role in origin activation.

Base Sequence↗
PubMed1996

Role for a Xenopus Orc2-related protein in controlling DNA replication.

The six-subunit origin recognition complex (ORC) is essential for the initiation of DNA replication at specific origins in the budding yeast Saccharomyces cerevisiae. An important issue is whether DNA replication in higher eukaryotes, in which the characteristics of replication origins are poorly defined, occurs by an ORC-dependent mechanism. We have identified a Xenopus laevis Orc2-related protein (XORC2) by its ability to rescue a mitotic-catastrophe mutant of the fission yeast Schizosaccharomyces pombe. We show that immunodepletion of XORC2 from Xenopus egg extracts abolishes the replication of chromosomal DNA but not elongation synthesis on a single-stranded DNA template. Indirect immunofluorescence indicates that XORC2 binds to chromatin well before the commencement of DNA synthesis, and even under conditions that prevent the association of replication licensing factor(s) with the DNA. These findings suggest that Orc2 plays an important role at an early step of chromosomal replication in animal cells.

Amino Acid Sequence↗
PubMed1999

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.

Animals↗
PubMed2005

Novel splicing variant of mouse Orc1 is deficient in nuclear translocation and resistant for proteasome-mediated degradation.

DNA replication is controlled by the stepwise assembly of the pre-replicative complex and the replication apparatus. Loading of the origin recognition complex (ORC) onto the chromatin is a prerequisite for the assembly of the pre-replicative complex. To define the physiological functions of the mammalian ORC, we cloned ORC subunit cDNAs from mouse NIH3T3 cells and found novel variant forms of Orc1, Orc2, and Orc3 each derived from alternative RNA splicing. The variant form of Orc1, Orc1B, lacks 35 amino acid residues in exon 5; the variant of Orc2, Orc2B, lacks 48 amino acid residues in exon 2. In the Orc3 variant, Orc3B, only 1 amino acid residue is deleted in exon 15. Reverse transcription-PCR analysis showed that the full-length Orc1-3 subunits, Orc1A, Orc2A, and Orc3A, as well as Orc2B and Orc3B, were widely expressed in various mouse cell lines and mouse tissues. In contrast, Orc1B was only expressed in the thymus and at an early embryonic stage. Overexpression of these Orc subunits in cultured cells revealed that Orc1A, Orc2A, Orc3A, Orc2B, and Orc3B are localized in the nucleus, whereas Orc1B remains exclusively in the cytoplasm. Moreover, fusion of the 35 amino acids spliced fragment from mOrc1A with beta-galactosidase resulted in its translocation into the nucleus. When Orc1B is expressed transiently, its degradation occurs in a proteasome-independent manner, whereas Orc1A is rapidly degraded by the ubiquitin-proteasome pathway. Taken together, we conclude that mouse Orc1, Orc2, and Orc3 each exist in two alternative-splicing variants and that naturally occurring Orc1B lacks a functional domain that is essential for nuclear translocation and proteasome-dependent degradation.

Active Transport, Cell Nucleus↗
PubMed2003

CpG methylation of DNA restricts prereplication complex assembly in Xenopus egg extracts.

In a Xenopus egg replication system, the origin recognition complex (ORC) does not bind to CpG methylated DNA and DNA replication is inhibited. Insertion of low density CpG DNA of at least 1.2 kb into methylated plasmids rescues both replication and ORC binding. Using this pseudo-origin, we find that ORC binding is restricted to low-CpG-density DNA; however, MCM is loaded onto both weakly and highly methylated DNA and occupies at least approximately 2 kb of DNA. Replication initiates coincident with MCM, and even the most distally bound MCM is associated with sites of replication initiation. These results suggest that in metazoans MCM is loaded onto and initiates replication over a large region distant from ORC.

Adenosine Triphosphate↗
PubMed2003

In vitro protein-DNA interactions at the human lamin B2 replication origin.

The complexity of mammalian origins of DNA replication has prevented, so far, the in vitro studies of the modalities of initiator protein binding and origin selection. We approached this problem by utilizing the human lamin B2 origin, wherein the precise start sites of replication initiation have been identified and known to be bound in vivo by the origin recognition complex (ORC). In order to analyze the in vitro interactions occurring at this origin, we have compared the DNA binding requirements and patterns of the human recombinant Orc4 with those of preparations of HeLa nuclear proteins containing the ORC complex. Here we show that both HsOrc4 alone and HeLa nuclear proteins recognize multiple sites within a 241-bp DNA sequence encompassing the lamin B2 origin. The DNA binding activity of HeLa cells requires the presence of ORC and can be reproduced in the absence of all the other proteins known to be recruited to origins by ORC. Both HsOrc4 alone and HeLa nuclear proteins exhibit cooperative and ATP-independent binding. This binding covers nucleotides 3853-3953 and then spreads outward. Because this region contains the start sites of DNA synthesis as well as the area protected in vivo and preserves protein binding capacity in vitro after removal of a fraction of the protected region, we suggest that it could contain the primary binding site. Thus the in vitro approach points to the sequence requirements for ORC binding as a key element for origin recognition.

Adenosine Triphosphate↗
PubMed2006

The spatial arrangement of ORC binding modules determines the functionality of replication origins in budding yeast.

In the quest to define autonomously replicating sequences (ARSs) in eukaryotic cells, an ARS consensus sequence (ACS) has emerged for budding yeast. This ACS is recognized by the replication initiator, the origin recognition complex (ORC). However, not every match to the ACS constitutes a replication origin. Here, we investigated the requirements for ORC binding to origins that carry multiple, redundant ACSs, such as ARS603. Previous studies raised the possibility that these ACSs function as individual ORC binding sites. Detailed mutational analysis of the two ACSs in ARS603 revealed that they function in concert and give rise to an initiation pattern compatible with a single bipartite ORC binding site. Consistent with this notion, deletion of one base pair between the ACS matches abolished ORC binding at ARS603. Importantly, loss of ORC binding in vitro correlated with the loss of ARS activity in vivo. Our results argue that replication origins in yeast are in general comprised of bipartite ORC binding sites that cannot function in random alignment but must conform to a configuration that permits ORC binding. These requirements help to explain why only a limited number of ACS matches in the yeast genome qualify as ORC binding sites.

Base Sequence↗
PubMedDate not supplied

Role for a Drosophila Myb-containing protein complex in site-specific DNA replication.

There is considerable interest in the developmental, temporal and tissue-specific patterns of DNA replication in metazoans. Site-specific DNA replication at the chorion loci in Drosophila follicle cells leads to extensive gene amplification, and the organization of the cis-acting DNA elements that regulate this process may provide a model for how such regulation is achieved. Two elements important for amplification of the third chromosome chorion gene cluster, ACE3 and Ori-beta, are directly bound by Orc (origin recognition complex), and two-dimensional gel analysis has revealed that the primary origin used is Ori-beta (refs 7-9). Here we show that the Drosophila homologue of the Myb (Myeloblastosis) oncoprotein family is tightly associated with four additional proteins, and that the complex binds site-specifically to these regulatory DNA elements. Drosophila Myb is required in trans for gene amplification, showing that a Myb protein is directly involved in DNA replication. A Drosophila Myb binding site, as well as the binding site for another Myb complex member (p120), is necessary in cis for replication of reporter transgenes. Chromatin immunoprecipitation experiments localize both proteins to the chorion loci in vivo. These data provide evidence that specific protein complexes bound to replication enhancer elements work together with the general replication machinery for site-specific origin utilization during replication.

Animals↗
PubMed2006

ORC is necessary at the interphase-to-mitosis transition to recruit cdc2 kinase and disassemble RPA foci.

The origin-recognition complex (ORC) has an essential role in defining DNA replication origins and in chromosome segregation. Recent studies in Drosophila orc2 mutants, and in human cells depleted of ORC2, have suggested that this factor is also implicated in mitotic chromosome assembly. We asked whether ORC was required for M phase chromosome assembly independently of its function in DNA replication. We performed depletion assays and reconstitution experiments in Xenopus egg extracts, in conditions of M phase chromosome assembly coupled or uncoupled from DNA replication. We show that, although ORC is dispensable for mitotic chromosome condensation, it is necessary at the interphase-mitosis transition for proper mitotic chromosome assembly to occur in a reaction not strictly dependent on DNA replication. This function involves the recruitment to chromatin of cdc2 kinase and the chromatin disassembly of interphasic replication protein A (RPA) foci. Furthermore, we show that mutations of RPA at the cdc2 kinase site prevents RPA dissociation from chromatin and impairs mitotic chromosome assembly without affecting DNA replication. Our results support the conclusion that in addition to its role in the assembly of prereplication complexes (pre-RCs), at the G1-S transition, ORC is also required for their disassembly at mitotic entry.

Animals↗
PubMed1995

Conserved initiator proteins in eukaryotes.

The origin recognition complex (ORC), a multisubunit protein identified in Saccharomyces cerevisiae, binds to chromosomal replicators and is required for the initiation of cellular DNA replication. Complementary DNAs (cDNAs) encoding proteins related to the two largest subunits of ORC were cloned from various eukaryotes. The cDNAs encoding proteins related to S. cerevisiae Orc1p were cloned from the budding yeast Kluyveromyces lactis, the fission yeast Schizosaccharomyces pombe, and human cells. These proteins show similarity to regulators of the S and M phases of the cell cycle. Genetic analysis of orc1+ from S. pombe reveals that it is essential for cell viability. The cDNAs encoding proteins related to S. cerevisiae Orc2p were cloned from Arabidopsis thaliana, Caenorhabditis elegans, and human cells. The human ORC-related proteins interact in vivo to form a complex. These studies studies suggest that ORC subunits are conserved and that the role of ORC is a general feature of eukaryotic DNA replication.

Amino Acid Sequence↗