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Results for “Origin Recognition Complex”

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PubMed2001

Replication origins in Xenopus egg extract Are 5-15 kilobases apart and are activated in clusters that fire at different times.

When Xenopus eggs and egg extracts replicate DNA, replication origins are positioned randomly with respect to DNA sequence. However, a completely random distribution of origins would generate some unacceptably large interorigin distances. We have investigated the distribution of replication origins in Xenopus sperm nuclei replicating in Xenopus egg extract. Replicating DNA was labeled with [(3)H]thymidine or bromodeoxyuridine and the geometry of labeled sites on spread DNA was examined. Most origins were spaced 5-15 kb apart. This regular distribution provides an explanation for how complete chromosome replication can be ensured although origins are positioned randomly with respect to DNA sequence. Origins were grouped into small clusters (typically containing 5-10 replicons) that fired at approximately the same time, with different clusters being activated at different times in S phase. This suggests that a temporal program of origin firing similar to that seen in somatic cells also exists in the Xenopus embryo. When the quantity of origin recognition complexes (ORCs) on the chromatin was restricted, the average interorigin distance increased, and the number of origins in each cluster decreased. This suggests that the binding of ORCs to chromatin determines the regular spacing of origins in this system.

Animals↗

PubMed2005

The requirement of yeast replication origins for pre-replication complex proteins is modulated by transcription.

The mini-chromosome maintenance proteins Mcm2-7 are essential for DNA replication. They are loaded onto replication origins during G1 phase of the cell cycle to form a pre-replication complex (pre-RC) that licenses each origin for subsequent initiation. We have investigated the DNA elements that determine the dependence of yeast replication origins on Mcm2-7 activity, i.e. the sensitivity of an origin to mcm mutations. Using chimaeric constructs from mcm sensitive and mcm insensitive origins, we have identified two main elements affecting the requirement for Mcm2-7 function. First, transcription into an origin increases its dependence on Mcm2-7 function, revealing a conflict between pre-RC assembly and transcription. Second, sequence elements within the minimal origin influence its mcm sensitivity. Replication origins show similar differences in sensitivity to mutations in other pre-RC proteins (such as Origin Recognition Complex and Cdc6), but not to mutations in initiation and elongation factors, demonstrating that the mcm sensitivity of an origin is determined by its ability to establish a pre-RC. We propose that there is a hierarchy of replication origins with respect to the range of pre-RC protein concentrations under which they will function. This hierarchy is both 'hard-wired' by the minimal origin sequences and 'soft-wired' by local transcriptional context.

Binding Sites↗

PubMed1995

Silencing and heritable domains of gene expression.

Silencing is a process that assembles particular regions of eukaryotic chromosomes into transcriptionally inactive chromatin structures. Silencing involves specialized regulatory sites known as silencers and a combination of general DNA-binding proteins and proteins dedicated to silencing. In the yeast Saccharomyces cerevisiae, these proteins include transcription factors and the origin recognition complex (ORC). Silencing has three recognizably separate phases: establishment, maintenance, and inheritance. At least some silencers are origins of replication, and the establishment of the silenced state requires an S phase-specific event. Once established, the silenced state is heritable, even in the absence of proteins required for its establishment. The silencing of mating-type genes bears many similarities to telomere position effects, and the two processes require many of the same proteins.

Alleles↗

PubMed2001

Multiple redundant sequence elements within the fission yeast ura4 replication origin enhancer.

BACKGROUND: Some origins in eukaryotic chromosomes fire more frequently than others. In the fission yeast, Schizosaccharomyces pombe, the relative firing frequencies of the three origins clustered 4-8 kbp upstream of the ura4 gene are controlled by a replication enhancer - an element that stimulates nearby origins in a relatively position-and orientation-independent fashion. The important sequence motifs within this enhancer were not previously localized. RESULTS: Systematic deletion of consecutive segments of approximately 50, approximately 100 or approximately 150 bp within the enhancer and its adjacent core origin (ars3002) revealed that several of the approximately 50-bp stretches within the enhancer contribute to its function in partially redundant fashion. Other stretches within the enhancer are inhibitory. Some of the stretches within the enhancer proved to be redundant with sequences within core ars3002. Consequently the collection of sequences important for core origin function was found to depend on whether the core origin is assayed in the presence or absence of the enhancer. Some of the important sequences in the core origin and enhancer co-localize with short runs of adenines or thymines, which may serve as binding sites for the fission yeast Origin Recognition Complex (ORC). Others co-localize with matches to consensus sequences commonly found in fission yeast replication origins. CONCLUSIONS: The enhancer within the ura4 origin cluster in fission yeast contains multiple sequence motifs. Many of these stimulate origin function in partially redundant fashion. Some of them resemble motifs also found in core origins. The next step is to identify the proteins that bind to these stimulatory sequences.

Journal Article↗

PubMed2000

Central role for cdc45 in establishing an initiation complex of DNA replication in Xenopus egg extracts.

BACKGROUND: In eukaryotes, chromosomal DNA is licensed to be replicated through the sequential loading of the origin recognition complex, Cdc6 and mini-chromosome maintenance protein complex (MCM) onto chromatin. However, how the replication machinery is assembled onto the licensed chromatin during initiation of replication is poorly understood. RESULTS: Using Xenopus egg extracts, we have investigated the role of Cdc45 in the loading of various replication proteins onto chromatin at the onset of S phase, and found that Cdc45, which required MCM for its loading, was essential for the sequential loading of replication protein A (RPA), DNA polymerase alpha and proliferating cell nuclear antigen (PCNA) onto chromatin. The assembly of DNA polymerase epsilon onto chromatin required Cdc45 but did not require DNA polymerase alpha. Analysis of nuclease-digested chromatin fractions shows that Cdc45 formed a stable complex with either MCM or DNA polymerase alpha on chromatin. CONCLUSIONS: These results demonstrate a central role for Cdc45 in activation of the licensed chromatin to form replication complexes at the onset of S phase, and suggest that Cdc45 has a dual role in the initiation of DNA replication: the unwinding of DNA and the recruiting of DNA polymerases onto DNA.

Animals↗

PubMed1997

Persistent initiation of DNA replication and chromatin-bound MCM proteins during the cell cycle in cdc6 mutants.

Faithful inheritance of genetic information requires that DNA be copied only once each cell cycle. Initiation of DNA replication involves the establishment of a prereplication complex (pre-RC) and subsequent activation by CDK/cyclins, converting the pre-RC to a post-RC. The origin recognition complex (ORC), Cdc6p, and the MCM proteins are required for establishing the pre-RC. We show that all six ORC subunits remain bound to chromatin throughout the cell cycle, whereas the MCM proteins cycle on and off, corresponding precisely to transitions of the RC. A newly isolated cdc6 mutant displays promiscuous initiation of DNA replication, increased nuclear DNA content, and constant MCM protein association with chromatin throughout the cell cycle. This gain-of-function cdc6 mutant ignores the negative controls imposed normally on initiation by the CDK/cyclins, suggesting that Cdc6p is a key mediator of once-per-cell-cycle control of DNA replication.

CDC28 Protein Kinase, S cerevisiae↗

PubMed1995

Chromatin-mediated transcriptional repression in yeast.

Chromatin is linked to two modes of transcriptional repression in yeast: silencing at the HM loci and telomeres, and repression by the global regulator complex Ssn6/Tup1. In both cases, unique chromatin configurations appear to be orchestrated by multiprotein complexes targeted to particular regions through interactions with specific DNA-binding proteins, such as RAP1, the origin-recognition complex, and alpha 2.

Chromatin↗

PubMed2002

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.

Blotting, Western↗

PubMed1998

Association of RPA with chromosomal replication origins requires an Mcm protein, and is regulated by Rad53, and cyclin- and Dbf4-dependent kinases.

Eukaryotic cells use multiple replication origins to replicate their large genomes. Some origins fire early during S phase whereas others fire late. In Saccharomyces cerevisiae, initiator sequences (ARSs) are bound by the origin recognition complex (ORC). Cdc6p synthesized at the end of mitosis joins ORC and facilitates recruitment of Mcm proteins, which renders origins competent to fire. However, origins fire only upon the subsequent activation of S phase cyclin-dependent kinases (S-CDKs) and Dbf4/Cdc7 at the G1/S boundary. We have used a chromatin immunoprecipitation assay to measure the association with ARS sequences of DNA primase and the single-stranded DNA binding replication protein A (RPA) when fork movement is inhibited by hydroxyurea (HU). RPA's association with origins requires S-CDKs, Dbf4/Cdc7 kinase and an Mcm protein. The recruitment of DNA primase depends on RPA. Furthermore, early- and late-firing origins differ not in the timing of their recruitment of an Mcm protein, but in the timing of RPA's recruitment. RPA is recruited to early but not to late origins in HU. We also show that Rad53 kinase is required to prevent RPA association with a late origin in HU. Our data suggest that the origin unwinding accompanied by RPA association is a key step, regulated by S-CDKs, Dbf4/Cdc7 and Rad53p. Thus, in the presence of active S-CDKs and Dbf4/Cdc7, Mcms may open origins and thereby facilitate the loading of RPA.

Adenosine Triphosphatases↗

PubMed1996

ORC- and Cdc6-dependent complexes at active and inactive chromosomal replication origins in Saccharomyces cerevisiae.

We have developed a genomic footprinting protocol which allows us to examine protein-DNA interactions at single copy chromosomal origins of DNA replication in the budding yeast Saccharomyces cerevisiae. We show that active replication origins oscillate between two chromatin states during the cell cycle: an origin recognition complex (ORC)-dependent post-replicative state and a Cdc6p-dependent pre-replicative state. Furthermore, we show that both post- and pre-replicative complexes can form efficiently on closely apposed replicators. Surprisingly, ARS301 which is active as an origin on plasmids but not in its normal chromosomal location, forms ORC- and Cdc6p-dependent complexes in both its active and inactive contexts. Thus, although ORC and Cdc6p are essential for initiation, their binding is not sufficient to dictate origin use.

Base Sequence↗

PubMed2004

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.

Binding Sites↗

PubMed2001

The B2 element of the Saccharomyces cerevisiae ARS1 origin of replication requires specific sequences to facilitate pre-RC formation.

The minimal requirements for a eukaryotic origin of replication are an initiator binding site and a region of helically unstable DNA [DNA unwinding element (DUE)]. Budding yeast origins consist of modular elements, and one of these elements, B2, has been proposed to act as a DUE. To test this hypothesis, we screened for sequences that function at the B2 element of ARS1. We found that the B2 element required A-rich sequences, but that the function of these identified sequences did not correlate with helical instability. Instead, the sequences that substituted fully for B2 function showed similarity to the ARS consensus sequence (ACS). The ACS is the binding site for the initiator origin recognition complex (ORC), but the selected sequences are not strong ORC binding sites in vitro. Nonfunctional B2 sequences show a corresponding loss in Mcm2-7p origin association. The function of these mutant sequences is rescued by Cdc6p overexpression. We propose that the B2 element requires specific sequences to bind a component of the pre-RC.

Binding Sites↗

PubMed2002

Essential role of human CDT1 in DNA replication and chromatin licensing.

Formation of pre-replicative complexes at origins is an early cell cycle event essential for DNA duplication. A large body of evidence supports the notion that Cdc6 protein, through its interaction with the origin recognition complex, is required for pre-replicative complex assembly by loading minichromosome maintenance proteins onto DNA. In fission yeast and Xenopus, this reaction known as the licensing of chromatin for DNA replication also requires the newly identified Cdt1 protein. We studied the role of hCdt1 protein in the duplication of the human genome by antibody microinjection experiments and analyzed its expression during the cell cycle in human non-transformed cells. We show that hCdt1 is essential for DNA replication in intact human cells, that it executes its function in a window of the cell cycle overlapping with pre-replicative complex formation and that it is necessary for the loading of minichromosome maintenance proteins onto chromatin. Intriguingly, we observed that hCdt1 protein, in contrast to other licensing factors, is already present in serum-deprived G0 arrested cells and its levels increase only marginally upon re-entry in the cell cycle.

Cell Cycle↗

PubMed1996

A novel role for Cdc5p in DNA replication.

DNA replication initiates from specific chromosomal sites called origins, and in the budding yeast Saccharomyces cerevisiae these sites are occupied by the origin recognition complex (ORC). Dbf4p is proposed to play a role in targeting the G1/S kinase Cdc7p to initiation complexes late in G1. We report that Dbf4p may also recruit Cdc5p to origin complexes. Cdc5p is a member of the Polo family of kinases that is required for the completion of mitosis. Cdc5p and Cdc7p each interact with a distinct domain of Dbf4p. cdc5-1 mutants have a plasmid maintenance defect that can be suppressed by the addition of multiple origins. cdc5-1 orc2-1 double mutants are synthetically lethal. Levels of Cdc5p were found to be cell cycle regulated and peaked in G2/M. These results suggest a role for Cdc5p and possibly Polo-like kinases at origin complexes.

Cell Cycle Proteins↗

PubMed2001

Inheritance of the replication complex: a unique or common phenomenon in the control of DNA replication?

Early models of the regulation of initiation of DNA replication by protein complexes predicted that binding of a replication initiator protein to a replicator region is required for initiation of each DNA replication round, since after the initiation event the replication initiator should dissociate from DNA. It was, therefore, assumed that binding of the replication initiator is a signal for triggering DNA replication. However, more recent investigations have revealed that in many replicons this is not the case. Studies on the regulation of the replication of plasmids derived from bacteriophage lambda demonstrated that, once assembled, the replication complex can be inherited by one of the two daughter plasmid copies after each replication round and may function in subsequent replication rounds. Since this DNA-bound protein complex bears information about specific initiation of DNA replication, this phenomenon has been called "protein inheritance." A similar phenomenon has recently been reported for oriJ-based plasmids. Moreover, the current model of the initiation of DNA replication in the yeast Saccharomyces cerevisiae proposes that the origin recognition complex (ORC) remains bound to one copy of the ori sequence (the ARS region) after initiation of DNA replication. Thus, it seems plausible that protein inheritance is not unique for lambda plasmids, but may be a common phenomenon in the control of DNA replication, at least in microbes.

Bacteriophage lambda↗

PubMed1998

Replication focus-forming activity 1 and the Werner syndrome gene product.

The initiation of DNA replication involves a minimum of four factors: a specific DNA sequence (origin), an initiator protein which binds to the origin, a helicase that unwinds the origin and a protein that binds single-stranded DNA that stabilizes the unwound origin. In eukaryotic cells, the origin recognition complex (ORC) is the initiator protein and replication protein A (RPA; ref. 3) is the single-stranded DNA-binding protein. However, the helicase has not been identified and the nature of origins remains elusive, except in the case of Saccharomyces cerevisiae. A unique feature of eukaryotic DNA replication is that it occurs at a few-hundred discrete foci. It has thus been proposed that a real origin must contain a specific DNA sequence and must be attached to replication foci. Using Xenopus laevis egg extracts, we have identified and purified a 170-kD protein, focus-forming activity 1 (FFA-1), which is required for the formation of replication foci. Here we report that FFA-1 has DNA-helicase activity. Moreover, it is a homologue of the human Werner syndrome gene product WRN, a protein associated with premature ageing in humans.

Adenosine Triphosphate↗

PubMed2003

The 'ORC cycle': a novel pathway for regulating eukaryotic DNA replication.

The function of the 'origin recognition complex' (ORC) in eukaryotic cells is to select genomic sites where pre-replication complexes (pre-RCs) can be assembled. Subsequent activation of these pre-RCs results in bi-directional DNA replication that originates at or close to the ORC DNA binding sites. Recent results have revealed that one or more of the six ORC subunits is modified during the G1 to S-phase transition in such a way that ORC activity is inhibited until mitosis is complete and a nuclear membrane is assembled. In yeast, Cdk1/Clb phosphorylates ORC. In frog eggs, pre-RC assembly destabilizes ORC/chromatin sites, and ORC is eventually hyperphosphorylated and released. In mammals, the affinity of Orc1 for chromatin is selectively reduced during S-phase and restored during early G1-phase. Unbound Orc1 is ubiquitinated during S-phase and in some cases degraded. Thus, most, perhaps all, eukaryotes exhibit some manifestation of an 'ORC cycle' that restricts the ability of ORC to initiate pre-RC assembly to the early G1-phase of the cell cycle, making the 'ORC cycle' the premier step in determining when replication begins.

Animals↗

PubMed2006

Perturbation of the activity of replication origin by meiosis-specific transcription.

We have determined the activity of all ARSs on the Saccharomyces cerevisiae chromosome VI as chromosomal replication origins in premeiotic S-phase by neutral/neutral two-dimensional gel electrophoresis. The comparison of origin activity of each origin in mitotic and premeiotic S-phase showed that one of the most efficient origins in mitotic S-phase, ARS605, was completely inhibited in premeiotic S-phase. ARS605 is located within the open reading frame of MSH4 gene that is transcribed specifically during an early stage of meiosis. Systematic analysis of relationships between MSH4 transcription and ARS605 origin activity revealed that transcription of MSH4 inhibited the ARS605 origin activity by removing origin recognition complex from ARS605. Deletion of UME6, a transcription factor responsible for repressing MSH4 during mitotic S-phase, resulted in inactivation of ARS605 in mitosis. Our finding is the first demonstration that the transcriptional regulation on the replication origin activity is related to changes in cell physiology. These results may provide insights into changes in replication origin activity in embryonic cell cycle during early developmental stages.

Cell Cycle Proteins↗