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PubMed2004

ATP hydrolysis by ORC catalyzes reiterative Mcm2-7 assembly at a defined origin of replication.

The origin recognition complex (ORC) is a six-subunit, ATP-regulated, DNA binding protein that is required for the formation of the prereplicative complex (pre-RC), an essential replication intermediate formed at each origin of DNA replication. In this study, we investigate the mechanism of ORC function during pre-RC formation and how ATP influences this event. We demonstrate that ATP hydrolysis by ORC requires the coordinate function of the Orc1 and Orc4 subunits. Mutations that eliminate ORC ATP hydrolysis do not support cell viability and show defects in pre-RC formation. Pre-RC formation involves reiterative loading of the putative replicative helicase, Mcm2-7, at the origin. Importantly, preventing ORC ATP hydrolysis inhibits this repeated Mcm2-7 loading. Our findings indicate that ORC is part of a helicase-loading molecular machine that repeatedly assembles Mcm2-7 complexes onto origin DNA and suggest that the assembly of multiple Mcm2-7 complexes plays a critical role in origin function.

Adenosine Triphosphate↗
PubMed2006

Identification of new human origins of DNA replication by an origin-trapping assay.

Metazoan genomes contain thousands of replication origins, but only a limited number have been characterized so far. We developed a two-step origin-trapping assay in which human chromatin fragments associated with origin recognition complex (ORC) in vivo were first enriched by chromatin immunoprecipitation. In a second step, these fragments were screened for transient replication competence in a plasmid-based assay utilizing the Epstein-Barr virus latent origin oriP. oriP contains two elements, an origin (dyad symmetry element [DS]) and the family of repeats, that when associated with the viral protein EBNA1 facilitate extrachromosomal stability. Insertion of the ORC-binding human DNA fragments in oriP plasmids in place of DS enabled us to screen functionally for their abilities to restore replication. Using the origin-trapping assay, we isolated and characterized five previously unknown human origins. The assay was validated with nascent strand abundance assays that confirm these origins as active initiation sites in their native chromosomal contexts. Furthermore, ORC and MCM2-7 components localized at these origins during G(1) phase of the cell cycle but were not detected during mitosis. This finding extends the current understanding of origin-ORC dynamics by suggesting that replication origins must be reestablished during the early stages of each cell division cycle and that ORC itself participates in this process.

Base Sequence↗
PubMed1995

ORC and Cdc6p interact and determine the frequency of initiation of DNA replication in the genome.

The origin recognition complex (ORC) binds replicators in the yeast S. cerevisiae in a manner consistent with it being an initiator protein for DNA replication. Two-dimensional (2D) gel techniques were used to examine directly initiation of chromosomal DNA replication in temperature-sensitive orc mutants. Unlike in wild-type cells, in orc2-1 and orc5-1 mutant cells, only a subset of replicators formed active origins of DNA replication at the permissive temperature. At the restrictive temperature, the number of active replicators was diminished further. Using a genetic screen, CDC6 was identified as a multicopy suppressor of orc5-1. 2D gel and biochemical analyses demonstrated that Cdc6p interacted functionally and physically with ORC. We suggest that ORC and Cdc6p form a prereplication complex at individual replicators and therefore cooperate to determine the frequency of initiation of DNA replication in the genome.

Cell Cycle Proteins↗
PubMed2005

A novel motif governs APC-dependent degradation of Drosophila ORC1 in vivo.

Regulated degradation plays a key role in setting the level of many factors that govern cell cycle progression. In Drosophila, the largest subunit of the origin recognition complex protein 1 (ORC1) is degraded at the end of M phase and throughout much of G1 by anaphase-promoting complexes (APC) activated by Fzr/Cdh1. We show here that none of the previously identified APC motifs targets ORC1 for degradation. Instead, a novel sequence, the O-box, is necessary and sufficient to direct Fzr/Cdh1-dependent polyubiquitylation in vitro and degradation in vivo. The O-box is similar to but distinct from the well characterized D-box. Finally, we show that O-box motifs in two other proteins, Drosophila Abnormal Spindle and Schizosaccharomyces pombe Cut2, contribute to Cdh1-dependent polyubiquitylation in vitro, suggesting that the O-box may mediate degradation of a variety of cell cycle factors.

Amino Acid Motifs↗
PubMed2002

Human replication protein Cdc6 is selectively cleaved by caspase 3 during apoptosis.

In eukaryotes, the initiation of DNA replication involves the ordered assembly on chromatin of pre-replicative complexes (pre-RCs), including the origin recognition complex (ORC), Cdc6, Cdt1 and the minichromosome maintenance proteins (MCMs). In light of its indispensable role in the formation of pre-RCs, Cdc6 binding to chromatin represents a key step in the regulation of DNA replication and cell proliferation. Here, we study the human Cdc6 (HuCdc6) protein during programmed cell death (apoptosis). We find that HuCdc6, but not HuOrc2 (a member of the ORC) or HuMcm5 (one of the MCMs), is specifically cleaved in several human cell lines induced to undergo apoptosis by a variety of stimuli. Expression of caspase-uncleavable mutant HuCdc6 attenuates apoptosis, delaying cell death. Therefore, an important function for cleavage of HuCdc6 is to prevent a wounded cell from replicating and to facilitate death.

Apoptosis↗
PubMed1999

A region of the Sir1 protein dedicated to recognition of a silencer and required for interaction with the Orc1 protein in saccharomyces cerevisiae.

Silencing of the cryptic mating-type loci HMR and HML requires the recognition of DNA sequence elements called silencers by the Sir1p, one of four proteins dedicated to the assembly of silenced chromatin in Saccharomyces cerevisiae. The Sir1p is thought to recognize silencers indirectly through interactions with proteins that bind the silencer DNA directly, such as the origin recognition complex (ORC). Eight recessive alleles of SIR1 were discovered that encode mutant Sir1 proteins specifically defective in their ability to recognize the HMR-E silencer. The eight missense mutations all map within a 17-amino-acid segment of Sir1p, and this segment was also required for Sir1p's interaction with Orc1p. The mutant Sir1 proteins could function in silencing if tethered to a silencer directly through a heterologous DNA-binding domain. Thus the amino acids identified are required for Sir1 protein's recognition of the HMR-E silencer and interaction with Orc1p, but not for its ability to function in silencing per se. The approach used to find these mutations may be applicable to defining interaction surfaces on proteins involved in other processes that require the assembly of macromolecular complexes.

Alleles↗
PubMed2003

Putative subunits of the maize origin of replication recognition complex ZmORC1-ZmORC5.

The finding in animal species of complexes homologous to the products of six Saccharomyces cerevisiae genes, origin of replication recognition complex (ORC), has suggested that ORC-related mechanisms have been conserved in all eukaryotes. In plants, however, the only cloned putative homologs of ORC subunits are the Arabidopsis ORC2 and the rice ORC1. Homologs of other subunits of plant origin have not been cloned and characterized. A striking observation was the absence from the Arabidopsis genome of an obvious candidate gene-homolog of ORC4. This fact raised compelling questions of whether plants, in general, and Arabidopsis, in particular, may have lost the ORC4 gene, whether ORC-homologous subunits function within a complex in plants, whether an ORC complex may form and function without an ORC4 subunit, whether a functional (but not sequence) protein homolog may have taken up the role of ORC4 in Arabidopsis, and whether lack of ORC4 is a plant feature, in general. Here, we report the first cloned and molecularly characterized five genes coding for the maize putative homologs of ORC subunits ZmORC1, ZmORC2, ZmORC3, ZmORC4 and ZmORC5. Their expression profiles in tissues with different cell-dividing activities are compatible with a role in DNA replication. Based on the potential of ORC-homologous maize proteins to bind each other in yeast, we propose a model for their possible assembly within a maize ORC. The isolation and molecular characterization of an ORC4-homologous gene from maize argues that, in its evolution, Arabidopsis may have lost the homologous ORC4 gene.

DNA Replication↗
PubMed1999

A role for a replicator dominance mechanism in silencing.

The role of the natural HMR-E silencer in modulating replication initiation and silencing by the origin recognition complex (ORC) was examined. When natural HMR-E was the only silencer controlling HMR, the silencer's ORC-binding site (ACS) was dispensable for replication initiation but essential for silencing, indicating that a non-silencer chromosomal replicator(s) existed in close proximity to the silencer. Further analysis revealed that regions flanking both sides of HMR-E contained replicators. In contrast to replication initiation by the intact silencer, initiation by the non-silencer replicator(s) was abolished in an orc2-1 mutant, indicating that these replicators were extremely sensitive to defects in ORC. Remarkably, the activity of one of the non-silencer replicators correlated with reduced silencing; inactivation of these replicators caused by either the orc2-1 mutation or the deletion of flanking sequences enhanced silencing. These data were consistent with a role for the ORC bound to the HMR-E silencer ACS in suppressing the function of neighboring ORC molecules capable of inhibiting silencing, and indicated that differences in ORC-binding sites within HMR itself had profound effects on ORC function. Moreover, replication initiation by natural HMR-E was inefficient, suggesting that closely spaced replicators within HMR contributed to an inhibition of replication initiation.

Alleles↗
PubMed2000

Nucleoplasmin-mediated chromatin remodelling is required for Xenopus sperm nuclei to become licensed for DNA replication.

During late mitosis and early G(1), a series of proteins are assembled onto replication origins, resulting in them becoming 'licensed' for replication in the subsequent S phase. Four factors have so far been identified that are required for chromatin to become functionally licensed: ORC (the origin recognition complex) and Cdc6, plus the two components of the replication licensing system RLF-M and RLF-B. Here we describe the first steps of a systematic fractionation of Xenopus egg extracts to identify all the components necessary for the assembly of licensed replication origins on Xenopus sperm nuclei (the physiological DNA substrate in this system). We have purified a new activity essential for this reaction, and have shown that it is nucleoplasmin, a previously known chromatin remodelling protein. Nucleoplasmin decondenses the sperm chromatin by removing protamines, and is required at the earliest known step in origin assembly to allow ORC to bind to the DNA. Sperm nuclei can be licensed by a combination of nucleoplasmin, RLF-M and a partially purified fraction that contains ORC, Cdc6 and RLF-B. This suggests that we are likely to have identified most of the proteins required for this assembly reaction.

Animals↗
PubMed2002

Fission yeast Cdc23 interactions with DNA replication initiation proteins.

Schizosaccharomyces pombe Cdc23 is an essential DNA replication protein, conserved in eukaryotes and functionally homologous with Saccharomyces cerevisiae Dna43 (Mcm10). We sought evidence for interactions between Cdc23 and the MCM2-7 complex, a component of both the pre-replicative complex and the replication fork. Cdc23 shows genetic interactions with four MCM subunits: cdc23-M36 and cdc23-1E2 alleles both show synthetic phenotypes with mcm2 (cdc19-P1) and mcm6 (mis5-268), and cdc23-M36 is synthetically lethal with mcm4 (cdc21-K46) and with mcm5 (nda4-108). The wild-type cdc23 gene on multicopy plasmids can partially suppress temperature-dependent defects in mcm5 (nda4-108). Two-hybrid analysis demonstrates interactions at the protein-protein level between Cdc23 and Mcm4, Mcm5 and Mcm6. Cdc23 also interacts with four subunits of the Schizosaccharomyces pombe origin recognition complex (ORC) in yeast two-hybrid assay: Orc1, Orc2, Orc5 and Orc6. We found no evidence for interaction between Cdc23 and the MCM recruitment factor Cdc18 (the homologue of Saccharomyces cerevisiae Cdc6). Unlike Cdc18, Cdc23 mRNA shows no significant fluctuation in level through the cell cycle. These data suggest that fission yeast Cdc23 is an MCM-associated factor which has a role in the initiation of DNA replication.

Anaphase-Promoting Complex-Cyclosome↗
PubMed2006

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

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

Amino Acid Sequence↗
PubMed2000

The replication capacity of intact mammalian nuclei in Xenopus egg extracts declines with quiescence, but the residual DNA synthesis is independent of Xenopus MCM proteins.

In eukaryotes, the initiation of DNA synthesis requires the assembly of a pre-replicative complex (pre-RC) at origins of replication. This involves the sequential binding of ORC (origin-recognition-complex), Cdc6 and MCM proteins, a process referred to as licensing. After origin firing, the Cdc6 and MCM proteins dissociate from the chromatin, and do not rebind until after the completion of mitosis, thereby restricting replication to a single round in each cell cycle. Although nuclei normally become licensed for replication as they enter G(1), the extent to which the license is retained when cells enter the quiescent state (G(0)) is controversial. Here we show that the replication capacity of nuclei from Swiss 3T3 cells, in Xenopus egg extracts, is not lost abruptly with the onset of quiescence, but instead declines gradually. The decline in replication capacity, which affects both the number of nuclei induced to replicate and their subsequent rate of DNA synthesis, is accompanied by a fall in the level of chromatin-bound MCM2. When quiescent nuclei are incubated in egg extracts, they do not bind further MCMs unless the nuclei are first permeabilized. The residual replication capacity of intact nuclei must therefore be dependent on the remaining endogenous MCMs. Although high levels of Cdk activity are known to block MCM binding, we show that the failure of intact nuclei in egg extracts to increase their bound MCMs is not due to their uptake and accumulation of Cdk complexes. Instead, the failure of binding must be due to exclusion of some other binding factor from the nucleus, or to the presence within nuclei of an inhibitor of binding other than Cdk activity. In contrast to the situation in Xenopus egg extracts, following serum stimulation of intact quiescent cells, the level of bound MCMs does increase before the cells reach S phase, without any disruption of the nuclear envelope.

3T3 Cells↗
PubMed2003

Two bipartite NLSs mediate constitutive nuclear localization of Mcm10.

Some components of the pre-replicative complex (pre-RC) are in the nucleus only during the G1 phase, while others are constitutively nuclear throughout the cell cycle. A nuclear localization signal (NLS) mediating the G1-specific nuclear localization has been characterized. We report here the identification of bipartite NLSs in constitutively nuclear pre-RC components, including the origin recognition complex, Mcm10 and Noc3. Interestingly, in a subgroup of these pre-RC components, two putative NLSs were found in each protein. To examine whether multiple NLSs are required for these proteins to be nuclear throughout the cell cycle, we characterized the two NLSs in Mcm10. We show that at least one of the two NLSs is required for directing Mcm10 into the nucleus and that either of them is sufficient to bring the green fluorescence protein into the nucleus. Thus, Mcm10 contains two functional NLSs and either is sufficient for the constitutive nuclear localization of Mcm10.

Blotting, Western↗
PubMed2000

Interactions between Mcm10p and other replication factors are required for proper initiation and elongation of chromosomal DNA replication in Saccharomyces cerevisiae.

BACKGROUND: MCM10 is essential for the initiation of chromosomal DNA replication in Saccharomyces cerevisiae. Previous work showed that Mcm10p interacts with the Mcm2-7 protein complex that may be functioning as the replication-licensing factor. In addition, Mcm10p is required during origin activation and disassembly of the prereplicative complex, which allows smooth passage of replication forks. RESULTS: We show that an mcm10 mutation causes a slow progression of DNA synthesis and a loss of chromosome integrity during the S phase and prevents entry into mitosis, despite apparent completion of chromosomal DNA replication at nonpermissive temperatures. Furthermore, Mcm10p interacts genetically with the origin recognition complex (ORC) and various replication elongation factors, including a subunit of DNA polymerases epsilon and delta. Mcm10p is an abundant protein (approximately 4 x 10(4) copies per haploid cell) that is almost exclusively localized in the chromatin and/or nuclear matrix fractions during all phases of the cell cycle. When it is visualized by the chromosome-spreading method followed by immunostaining, Mcm10p forms punctate foci on chromatin throughout the cell cycle and these foci mostly overlap with those of Orc1p, a component of ORC. CONCLUSIONS: These results suggest that Mcm10p, like the Mcm2-7 proteins, is a critical component of the prereplication chromatin and acts together with ORC during the initiation of chromosomal DNA replication; in addition, Mcm10p plays an important role during the elongation of DNA replication.

Cell Cycle Proteins↗
PubMed1997

Functional equivalency and diversity of cis-acting elements among yeast replication origins.

The DNA replication origins of the yeast Saccharomyces cerevisiae require several short functional elements, most of which are not conserved in sequence. To better characterize ARS305, a replicator from a chromosomal origin, we swapped functional DNA elements of ARS305 with defined elements of ARS1. ARS305 contains elements that are functionally exchangeable with ARS1 A and B1 elements, which are known to bind the origin recognition complex; however, the ARS1 A element differs in that it does not require a 3' box adjacent to the essential autonomously replicating sequence consensus. At the position corresponding to ARS1 B3, ARS305 has a novel element, B4, that can functionally substitute for every type of short element (B1, B2, and B3) in the B domain. Unexpectedly, the replacement of element B4 by ARS1 B3, which binds ABF1p and is known as a replication enhancer, inhibited ARS305 function. ARS305 has no short functional element at or near positions corresponding to the B2 elements in ARS1 and ARS307 but contains an easily unwound region whose functional importance was supported by a broad G+C-rich substitution mutation. Surprisingly, the easily unwound region can functionally substitute for the ARS1 B2 element, even though ARS1 B2 was found to possess a distinct DNA sequence requirement. The functionally conserved B2 element in ARS307 contains a known sequence requirement, and helical stability analysis of linker and minilinker mutations suggested that B2 also contains a DNA unwinding element (DUE). Our findings suggest that yeast replication origins employ a B2 element or a DUE to mediate a common function, DNA unwinding during initiation, although not necessarily through a common mechanism.

DNA Helicases↗
PubMed1998

Roles for ORC in M phase and S phase.

The origin recognition complex (ORC), a six-subunit protein, functions as the replication initiator in the yeast Saccharomyces cerevisiae. Initiation depends on the assembly of the prereplication complex in late M phase and activation in S phase. One subunit of ORC, Orc5p, was required at G1/S and in early M phase. Asynchronous cells with a temperature-sensitive orc5-1 allele arrested in early M phase. In contrast, cells that were first synchronized in M phase, shifted to the restrictive temperature, and then released from the block arrested at the G1/S boundary. The G1/S arrest phenotype could not be suppressed by introducing wild-type Orc5p during G1. Although all orc2 and orc5 mutations were recessive in the conventional sense, this dominant phenotype was shared with other orc5 alleles and an orc2 allele. The dominant inhibition to cell-cycle progression exhibited by the orc mutants was restricted to the nucleus, suggesting that chromosomes with mutant ORC complexes were capable of sending a signal that blocked initiation on chromosomes containing functional origins.

CDC28 Protein Kinase, S cerevisiae↗
PubMed2006

DNA replication in protein extracts from human cells requires ORC and Mcm proteins.

We used protein extracts from proliferating human HeLa cells to support plasmid DNA replication in vitro. An extract with soluble nuclear proteins contains the major replicative chain elongation functions, whereas a high salt extract from isolated nuclei contains the proteins for initiation. Among the initiator proteins active in vitro are the origin recognition complex (ORC) and Mcm proteins. Recombinant Orc1 protein stimulates in vitro replication presumably in place of endogenous Orc1 that is known to be present in suboptimal amounts in HeLa cell nuclei. Partially purified endogenous ORC, but not recombinant ORC, is able to rescue immunodepleted nuclear extracts. Plasmid replication in the in vitro replication system is slow and of limited efficiency but robust enough to serve as a basis to investigate the formation of functional pre-replication complexes under biochemically defined conditions.

Animals↗
PubMed2005

Replication of heterochromatin: insights into mechanisms of epigenetic inheritance.

Heterochromatin is composed of tightly condensed chromatin in which the histones are deacetylated and methylated, and specific nonhistone proteins are bound. Additionally, in vertebrates and plants, the DNA within heterochromatin is methylated. As the heterochromatic state is stably inherited, replication of heterochromatin requires not only duplication of the DNA but also a reinstallment of the appropriate protein and DNA modifications. Thus replication of heterochromatin provides a framework for understanding mechanisms of epigenetic inheritance. In recent studies, roles have been identified for replication factors in reinstating heterochromatin, particularly functions for origin recognition complex, proliferating cell nuclear antigen, and chromatin-assembly factor 1 in recruiting the heterochromatin binding protein HP1, a histone methyltransferase, a DNA methyltransferase, and a chromatin remodeling complex. Potential mechanistic links between these factors are discussed. In some cells, replication of the heterochromatin is blocked, and in Drosophila this inhibition is mediated by a chromatin binding protein SuUR.

Animals↗