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PubMed2001

Two compound replication origins in Saccharomyces cerevisiae contain redundant origin recognition complex binding sites.

While many of the proteins involved in the initiation of DNA replication are conserved between yeasts and metazoans, the structure of the replication origins themselves has appeared to be different. As typified by ARS1, replication origins in Saccharomyces cerevisiae are <150 bp long and have a simple modular structure, consisting of a single binding site for the origin recognition complex, the replication initiator protein, and one or more accessory sequences. DNA replication initiates from a discrete site. While the important sequences are currently less well defined, metazoan origins appear to be different. These origins are large and appear to be composed of multiple, redundant elements, and replication initiates throughout zones as large as 55 kb. In this report, we characterize two S. cerevisiae replication origins, ARS101 and ARS310, which differ from the paradigm. These origins contain multiple, redundant binding sites for the origin recognition complex. Each binding site must be altered to abolish origin function, while the alteration of a single binding site is sufficient to inactivate ARS1. This redundant structure may be similar to that seen in metazoan origins.

Base Sequence↗

PubMed1993

Yeast origin recognition complex functions in transcription silencing and DNA replication.

The genes encoding two of the subunits of the Saccharomyces cerevisiae origin recognition complex (ORC) have been isolated. Characterization of a temperature-sensitive mutation in the gene encoding the 72-kD subunit of ORC (ORC2) indicates that this protein complex functions early in the DNA replication process. Moreover, ORC derived from orc2ts cells is defective for DNA binding. Others have shown a defect in orc2ts cells in transcriptional silencing at the silent mating-type loci. Consistent with this finding, ORC specifically binds to each of the four mating-type silencers identified in yeast. These findings support the hypothesis that ORC acts as an initiator protein at yeast origins of DNA replication and suggest that ORC also functions in the determination of transcriptional domains.

Amino Acid Sequence↗

PubMed2000

Subsets of human origin recognition complex (ORC) subunits are expressed in non-proliferating cells and associate with non-ORC proteins.

The origin recognition complex (ORC) in yeast is a complex of six tightly associated subunits essential for the initiation of DNA replication. Human ORC subunits are nuclear in proliferating cells and in proliferative tissues like the testis, consistent with a role of human ORC in DNA replication. Orc2, Orc3, and Orc5 also are detected in non-proliferating cells like cardiac myocytes, adrenal cortical cells, and neurons, suggesting an additional role of these proteins in non-proliferating cells. Although Orc2-5 co-immunoprecipitate with each other under mild extraction conditions, a holo complex of the subunits is difficult to detect. When extracted under more stringent extraction conditions, several of the subunits co-immunoprecipitate with stoichiometric amounts of other unidentified proteins but not with any of the known ORC subunits. The variation in abundance of individual ORC subunits (relative to each other) in several tissues, expression of some subunits in non-proliferating tissues, and the absence of a stoichiometric complex of all the subunits in cell extracts indicate that subunits of human ORC in somatic cells might have activities independent of their role as a six subunit complex involved in replication initiation. Finally, all ORC subunits remain consistently nuclear, and Orc2 is consistently phosphorylated through all stages of the cell cycle, whereas Orc1 is selectively phosphorylated in mitosis.

Blotting, Northern↗

PubMed2003

Analysis on origin recognition complex containing Orc5p with defective Walker A motif.

Orc5p is one of six proteins that make up the origin recognition complex (ORC), a candidate initiator of chromosomal DNA replication in eukaryotes. To investigate the role of ATP binding to Orc5p in cells, we constructed orc5-A, a strain of Saccharomyces cerevisiae having a mutation in the Walker A motif of Orc5p (K43E). The strain showed temperature-sensitive growth. Incubation at a nonpermissive temperature (37 degrees C) caused accumulation of cells with nearly 2C DNA content. Overproduction of Orc4p, another subunit of ORC, suppresses this temperature sensitivity, but overproduction of other subunits did not. Overproduction of Orc4p did not suppress the temperature sensitivity of another orc5 mutant, orc5-1, whose mutation, L331P, is outside the ATP-binding motif. These results suggest that Orc4p is specifically involved in ATP binding to Orc5p itself or its function in DNA replication. Immunoblotting experiments revealed that in the orc5-A strain at a nonpermissive temperature, all ORC subunits gradually disappeared, suggesting that ORC5-A becomes degraded at nonpermissive temperatures. We therefore consider that ATP binding to Orc5p is involved in efficient ORC formation and that Orc4p is involved in this process.

Adenosine Triphosphate↗

PubMed1998

Mouse homolog of the yeast origin recognition complex subunit ORC1 and chromosomal localization of the cognate mouse gene Orc1.

ORC1 encodes a subunit of the eukaryotic origin recognition complex in yeast, which has been shown to play a crucial role in chromosomal DNA replication and transcriptional silencing. We have identified a mouse protein closely related to Saccharomyces cerevisiae ORC1 as well as to the human, Xenopus and Drosophila homologs. MmORC1 has putative cyclin-dependent phosphorylation sites and a nucleotide triphosphate-binding motif. We have mapped the gene locus of the murine Orc1 gene to chromosome 4, band D. Conservation of structures among members of the ORC1-related proteins suggests that these proteins play a key role in the intiation of DNA replication in all eukaryotes.

Amino Acid Sequence↗

PubMed1995

Separation of origin recognition complex functions by cross-species complementation.

Transcriptional silencing at the HMRa locus of Saccharomyces cerevisiae requires the function of the origin recognition complex (ORC), the replication initiator of yeast. Expression of a Drosophila melanogaster Orc2 complementary DNA in the yeast orc2-1 strain, which is defective for replication and silencing, complemented the silencing defect but not the replication defect; this result indicated that the replication and silencing functions of ORC were separable. The orc2-1 mutation mapped to the region of greatest homology between the Drosophila and yeast proteins. The silent state mediated by DmOrc2 was epigenetic; it was propagated during mitotic divisions in a relatively stable way, whereas the nonsilent state was metastable. In contrast, the silent state was erased during meiosis.

Animals↗

PubMed2000

Chromatin association of human origin recognition complex, cdc6, and minichromosome maintenance proteins during the cell cycle: assembly of prereplication complexes in late mitosis.

Evidence obtained from studies with yeast and Xenopus indicate that the initiation of DNA replication is a multistep process. The origin recognition complex (ORC), Cdc6p, and minichromosome maintenance (MCM) proteins are required for establishing prereplication complexes, upon which initiation is triggered by the activation of cyclin-dependent kinases and the Dbf4p-dependent kinase Cdc7p. The identification of human homologues of these replication proteins allows investigation of S-phase regulation in mammalian cells. Using centrifugal elutriation of several human cell lines, we demonstrate that whereas human Orc2 (hOrc2p) and hMcm proteins are present throughout the cell cycle, hCdc6p levels vary, being very low in early G(1) and accumulating until cells enter mitosis. hCdc6p can be polyubiquitinated in vivo, and it is stabilized by proteasome inhibitors. Similar to the case for hOrc2p, a significant fraction of hCdc6p is present on chromatin throughout the cell cycle, whereas hMcm proteins alternate between soluble and chromatin-bound forms. Loading of hMcm proteins onto chromatin occurs in late mitosis concomitant with the destruction of cyclin B, indicating that the mitotic kinase activity inhibits prereplication complex formation in human cells.

Antibodies, Monoclonal↗

PubMed2003

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

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

Adenosine Triphosphate↗

PubMed2001

DNA replication control through interaction of E2F-RB and the origin recognition complex.

The E2F transcription factor and retinoblastoma protein control cell-cycle progression and DNA replication during S phase. Mutations in the Drosophila dE2F1 and dDP genes affect the origin recognition complex (DmORC) and initiation of replication at the chorion gene replication origin. Here we show that mutants of Rbf (an retinoblastoma protein homologue) fail to limit DNA replication. We also show that the dDP, dE2F1 and Rbf proteins are located in a complex with DmORC, and that dE2F1 and DmORC are bound to the chorion origin of replication in vivo. Our results indicate that dE2F1 and Rbf function together at replication origins to limit DNA replication through interactions with DmORC.

Animals↗

PubMed2006

Nucleotide-dependent conformational changes in the DnaA-like core of the origin recognition complex.

Structural details of initiator proteins for DNA replication have provided clues to the molecular events in this process. EM reconstructions of the Drosophila melanogaster origin recognition complex (ORC) reveal nucleotide-dependent conformational changes in the core of the complex. All five AAA+ domains in ORC contain a conserved structural element that, in DnaA, promotes formation of a right-handed helix, indicating that helical AAA+ substructures may be a feature of all initiators. A DnaA helical pentamer can be docked into ORC, and the location of Orc5 uniquely positions this core. The results suggest that ATP-dependent conformational changes observed in ORC derive from reorientation of the AAA+ domains. By analogy to the DNA-wrapping activity of DnaA, we posit that ORC together with Cdc6 prepares origin DNA for helicase loading through mechanisms related to the established pathway of prokaryotes.

Adenosine Triphosphate↗

PubMed2004

The origin recognition complex links replication, sister chromatid cohesion and transcriptional silencing in Saccharomyces cerevisiae.

Mutations in genes encoding the origin recognition complex (ORC) of Saccharomyces cerevisiae affect initiation of DNA replication and transcriptional repression at the silent mating-type loci. To explore the function of ORC in more detail, a screen for genetic interactions was undertaken using large-scale synthetic lethal analysis. Combination of orc2-1 and orc5-1 alleles with the complete set of haploid deletion mutants revealed synthetic lethal/sick phenotypes with genes involved in DNA replication, chromatin structure, checkpoints, DNA repair and recombination, and other genes that were unexpected on the basis of previous studies of ORC. Many of these genetic interactions are shared with other genes that are involved in initiation of DNA replication. Strong synthetic interactions were demonstrated with null mutations in genes that contribute to sister chromatid cohesion. A genetic interaction between orc5-1 and the cohesin mutant scc1-73 suggested that ORC function contributes to sister chromatid cohesion. Thus, comprehensive screening for genetic interactions with a replication gene revealed a connection between initiation of DNA replication and sister chromatid cohesion. Further experiments linked sister chromatid cohesion genes to silencing at mating-type loci and telomeres.

DNA Replication↗

PubMed1999

Assembly of functionally active Drosophila origin recognition complex from recombinant proteins.

In eukaryotes the sites for the initiation of chromosomal DNA replication are believed to be determined in part by the binding of a heteromeric origin recognition complex (ORC) to DNA. We have cloned the genes encoding the subunits of the Drosophila ORC. Each of the genes is unique and can be mapped to discrete chromosomal locations implying that the pattern and developmental regulation of origin usage in Drosophila is not regulated solely by a large family of different ORC proteins. The six-subunit ORC can be reconstituted with recombinant proteins into a complex that restores DNA replication in ORC-depleted Drosophila or Xenopus egg extracts.

Amino Acid Sequence↗

PubMed1997

The origin recognition complex, SIR1, and the S phase requirement for silencing.

Silencing of transcription in Saccharomyces cerevisiae has several links to DNA replication, including a role for the origin recognition complex (ORC), the DNA replication initiator, in both processes. In addition, the establishment of silencing at the HML and HMR loci requires cells to pass through the S phase of the cell cycle. Passage through S phase was required for silencing of HMR even under conditions in which ORC itself was no longer required. The requirement for ORC in silencing of HMR could be bypassed by tethering the Sir1 protein to the HMR-E silencer. However, ORC had a Sir1-independent role in transcriptional silencing at telomeres. Thus, the role of ORC in silencing was separable from its role in initiation, and the role of S phase in silencing was independent of replication initiation at the silencers.

Aldose-Ketose Isomerases↗

PubMed2001

Mutation analysis of the origin recognition complex subunit 5 (ORC5L) gene in adult patients with myeloid leukemias exhibiting deletions of chromosome band 7q22.

The ORC5L gene encoding a subunit of the human origin recognition complex (ORC) maps to chromosome band 7q22, a region frequently deleted in adult acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). Because of its localization within a region that is commonly deleted in patients with myeloid malignancies and because of the implication of its protein product in cell cycle control (DNA replication) and regulation of gene expression (transcriptional silencing), ORC5L appeared to be a candidate tumor suppressor gene for myeloid disorders associated with 7q22 deletions. Polymerase chain reaction amplification and sequencing analysis of the coding region of the remaining ORC5L allele has not revealed any mutations in nine patients with AML or MDS exhibiting 7q22 deletions. Allelic expression analysis indicates that ORC5L is not imprinted. These data suggest that ORC5L does not function as a tumor suppressor in patients with myeloid neoplasms.

Acute Disease↗

PubMed2001

Control of DNA rereplication via Cdc2 phosphorylation sites in the origin recognition complex.

Cdc2 kinase is a master regulator of cell cycle progression in the fission yeast Schizosaccharomyces pombe. Our data indicate that Cdc2 phosphorylates replication factor Orp2, a subunit of the origin recognition complex (ORC). Cdc2 phosphorylation of Orp2 appears to be one of multiple mechanisms by which Cdc2 prevents DNA rereplication in a single cell cycle. Cdc2 phosphorylation of Orp2 is not required for Cdc2 to activate DNA replication initiation. Phosphorylation of Orp2 appears first in S phase and becomes maximal in G(2) and M when Cdc2 kinase activity is required to prevent reinitiation of DNA replication. A mutant lacking Cdc2 phosphorylation sites in Orp2 (orp2-T4A) allowed greater rereplication of DNA than congenic orp2 wild-type strains when the limiting replication initiation factor Cdc18 was deregulated. Thus, Cdc2 phosphorylation of Orp2 may be redundant with regulation of Cdc18 for preventing reinitiation of DNA synthesis. Since Cdc2 phosphorylation sites are present in Orp2 (also known as Orc2) from yeasts to metazoans, we propose that cell cycle-regulated phosphorylation of the ORC provides a safety net to prevent DNA rereplication and resulting genetic instability.

Amino Acid Sequence↗

PubMed2005

The highly conserved tRNAHis guanylyltransferase Thg1p interacts with the origin recognition complex and is required for the G2/M phase transition in the yeast Saccharomyces cerevisiae.

Here we show that the Saccharomyces cerevisiae tRNA(His) guanylyltransferase Thg1p interacts with the origin recognition complex in vivo and in vitro and that overexpression of hemagglutinin-Thg1p selectively impedes growth of orc2-1(Ts) cells at the permissive temperature. Studies with conditional mutants indicate that Thg1p couples nuclear division and migration to cell budding and cytokinesis in yeast.

Cell Division↗

PubMed2007

Mechanism for the degradation of origin recognition complex containing Orc5p with a defective Walker A motif and its suppression by over-production of Orc4p in yeast cells.

Orc5p is one of six subunits constituting the ORC (origin recognition complex), a possible initiator of chromosomal DNA replication in eukaryotes. Orc5p contains a Walker A motif. We recently reported that a strain of Saccharomyces cerevisiae having a mutation in Orc5p's Walker A motif (orc5-A), showed cell-cycle arrest at G2/M and degradation of ORC at high temperatures (37 degrees C). Over-production of Orc4p, another subunit of ORC, specifically suppressed these phenotypes [Takahashi, Yamaguchi, Yamairi, Makise, Takenaka, Tsuchiya and Mizushima (2004) J. Biol. Chem. 279, 8469-8477]. In the present study, we examined the mechanisms of ORC degradation and of its suppression by Orc4p over-production. In orc5-A, at high temperatures, ORC is degraded by proteasomes; either addition of a proteasome inhibitor, or introduction of a mutation of either tan1-1 or nob1-4 that inhibits proteasomes, prevented ORC degradation. Introduction of the tan1-1 mutation restored cell cycle progression, suggesting that the defect was due to ORC degradation by proteasomes. Yeast two-hybrid and co-immunoprecipitation analyses suggested that Orc5p interacts preferentially with Orc4p and that the orc5-A mutation diminishes this interaction. We suggest that this interaction is mediated by the C-terminal region of Orc4p, and the N-terminal region of Orc5p. Based on these observations, we consider that ATP binding to Orc5p is required for efficient interaction with Orc4p and that, in orc5-A, loss of this interaction at higher temperatures allows proteasomes to degrade ORC, causing growth defects. This model could also explain why over-production of Orc4p suppresses the orc5-A strain's phenotype.

Adenosine Triphosphate↗

PubMed1999

latheo encodes a subunit of the origin recognition complex and disrupts neuronal proliferation and adult olfactory memory when mutant.

The Drosophila latheo (lat) gene was identified in a behavioral screen for olfactory memory mutants. The original hypomorphic latP1 mutant (Boynton and Tully, 1992) shows a structural defect in adult brain. Homozygous lethal lat mutants lack imaginal discs, show little cell proliferation in the CNS of third instar larvae, and die as early pupae. latP1 was cloned, and all of the above mentioned defects of hypomorphic or homozygous lethal lat mutants were rescued with a lat+ transgene. lat encodes a novel protein with homology to a subunit of the origin recognition complex (ORC). Human and Drosophila LAT both associate with ORC2 and are related to yeast ORC3, suggesting that LAT functions in DNA replication during cell proliferation.

Amino Acid Sequence↗