Origin recognition complex subunit 2
The Xenopus Cdc6 protein is essential for the initiation of DNA replication. The Xenopus homolog of ORC2, Xorc2 cannot bind to chromatin at M phase in Xenopus extracts, suggesting that its ability to bind to the origin DNA is regulated by the cell cycle. This difference may be due to the fact that chromosomes of Xenopus and other vertebrates undergo extensive condensation at mitosis. At the end of mitosis, when the chromatin decondenses and the Cdc2-cyclin B complex (for the fly homolog see Cyclin B) undergoes inactivation, both Xorc2 and Xcdc6 can reassociate with chromatin. Binding of Xcdc6 to chromatin requires Xorc2. Following the association of Xorc2 with Xenopus chromatin, the Xcdc6 and Xmcm3 (for a fly homolog see Disc proliferation abnormal) proteins bind shortly thereafter. Xmcm3 cannot bind to chromatin lacking Xcdc6. At some point, S-phase promoting factor (SPF) triggers the firing of the replication origins. In Xenopus, the Cdck2-cyclinE (for a fly homolog see CyclinE) complex is necessary and perhaps sufficient to fulfill the role of SPF. The critical targets of Cdk2-cyclinE may be Xcdc6 and/or components of the ORC. Phosphorylation of Xcdc6 by Cdk2-cyclinE could lead to both the firing of the origin and the subsequent elimination of Xcdc6 from the origin. In postreplicative nuclei, Xcdc6 is associated with the nuclear envelope (Coleman, 1996 and references).
The Xenopus Origin Recognition Complex interacts with the components of the replication licensing system. RLF-M binds to chromatin early in the cell cycle and licenses DNA for replication in the subsequent S phase. Immunodepletion of XOrc1 from Xenopus egg extracts blocks the initiation of DNA replication. In Xenopus egg extracts, ORC associates with chromatin throughout G1 and S phases. RLF-M also associates with chromatin early in the cell cycle but dissociates during S phase. The assembly of RLF-M into chromatin is dependent on the presence of chromatin-bound ORC, leading to sequential assembly of initiation proteins onto replication origins during the cell cycle (Rowles, 1996).
A Xenopus laevis Orc2-related protein (XORC2) has been identified by its ability to rescue a
mitotic-catastrophe mutant of the fission yeast S. pombe. Immunodepletion of XORC2 from Xenopus egg extracts abolishes the replication of chromosomal DNA but not elongation synthesis on a single-stranded DNA template. XORC2 binds to chromatin well before the commencement of DNA synthesis, 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 (Carpenter, 1996).
Before initiation of DNA replication, origin recognition complex (ORC) proteins [cdc6, and
minichromosome maintenance (MCM) proteins] bind to chromatin sequentially and form preinitiation
complexes. After the formation of these complexes in Xenopus laevis egg extracts,
and before initiation of DNA replication, cdc6 is rapidly removed from chromatin, possibly degraded by a cdk2-activated, ubiquitin-dependent proteolytic pathway. If this displacement is inhibited, DNA
replication fails to initiate. After assembly of MCM proteins into preinitiation
complexes, removal of the ORC from DNA does not block the subsequent initiation of replication.
Importantly, under conditions in which both ORC and cdc6 protein are absent from preinitiation
complexes, DNA replication is still dependent on cdk2 activity. Therefore, the final steps in the process
leading to initiation of DNA replication during S phase of the cell cycle are independent of ORC and
cdc6 proteins, but dependent on cdk2 activity. Potential targets for this second cdk2 step include phosphorylation of MCM proteins by cdk2, or the cdk2-dependent activation of other proteins essential for initiation, such as cdc7, or an as yet to be identified helicase (Hua, 1998).
Replication licensing factor (RLF) is an essential initiation factor that can prevent re-replication of
DNA in a single cell cycle. It is required for the initiation of DNA replication, binds to chromatin
early in the cell cycle, is removed from chromatin as DNA replicates and is unable to re-bind replicated
chromatin until the following mitosis. Chromatography of RLF from Xenopus extracts has shown that it
consists of two components termed RLF-B and RLF-M. The RLF-M component consists of
complexes of all six Xenopus minichromosome maintenance (MCM/P1) proteins (XMcm2-7), which
bind to chromatin in late mitosis and are removed as replication occurs. The identity
of RLF-B is currently unknown. At least two factors must be present on chromatin before licensing
can occur: the Xenopus origin recognition complex (XORC) and Xenopus Cdc6 (XCdc6).
XORC saturates Xenopus sperm chromatin at approximately one copy per replication origin whereas
XCdc6 binds to chromatin only if XORC is bound first. Although XORC has been shown
to be a distinct activity from RLF-B, the relationship between XCdc6 and RLF-B is currently
unclear. Active XCdc6 is loaded onto chromatin in extracts with defective RLF,
and both RLF-M and RLF-B are still required for the licensing of XCdc6-containing chromatin.
Furthermore, RLF-B can be separated from XCdc6 by immunoprecipitation and standard
chromatography. These experiments demonstrate that RLF-B is both functionally and physically
distinct from XCdc6, and that XCdc6 is loaded onto chromatin before RLF-B function is executed (Tada, 1999).
A new member of human origin recognition complex (ORC) has been cloned and identified as the human homologue of Saccharomyces cerevisiae ORC4. HsORC4
is a 45-kDa protein encoded by a 2.2-kilobase mRNA whose amino acid sequence is 29% identical to ScORC4. HsORC4 has a putative nucleotide triphosphate
binding motif that is not seen in ScORC4. HsORC4P also reveals an unsuspected homology to the ORC1-Cdc18 family of proteins. HsORC4 mRNA expression
and protein levels remain constant through the cell cycle. HsORC4P is coimmunoprecipitated from cell extracts with another subunit of human ORC, HsORC2P,
consistent with it being a part of the putative human origin recognition complex (Quintana, 1997).
A new member of the human origin recognition complex (ORC) was cloned and identified as ORC5L. HsORC5p is a 50-kDa protein whose sequence is 38%
identical and 62% similar to ORC5p from Drosophila melanogaster. Two alleles of ORC5L were identified, one with and one without an evolutionarily conserved
purine nucleotide binding motif. HsORC5p is precipitated from cell extracts with HsORC2p and HsORC4p, indicating that it is part of the putative human ORC.
The bulk of HsORC5p is in an insoluble nuclear fraction, whereas the other known human ORC subunits (HsORC1p, HsORC2p, and HsORC4p) are easily
extracted in the nuclear-soluble fractions and in S100 (HsORC1p). In addition, an alternatively spliced mRNA has been identified from the same locus (HsORC5T).
HsORC5Tp also forms a complex with HsORC4p but not with HsORC2p, suggesting it may play a regulatory role in the assembly of different ORC
subcomplexes. HsORC5, HsORC5T, and HsORC4 transcripts are abundant in spleen, ovary, and prostate in addition to tissues with high levels of DNA replication
like testes and colon mucosa, implicating the human ORC proteins in functions besides DNA replication. Finally, the gene for ORC5L is located at chromosome 7
band q22, in the minimal region deleted in 10% of uterine leiomyomas and in 10-20% of acute myeloid leukemias and myelodysplastic syndromes (Quintana, 1998).
Constituents of the human replication ORC are in the process of being identified. Of the six subunits of ORC
identified in Saccharomyces cerevisiae, homologs of four have been molecularly cloned from human cells and are known to
associate with each other. A fifth, 80 kDa protein consistently coimmunopreciptiates with human ORC2 from human
cell extracts. The immunoprecipitation reaction was scaled up, and the 80 kDa
protein was identified by ion trap mass spectrometry. Multiple peptides were identical to the sequence of the protein encoded
by the human EST clone. Additional peptides were identified, however, suggesting a 5' truncation of the clone. A complete
cDNA was obtained by RACE-PCR and from other EST clones in the database. The
sequence of the 80 kDa protein deduced from the full-length cDNA reveals that it is 30% identical (42% similar) to
Drosophila melanogaster Latheo. Accordingly, the 80 kDa protein Homo sapiens has been termed Lat
(HsLat). HsLat and DmLat also appear related to S. cerevisiae ORC3. When the human protein is compared
with all translated ORFs in the S. cerevisiae genome database, ScORC3 emerges as the closest homolog in the yeast
genome, with one region of the human protein (residues 216 to 559) 23% identical (43% similar) to ScORC3 (Pinto, 1999 and references).
Immunoprecipitation of human ORC2 with anti-HsORC2 antibodies coprecipitates metabolically labeled
HsORC4 (45 kDa) and an unknown protein of 80 kDa (Quintana, 1997). In a complementary experiment, anti-HsLat immunoprecipitates were probed with
anti-HsORC2 or anti-HsLat. Both proteins were present in the anti-HsLat immunoprecipitate, confirming that HsORC2
and HsLat associate with each other in cell extracts. The interaction between HsORC2 and HsLat is not mediated by association of the two
proteins with DNA. The presence of 200 µg/ml ethidium bromide, which intercalates with DNA and disrupts protein-DNA
interactions does not dissociate the HsORC2-HsLat complex (Pinto, 1999).
HsORC4 and HsORC5 were expressed in mammalian cells with an N-terminal glutathione S-transferase (GST) epitope tag. Both GST-HsORC4 and GST-HsORC5 expressed in mammalian cells copurify
with human HsORC2 (Quintana, 1997; Quintana, 1998). When GST-HsORC4 or
GST-HsORC5 is isolated from human embryonic kidney 293T cells by affinity purification with glutathione agarose
beads, HsLat copurifies. GST alone expressed in mammalian cells failed to copurify with
HsLat, suggesting that HsLat forms a complex in mammalian cells with HsORC4 and HsORC5, as it does with
HsORC2. GST-HsLat also was
expressed in mammalian cells and shown to copurify specifically with the 72 kDa HsORC2 protein on glutathione agarose
beads. At least a portion of cellular HsLat, HsORC2, HsORC4, and HsORC5, therefore, are
associated with each other, which is consistent with the notion that Lat functions as a subunit of the ORC (Pinto, 1999).
The origin recognition complex (ORC) is an initiator protein for DNA replication, but also effects transcriptional silencing in
Saccharomyces cerevisiae and heterochromatin function in Drosophila. It is not known, however, whether any of these
functions of ORC is conserved in mammals. A novel protein, HBO1 (histone
acetyltransferase binding to ORC), has been identified that interacts with human ORC1 protein, the largest subunit of ORC. The cDNA sequence encodes a 611-amino acid protein with a
predicted molecular mass of 83 kDa. HBO1 exists as part of a multisubunit
complex that possesses histone H3 and H4 acetyltransferase activities. A fraction of the relatively abundant HBO1 protein
associates with ORC1 in human cell extracts. HBO1 is a member of the MYST domain family that includes S. cerevisiae Sas2p, a protein involved in control of
transcriptional silencing that also has been genetically linked to ORC function. Thus the interaction between ORC and a MYST domain acetyltransferase is widely
conserved. Roles for ORC-mediated acetylation of chromatin in control of both DNA replication and gene expression are suggested by these results (Iizuka, 1999).
Two sequence motifs have been noted in HBO1. One is a serine-rich
region located at the amino terminus of the protein. The other feature,
revealed by a BLASTP search of the nonredundant protein data base, is a
270-amino acid carboxyl-terminal region known as a MYST domain. This motif contains a putative acetyl-coenzyme A (acetyl-CoA)
binding domain. Throughout the MYST domain,
HBO1 is approximately 50% identical and 70% similar to several other
MYST domain proteins, many of which have been
implicated in transcriptional regulation. One particular protein of
interest, Sas2p, promotes silencing of the silent mating type gene at
HMLalpha and genes placed near a telomere. In contrast,
Sas2p (and other SAS gene products) antagonizes silencing at
the HMR-a locus when the essential HMR-E silencer element is mutated. Interestingly, the latter
function of Sas2p is mediated through ORC (Iizuka, 1999).
Of the other MYST domain proteins, the Drosophila
'males-absent on the first' (mof) gene has been shown to
be involved in dosage compensation, a process that results in a
two-fold increase in transcription of the single X chromosome in male
flies and is accompanied by histone H4 hyperacetylation at lysine 16 (Hilfiker, 1997). Interestingly, the mof mutant in which both dosage
compensation and H4 acetylation at position 16 are reduced has a single
amino acid substitution from Gly to Glu at position 691. This glycine residue is absolutely conserved among the putative acetyl-CoA-binding motifs of the various MYST domain family members and other histone acetyltransferases. The human MOZ gene is
rearranged and fused to the gene encoding the CREB-binding protein in a
recurrent chromosomal translocation characteristic of acute monocytic
leukemia. Tip60, a human MYST domain protein interacts with the human immunodeficiency virus Tat protein and augments expression from the HIV-1 promoter. Northern blot analysis reveals that the HBO1 mRNA is expressed in
all human tissues tested. HBO1 mRNA abundance is not strictly correlated with cell proliferation and is
particularly high in ovarian tissue (Iizuka, 1999).
Attempts were made to determine whether HBO1 binds to the multisubunit ORC
protein complex rather than to hORC1 alone. Unfortunately, the only other human
ORC subunit against which antibodies were currently available is hORC2. No co-immunoprecipitate hORC2 and HBO1 from
human 293 cell nuclear extracts or from 293 cells overexpressing the
T7-hORC1 polypeptide could be detected. In addition to the other ORC subunits, the two
ORC1-interacting proteins that have been isolated play a role in
heterochromatin function. The budding yeast silencing protein Sir1p has
been shown to bind to the NH2-terminal portion (amino acid
residues 5-228) of S. cerevisiae Orc1p. Similarly,
Drosophila HP1 also binds to the NH2-terminal
domain of dORC1 (amino acid residues 161-319). Thus, the
NH2-terminal domain of ORC1, although it is not
structurally conserved through evolution, may have a generally
conserved function in recruitment of heterochromatin proteins. The region of hORC1 responsible for binding to HBO1, is found between amino acids 210 and 861 of hORC1 (Iizuka, 1999).
Based upon the presence of a putative acetyl-CoA binding motif shared
by many acetyltransferases and the finding that
the Drosophila mof mutant displays reduced acetylation of histone H4 at lysine 16 on the male X chromosome (Hilfiker, 1997), it has been
proposed that MYST domain proteins may generally act as HATs. Both human Tip60 and yeast Esa1p proteins are histone H3
and H4 acetyltransferases. To test whether HBO1 might have HAT
activity, the protein was immunoprecipitated from 293 cell nuclear
extracts and assayed for HAT activity using recombinant core histones
as substrates. Immunoprecipitates from 293 cell nuclear extracts with
polyclonal anti-HBO1 antibodies have an activity capable of acetylating
histones H3 and H4 and, more weakly, H2A,
suggesting that the immune complex containing the HBO1 protein have HAT
activity. Recombinant HBO1 protein has no detectable HAT activity under a
variety of pH (6.5-9.0) and divalent zinc ion concentrations. It is
likely that additional polypeptides associated with HBO1 in human
cells are required for HAT activity. For instance, the HAT activity of
S. cerevisiae and human HAT1 are stimulated by association
with a core histone-binding subunit known as HAT2 and the
transcriptional activator GCN5 only acetylates nucleosomes efficiently
when the GCN5 catalytic subunit is part of a large multisubunit complex (Iizuka, 1999).
Histone acetylation has been generally implicated in transcriptional
activation. Thus, one obvious possibility from these studies is that
HBO1 acetylation of histones or another protein may activate DNA
replication. This could facilitate access to DNA replication initiation
sites for DNA replication proteins or plasticity of nucleosomes as DNA
replication occurs. Alternatively, because MYST domain proteins have
been shown to be involved in transcriptional silencing (Sas2p and
Sas3p), dosage compensation (mof), and
transcriptional activation (Tip60), HBO1 may play a role in the
control of gene expression mediated by the interaction with hORC1. It is
noted that Drosophila ORC
has been implicated in position
effect variegation of gene expression and heterochromatin function. It may be that ORC bound to heterochromatin could facilitate
specific histone acetylation patterns, thereby ensuring the silencing
of gene transcription in these regions of the chromosomes. In this regard, genetic studies with SAS2 in S. cerevisiae suggest that this HBO1-related MYST domain protein
antagonizes the function of ORC in DNA replication and effects
ORC-mediated transcriptional silencing of the mating-type genes.
Disruption of the SAS2 gene partially suppresses the
temperature sensitivity of orc2-1 and orc5-1
mutants. Sas2p also antagonizes ORC mediated silencing at an altered
HMR silencer, but helps silencing at HML. Sas2p may exert such
functions by acetylation of histones, by acetylation of one of the ORC
subunits, or more simply, by binding to ORC. It is not yet clear
whether SAS proteins bind directly to Orc1p in S. cerevisiae. None of the three budding yeast Sas2p-like proteins
(Sas2p, Sas3p, and Esa1p) that were tested bind to S. cerevisiae Orc1p in two-hybrid assays (Iizuka, 1999 and references).
MYST domain proteins have been proposed to regulate transcription through protein acetylation, although this has not been shown directly for any member of this family. Among the various members of the GCN5-related Nepsilon-acetyltransferase superfamily, GCN5-related HATs, P/CAF, Hat1, and GCN5 all contain three amino acid sequence motifs, named A, B, and D. In contrast, MYST domain proteins only have motif A. Because HBO1 protein is a MYST domain protein and protein complexes containing the HBO1 exhibit histone acetyltransferase activity, it is suggested that HBO1 is an acetyltransferase. Unlike Esa1p, HBO1 seems to require other as yet unidentified stimulatory subunits. Sequence comparisons show that of the yeast MYST domain proteins, Esa1p is closest in primary sequence to human HBO1, but interpretation of functional homology based on sequence similarity within a family of proteins is risky. Nevertheless, genetic interactions between the genes encoding Sas2p and the ORC subunits suggest that HBO1 might be functionally related to Sas2p protein (Iizuka, 1999 and references).
To investigate the events leading to initiation of DNA replication in mammalian chromosomes, the time when hamster origin recognition complexes (ORCs) become functional was related to the time when Orc1, Orc2 and Mcm3 proteins become stably bound to hamster chromatin. Functional ORCs, defined as those able to initiate DNA replication, are absent during mitosis and early G1 phase, and reappear as cells progress through G1 phase. Immunoblotting analysis reveals that hamster Orc1 and Orc2 proteins are present in nuclei at equivalent concentrations throughout the cell cycle, but only Orc2 is stably bound to chromatin. Orc1 and Mcm3 are easily eluted from chromatin during mitosis and early G1 phase, but become stably bound during mid-G1 phase, concomitant with the appearance of a functional pre-replication complex at a hamster replication origin. Since hamster Orc proteins are closely related to their human and mouse homologs, the unexpected behavior of hamster Orc1 provides a novel mechanism in mammals for delaying assembly of pre-replication complexes until mitosis is complete and a nuclear structure has formed (Natale, 2000).
One difference that may exist between yeast and metazoan cells is the way in which the ORC interacts with chromatin, a step that is critical to understanding how initiation sites are selected. In yeast, both DNA footprinting and immunoprecipitation analyses reveal that a complete ORC binds to yeast replication origins immediately after initiation of replication occurs and remains there throughout the cell division cycle. In metazoa, however, the situation is less clear. Some evidence suggests that the ORC dissociates from chromatin during mitosis. G1 phase nuclei from mammalian cells can initiate DNA replication when incubated in a Xenopus egg extract that has been depleted of Xenopus laevis Orc (XlOrc) proteins, whereas metaphase chromatin from mammalian cells replicates poorly under these conditions. These data suggest that mammalian Orc proteins are absent from chromatin during metaphase and then rebind at some time before G1 phase begins. Consistent with this conclusion, Orc proteins in activated Xenopus eggs bind to sperm chromatin, whereas Orc proteins in mitotic Xenopus eggs do not, and both Orc1 and Orc2 proteins are present on chromatin in cultured Xenopus cells during interphase but not during metaphase. However, in Drosophila, Orc2 is present in both interphase and metaphase, and human Orc2 is present on cells throughout the cell cycle, although metaphase cells per se were not examined. These data appear to contradict the work in Xenopus, suggesting that the behavior of ORC may vary even among metazoa. Therefore, it is difficult to relate the presence of Orc activity to the presence of Orc proteins in metazoa, because different assays for ORC function or Orc proteins have been carried out in different organisms. Moreover, the hypothesis that functional, chromatin-bound ORCs exist on mammalian chromatin throughout G1 phase is difficult to reconcile with the presence of an 'origin decision point' (ODP) in mammalian cells; initiation of DNA replication can be induced by a Xenopus egg extract in all G1 nuclei, but initiation events in late G1 nuclei occur at the same replication origins used in vivo whereas initiation events in early G1 nuclei are randomly distributed along the genome (Natale, 2000 and references therein).
A solution to this paradox is provided by showing that the mammalian ORC disassembles, at least in part, during mitosis and then reassembles completely in early G1 nuclei. In Chinese hamster ovary (CHO) cells, CgOrc2 (where Cg indicates Cricetulus griseus, the Chinese hamster) remains stably bound to chromatin throughout the cell cycle, consistent with studies of Drosophila and human cells, but CgOrc1 becomes unstably bound to chromatin during mitosis, consistent with studies in Xenopus cells. Moreover, the ability to initiate DNA synthesis in hamster cells and the ability to activate specific mammalian replication origins selectively by incubation in Xenopus egg extract are directly related to the stability of CgOrc1 binding to chromatin. These results support the general concept that metazoan ORCs, in contrast to yeast ORCs, dissociate from chromatin during mitosis. In addition, they reveal that mammals selectively destabilize Orc1 protein during the mitotic to G1 transition, thus providing a novel mechanism in mammalian cells for delaying assembly of pre-replication complexes (pre-RCs), consisting of ORC, Cdc6 and one or more copies of Mcm proteins 2-7, until mitosis is complete and a nuclear membrane has reformed. Finally, they strongly suggest that mammalian ORCs are assembled at specific chromosomal sites (Natale, 2000).
Several observations suggest that pre-RCs are assembled at specific chromosomal loci in differentiated mammalian cells. (1) Orc-depleted Xenopus egg extract can selectively activate ori-ß and ori-ß' in hamster G1 phase nuclei, two origins of bidirectional replication that are activated at the beginning of the hamster S phase). Therefore, pre-RCs must have been assembled at these initiation sites during G1 phase in vivo. (2) The appearance of these pre-RCs coincides with the strong binding of CgOrc1 and CgMcm3 to chromatin. (3) Since CgOrc2, and possibly other Orc proteins, remain bound to the chromatin during mitosis and G1 phase, at least some Orc2 must be bound at specific sites such as ori-ß in order for Orc1 to form a functional ORC. In fact, Drosophila ORC binds specifically to ACE3, an element controlling the origin of DNA replication. Thus, the release and reassociation of Orc1 offer a novel mechanism to select which initiation sites will be used during the subsequent round of DNA replication. While this role for Orc1 may have little significance in cultured cells where the same origins are used each cell cycle, it could provide a mechanism for reprogramming the numbers and locations of pre-RCs during animal development. Because the cellular ratio of Orc proteins to DNA changes during animal development, simply limiting the amount of Orc1 present would reduce the number of ORCs that could form. ORCs will be assembled at those origins that are most accessible and that bind Orc proteins most tightly. Incomplete ORCs that remain may be displaced from chromatin by replication forks or transcription passing through the origin. Such a mechanism may account for the observations that the amount of DmOrc1 per embryo decreases dramatically during Drosophila development, and that expression of ectopic DmOrc1 promotes rather than inhibits DNA synthesis, suggesting that the level of Orc1 protein governs the activity of replication origins (Natale, 2000 and references therein).
In Drosophila melanogaster, Orc1p abundance is developmentally regulated (Asano, 1999). In the embryo, it accumulates mostly in proliferating cells, and it becomes cell cycle regulated in the eye imaginal disc, being abundant from late G1 until the end of S phase. In the ovary, DmOrc1p is directed to subnuclear foci at the time of the switch from endoreplication to amplification of the chorion gene clusters (Asano, 1999). Eukaryotic cells possess overlapping mechanisms to ensure that DNA replication is restricted to the S phase of the cell cycle. The levels of hOrc1p, the largest subunit of the human origin recognition complex, vary during the cell division cycle. In rapidly proliferating cells, hOrc1p is expressed and targeted to chromatin as cells exit mitosis and prereplicative complexes are formed. Later, as cyclin A accumulates and cells enter S phase, hOrc1p is ubiquitinated on chromatin and then degraded. hOrc1p destruction occurs by means of the proteasome and is signaled in part by the SCFSkp2 ubiquitin-ligase complex. Other hORC subunits are stable throughout the cell cycle. The regulation of hOrc1p may be an important mechanism in maintaining the ploidy in human cells (Méndez, 2002).
The c-myc oncogene product (c-Myc) is a transcription factor that forms a complex with Max and recognizes the E-box sequence. c-Myc plays key functions in cell proliferation, differentiation and apoptosis. As for its activity towards cell proliferation, it is generally thought that c-Myc transactivates the E-box-containing genes that encode proteins essential to cell-cycle progression. Despite the characterization of candidate genes regulated by c-Myc in culture cells, these have still not been firmly recognized as real target genes for c-Myc. c-Myc has been found to directly bind to the N-terminal region of origin recognition complex-1 (ORC1), a region that is responsible for gene silencing, in a state of complex containing other ORC subunits and Max in vivo and in vitro. Furthermore, ORC1 inhibits E-box-dependent transcription activity of c-Myc by competitive binding to the C-terminal region of c-Myc with SNF5, a component of chromatin remodelling complex SNF/Swi1. These results suggest that ORC1 suppresses the transcription activity of c-Myc by its recruitment into an inactive form of chromatin during some stage of the cell cycle (Takayama, 2000).
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 (Thome, 2000).
It is proposed that the full ORC complex, including Orc1, is formed in proliferating cells but only during a small window of time in the cell cycle or in a sub-nuclear fraction that is difficult to extract in lysates. The co-precipitation and gel filtration experiments in 0.1% Nonidet P-40 buffer indicate that at least a sub-complex of Orc2, Orc3, Orc4, and Orc5 can be detected in cell extracts. In addition several of the ORC subunits are detected in non-proliferating cells. Upon induction of the cell cycle, Orc1 is expressed from an E2F-regulated promoter. The newly synthesized Orc1 is better extracted than mature Orc1 but is not associated with other ORC subunits. It is speculate that the new Orc1 protein subsequently associates with the other ORC subunits (most likely in a cellular fraction that is tightly associated with the chromatin) and renders ORC competent for initiating DNA replication. This suggestion is consistent with a recent report that in Chinese hamster ovary cells Orc2 remains firmly associated with chromatin in all phases of the cell cycle, whereas Orc1 selectively associates with chromatin in G1, thereby making the origins competent to replicate (Thome, 2000).
The association of human origin recognition complex (ORC) proteins hOrc1p and hOrc2p with chromatin was investigated in HeLa cells. Independent procedures including limited nuclease digestion and differential salt extraction of isolated nuclei show that a complex containing hOrc1p and hOrc2p occurs in a nuclease-resistant compartment of chromatin and can be eluted with moderate high salt concentrations. A second fraction of hOrc2p that dissociates in vitro at low salt conditions was found to occur in a chromatin compartment characterized by its high accessibility to micrococcal nuclease. Functional differences between these two sites become apparent in HeLa cells that synchronously enter the S phase after a release from a double-thymidine block. The hOrc1p/hOrc2p-containing complexes dissociate from their chromatin sites during S phase and reassociate at the end of mitosis. In contrast, the fraction of hOrc2p in nuclease-accessible, more open chromatin remains bound during all phases of the cell cycle. It is proposed that the hOrc1p/hOrc2p-containing complexes are components of the human origin recognition complex. Thus, the observed cell cycle-dependent release of the hOrc1p/hOrc2p-containing complexes is in line with previous studies with Xenopus and Drosophila systems, which indicate that a change in ORC stability occurs after prereplication complex formation. This could be a powerful mechanism that prevents the rereplication of already replicated chromatin in the metazoan cell cycle (Kreitz, 2001).
Origin recognition complex (ORC), CDC6, and MCM proteins assemble sequentially to form prereplication chromatin. However, their organization remains largely unclear in mammalian cells. Using an in vivo chemical cross-linking method, is has been shown that ORC1 proteins are associated with non-chromatin nuclear structures and assemble in nuclear foci in mammalian cells. CDC6 proteins also assemble in nuclear foci on non-chromatin nuclear structures, although their physical association with ORC1 has been undetectable. In contrast to the situation in yeast cells, CDC6 was found to remain associated with non-chromatin nuclear structures even after cells entered into S phase. Instead, ORC1 proteins were found to be degraded by a proteasome-dependent pathway during S phase. Some ORC2 proteins are associated with non-chromatin nuclear structures like ORC1, although the remainder bind to nuclease-sensitive chromatin. Further analyses indicate that ORC2 physically interacts with ORC1 on non-chromatin nuclear structures. However, the results suggest that although a small proportion of MCM complexes are loaded onto chromatin regions near ORC foci, most of them are more widely distributed. Possible relations between such organization of prereplication chromatin and complicated origin specification in higher eukaryotic cells are discussed (Fujita, 2002).
The levels of hemagglutinin tagged ORC1 (HA-ORC1) proteins were examined during the cell cycle. A clear decrease was observed in the S-phase cells. This is strikingly different from the previously reported finding that ORC1 protein levels are relatively constant during the cell cycle. The reason for the difference is not clear at present. Because an HA-tagged system was used, the specificity of results with immunoassays could be higher. When MG132, a peptide aldehyde that binds and inactivates 26 S proteasomes, was added to the culture, the S-phase degradation of ORC1 was decreased, and a ladder of ORC1 bands with much retarded mobility, presumably due to ubiquitinylated intermediates, was detected. No obvious difference in MCM7 or ORC2 protein levels was observed among the samples. Together, the available data suggest that human ORC1 proteins are degraded during S phase via the ubiquitin-proteasome system (Fujita, 2002).
The binding regions were examined of components of the origin recognition complex (ORC) in the human genome. For this purpose, chromatin immunoprecipitation assays were performed with antibodies against human Orc1 and Orc2 proteins. A binding region for human Orc proteins 1 and 2 was identified in a <1-kbp segment between two divergently transcribed human genes. The region is characterized by CpG tracts and a central sequence rich in AT base pairs. Both, Orc1 and Orc2 proteins are found at the intergenic region in the G1 phase, but S-phase chromatin contains only Orc2 protein, supporting the notion that Orc1p dissociates from its binding site in the S phase. Sequences corresponding to the intergenic region are highly abundant in a fraction of nascent DNA strands, strongly suggesting that this region not only harbors the binding sites for Orc1 protein and Orc2 protein but also serves as an origin of bidirectional DNA replication (Ladenberger, 2002).
Changes in the affinity of the Orc1 protein for chromatin in hamsters during the M-to-G(1) transition have been shown to correlate with the activity of hamster origin recognition complexes (ORCs) and the appearance of prereplication complexes at specific sites. Orc1 is shown to be selectively released from chromatin as cells enter S phase, converted into a mono- or diubiquitinated form, and then deubiquitinated and re-bound to chromatin during the M-to-G(1) transition. Orc1 is degraded by the 26S proteasome only when released into the cytosol, and peptide additions to Orc1 make it hypersensitive to polyubiquitination. In contrast, Orc2 remains tightly bound to chromatin throughout the cell cycle and is not a substrate for ubiquitination. Since the concentration of Orc1 remains constant throughout the cell cycle, and its half-life in vivo is the same as that of Orc2, ubiquitination of non-chromatin-bound Orc1 presumably facilitates the inactivation of ORCs by sequestering Orc1 during S phase. Thus, in contrast to yeast (Saccharomyces cerevisiae and Schizosaccharomyces pombe), mammalian ORC activity appears to be regulated during each cell cycle through selective dissociation and reassociation of Orc1 from chromatin-bound ORCs (Li, 2002).
The results support the existence of a novel regulatory pathway in the initiation of mammalian DNA replication. In mammals, Orc1 and Orc2 are both tightly bound to chromatin during late G1 phase, and late G1-phase nuclei contain active ORC-chromatin sites by virtue of the fact that they can initiate DNA replication at specific genomic sites when incubated in an Orc-depleted Xenopus egg extract. In contrast to yeast in which all six ORC subunits are stably bound to chromatin throughout the cell cycle, the affinity of mammalian Orc1 for chromatin is selectively reduced during S phase, such that lysis of cells in 0.1% Triton X-100, 0.15 M NaCl, and 1 mM Mg2+-ATP releases Orc1, but not Orc2, into the non-chromatin-bound fraction. Moreover, the Orc1 that is released during S phase is rapidly ubiquitinated (Li, 2002).
Although Orc2 was generally not detected in the non-chromatin-bound fraction, a small amount was sometimes observed. Whether this resulted from chromatin fragmentation or the release of a special Orc1/Orc2-containing complex is not known. However, the amount of Orc2 tightly bound to chromatin remains essentially constant throughout the cell cycle, and neither endogenous Orc2 nor FLAG-Orc2 was a substrate for ubiquitination, suggesting that only Orc1 is released from chromatin-bound ORCs. Orc4p also remains tightly bound to chromatin throughout the cell cycle (Li, 2002).
The proteins bound in vivo at the human lamin B2 DNA replication origin and their precise sites of binding were investigated along the cell cycle utilizing two novel procedures based on immunoprecipitation following UV irradiation with a pulsed laser light source. In G(1), the pre-replicative complex contains CDC6, MCM3, ORC1 and ORC2 proteins; of these, the post-replicative complex in S phase contains only ORC2; in M phase none of them are bound. The precise nucleotide of binding was identified for the two ORC and the CDC6 proteins near the start sites for leading-strand synthesis; the transition from the pre- to the post-replicative complex is accompanied by a 17 bp displacement of the ORC2 protein towards the start site (Abdurashidova, 2003).
ORC is involved in formation of prereplicative complexes (pre-RCs) on replication origins in the G1 phase. At the G1/S transition, elevated cyclin E-CDK2 activity triggers 1DNA replication to enter S phase. The CDK cycle works as an engine that drives progression of cell cycle events by successive activation of different types of cyclin-CDK. However, how the CDK cycle is coordinated with replication initiation remains elusive. This study report that acute depletion of ORC2 by RNA interference (RNAi) arrests cells with low cyclin E-CDK2 activity. This result suggests that loss of a replication initiation protein prevents progression of the CDK cycle in G1. p27 and p21 proteins accumulate following ORC2 RNAi and are required for the CDK2 inhibition. Restoration of CDK activity by co-depletion of p27 and p21 allows many ORC2-depleted cells to enter S phase and go on to mitosis. However, in some cells the release of the CDK2 block causes catastrophic events like apoptosis. Therefore, the CDK2 inhibition observed following ORC2 RNAi seems to protect cells from premature S phase entry and crisis in DNA replication. These results demonstrate an unexpected role of ORC2 in CDK2 activation, a linkage that could be important for maintaining genomic stability (Machida, 2005).
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. It was asked whether ORC is required for M phase chromosome assembly independently of its function in DNA replication. Depletion assays and reconstitution experiments were performed in Xenopus egg extracts, in conditions of M phase chromosome assembly coupled or uncoupled from DNA replication. 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, mutations of RPA at the cdc2 kinase site prevents RPA dissociation from chromatin and impairs mitotic chromosome assembly without affecting DNA replication. These 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 (Cuvier, 2006).
Eukaryotic DNA replication begins with the binding of a six subunit origin recognition complex (ORC) to DNA. To study the assembly and function of mammalian ORC proteins in their native environment, HeLa cells were constructed that constitutively expressed an epitope-tagged, recombinant human Orc2 subunit that had been genetically altered. Analysis of these cell lines revealed that Orc2 contains a single ORC assembly domain that is required in vivo for interaction with all other ORC subunits, as well as two nuclear localization signals (NLSs) that are required for ORC accumulation in the nucleus. The recombinant Orc2 existed in the nucleus either as an ORC-(2-5) or ORC-(1-5) complex; no other combinations of ORC subunits were detected. Moreover, only ORC-(1-5) was bound to the chromatin fraction, suggesting that Orc1 is required in vivo to load ORC-(2-5) onto chromatin. Surprisingly, recombinant Orc2 suppresses expression of endogenous Orc2, revealing that mammalian cells limit the intracellular level of Orc2, and thereby limit the amount of ORC-(2-5) in the nucleus. Because this suppression requires only the ORC assembly and NLS domains, these domains appear to constitute the functional domain of Orc2 (Radichev, 2006).
Previous studies have suggested that the activity of the mammalian origin recognition complex (ORC) is regulated by cell-cycle-dependent changes in its Orc1 subunit. This study shows that Orc1 modifications such as mono-ubiquitylation and hyperphosphorylation that occur normally during S and G2-M phases, respectively, can cause Orc1 to accumulate in the cytoplasm. This would suppress reassembly of pre-replication complexes until mitosis is complete. In the absence of these modifications, transient expression of Orc1 rapidly induces p53-independent apoptosis, and Orc1 accumulates perinuclearly rather than uniformly throughout the nucleus. This behavior mimics the increased concentration and perinuclear accumulation of endogenous Orc1 in apoptotic cells that arise spontaneously in proliferating cell cultures. Remarkably, expression of Orc1 in the presence of an equivalent amount of Orc2, the only ORC subunit that does not induce apoptosis, prevents induction of apoptosis and restores uniform nuclear localization of Orc1. This would promote assembly of ORC-chromatin sites, such as occurs during the transition from M to G1 phase. These results provide direct evidence in support of the regulatory role proposed for Orc1, and suggest that aberrant DNA replication during mammalian development could result in apoptosis through the appearance of 'unmodified' Orc1 (Saha, 2006).
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 does, however, facilitate reassociation of Orc1 with chromosomes during the M to G1-phase transition, and it is required for binding Orc1 to the Epstein-Barr virus oriP and stimulating oriP-dependent plasmid DNA replication. Moreover, the BAH domain affects 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 (Noguchi, 2006).
Eukaryotic DNA replication initiates at a large number of chromosomal origins, controlled by the ordered assembly of multiprotein replication complexes and the cell cycle-dependent activity of kinases that phosphorylate them. In cases where origins have been transposed to other chromosomal locations, they have been found to colocalize with genetically defined replicators, i.e., sequences capable of promoting DNA replication at ectopic genomic sites. In metazoan systems, replication origins or replicators are bound by homologues of proteins first characterized for the yeast Saccharomyces cerevisiae, suggesting that the basic mechanisms controlling replication initiation are conserved among eukaryotes. In S. cerevisiae, replicators typically comprise a binding site for the hexameric origin recognition complex ORC and a DNA unwinding element (DUE). ORC enables the Cdc6-, Cdt1-dependent recruitment of the MCM helicase complex to replication origins, forming a prereplication complex (pre-RC) early during the G1 phase of the cell cycle. Cyclin-dependent kinase and DDK activities promote the binding of Mcm10, Cdc45, and RPA to form preinitiation complexes and unwind the DNA template in advance of replication. The effect of kinase activity on the pre-RC is partially to disassemble ORC and release MCMs and Cdc6 from chromatin (Noguchi, 2006 and references therein).
The 2.4-kb 5' region of the human c-myc gene contains multiple transcription factor binding sites and a DUE that is unwound in vivo. The DUE is situated in a 100-bp zone containing three 10/11 matches to the S. cerevisiae ARS consensus sequence. It was initially reported that replication initiates in this region, and quantitative PCR (qPCR) has been used to define the replication initiation zone. Subsequent work has confirmed that replication initiates in the 5' flanking DNA of the c-myc gene in multiple species. The 2.4-kb c-myc core origin endows plasmids with ARS activity in vitro and shows replicator activity when moved to an ectopic chromosomal location. This region displays an ordered chromatin structure stable to chromosomal translocation, and mutational analyses have identified regions of the replicator essential for replication initiation, including the DUE (Noguchi, 2006 and references therein).
Chromatin immunoprecipitation (ChIP) was used in this work to show that the human analogs of the yeast ORC, MCM, and Cdc6 proteins bind preferentially and selectively to the c-myc replicator. The distributions of Mcm3 and Mcm7 are similar in asynchronous cells, with the greatest ChIP signal at, and upstream of, the DNA unwinding element. These distributions change in parallel in cells synchronized in G1 or M phases. By contrast, Orc1, Orc2, and Cdc6 appear to be least abundant at the DUE and each displays a different temporal pattern of replicator binding. The DNA unwinding element binding protein DUE-B, identified using the c-myc DUE as bait in a yeast one-hybrid assay, preferentially binds near the c-myc DUE in a pattern comparable to that of the MCMs in asynchronous and G1-phase cells. Furthermore, at an ectopic locus, c-myc replicator deletions that removed the DUE or altered chromatin structure suppressed DUE-B or Mcm3 binding, respectively, and eliminated origin activity. The relationship between chromatin structure, MCM binding, and origin activity is supported by the demonstration that inhibition of histone deacetylase activity by trichostatin A (TSA) causes a redistribution of Mcm3 binding similar to the broadening of the c-myc replication initiation zone. These results suggest that pre-RC proteins bind nonrandomly to the c-myc replicator and that c-myc origin activity is a function of ORC, MCM, Cdc6, and DUE-B binding to c-myc chromatin (Noguchi, 2006).
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