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J. Biol. Chem., Vol. 276, Issue 28, 26666-26673, July 13, 2001
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§,,**
From the Department of Molecular Biology and
Genetics, Johns Hopkins University School of Medicine, Baltimore,
Maryland 21205 and ¶ Laboratory of Viral Diseases, NIAID, National
Institutes of Health, Bethesda, Maryland 20892
Received for publication, March 20, 2001
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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The six-subunit origin recognition
complex (ORC) was originally identified in the yeast
Saccharomyces cerevisiae. Yeast ORC binds specifically to
origins of replication and serves as a platform for the assembly of
additional initiation factors, such as Cdc6 and the Mcm proteins. Human
homologues of all six ORC subunits have been identified by sequence
similarity to their yeast counterparts, but little is known about the
biochemical characteristics of human ORC (HsORC). We have extracted
HsORC from HeLa cell chromatin and probed its subunit composition using
specific antibodies. The endogenous HsORC, identified in these
experiments, contained homologues of Orc1-Orc5 but lacked a putative
homologue of Orc6. By expressing HsORC subunits in insect cells using
the baculovirus system, we were able to identify a complex containing
all six subunits. To explore the subunit-subunit interactions that are required for the assembly of HsORC, we carried out extensive
co-immunoprecipitation experiments with recombinant ORC subunits
expressed in different combinations. These studies revealed the
following binary interactions: HsOrc2-HsOrc3, HsOrc2-HsOrc4,
HsOrc3-HsOrc4, HsOrc2-HsOrc6, and HsOrc3-HsOrc6. HsOrc5 did not form
stable binary complexes with any other HsORC subunit but interacted
with sub-complexes containing any two of subunits HsOrc2, HsOrc3, or
HsOrc4. Complex formation by HsOrc1 required the presence of HsOrc2,
HsOrc3, HsOrc4, and HsOrc5 subunits. These results suggest that the
subunits HsOrc2, HsOrc3, and HsOrc4 form a core upon which the ordered
assembly of HsOrc5 and HsOrc1 takes place. The characterization of
HsORC should facilitate the identification of human origins of DNA replication.
The initiation of DNA replication in lower eukaryotes is similar
to that observed in bacteria in that it occurs at well defined origins
of DNA replication that are recognized by specific initiator proteins
(1-5). Saccharomyces cerevisiae origins of DNA replication are short, ~100-base pair
(bp)1 segments that consist
of two essential regions: the A domain, which contains a conserved
11-bp consensus sequence (ACS) that is required for origin function,
and the B domain, which contains several stimulatory elements (6, 7).
The ACS is recognized specifically by the S. cerevisiae
initiator protein, the origin recognition complex (ORC) (8, 9).
S. cerevisiae ORC (ScORC) is comprised of six proteins
(Orc1-6), each of which is essential for viability in S. cerevisiae (10-13). A number of lines of evidence indicate that
ScORC is required for initiation of DNA replication in vivo
and functions in part to recruit other initiation factors, such as Cdc6
and the minichromosome maintenance proteins, to replication origins
(13-15).
Homologues of ORC subunits have been identified in a variety of other
eukaryotic species (2, 16), and ORC has been purified from the fission
yeast Schizosaccharomyces pombe and the metazoans Drosophila melanogaster and Xenopus laevis
(17-19). Genetic and biochemical data indicate that specific ORC
subunits from the latter organisms, like those of S. cerevisiae, are required for the initiation of chromosomal DNA
replication (20-22). The conservation of ORC as well as other
initiation factors strongly suggests that there are common mechanisms
for initiating DNA replication shared by all eukaryotes. However,
despite this high degree of evolutionary conservation, the
identification in other eukaryotes of ORC binding sequences comparable
with the S. cerevisiae ACS elements has so far proved
elusive. Origins of DNA replication in S. pombe are large,
AT-rich elements that lack a common consensus sequence (23-25). The
binding of S. pombe ORC to origins of DNA replication appears to be mediated in large part by a unique N-terminal domain of
SpOrc4 that contains multiple copies of a motif that binds to AT tracts
(26). Purified D. melanogaster ORC has been shown to
interact with multiple sequences in ACE3, a cis-acting
element that is required for the specialized DNA replication process
that occurs during chorion gene amplification, but it is not yet clear that this interaction is relevant to initiation of DNA replication at
origins of DNA replication in somatic cells (27, 28). These observations raise the possibility that there are functionally important differences in the way ORC mediates initiation of DNA replication in different eukaryotes.
Human homologues of all six ORC subunits have been identified
by sequence similarity searches (29-34). HsOrc1, HsOrc2, HsOrc4, and
HsOrc5 exhibit considerable homology with their yeast and metazoan
counterparts (28, 29, 31-33). Orc3 and Orc6 appear to have evolved
faster than the other ORC subunits. HsOrc3 has significant similarity
to metazoan and S. pombe Orc3 but barely detectable
similarity to S. cerevisiae Orc3 (17, 30). HsOrc6 has
significant similarity to its D. melanogaster counterpart but no detectable similarity to S. cerevisiae Orc6 (34).
Indeed, Orc6 is the least conserved of all of the ORC subunits.
S. cerevisiae Orc6 is very weakly related to S. pombe Orc6 but not to any known metazoan Orc6 (17). It has been
demonstrated that S. cerevisiae Orc6 is not required either
for specific binding of ScORC to the ACS or for formation of
multi-subunit complexes containing the ScOrc1-ScOrc5 (35). It has also
been reported that HsOrc6 cannot be co-immunoprecipitated with HsOrc2,
suggesting that HsOrc6 may be only weakly associated with other human
ORC subunits (34).
Although immunoprecipitation experiments from several laboratories have
demonstrated physical interactions between several subunits of HsORC
(29, 36, 37), a holo-complex of all six proteins has not been
identified and characterized. Given the possibility of differences in
ORC function among eukaryotes it will be important to identify and
characterize HsORC. We report here that endogenous HsORC consisting of
a complex of five subunits (HsOrc1-HsOrc5) can be extracted from HeLa
cell chromatin. We have also expressed the HsORC subunits in insect
cells using the baculovirus system and have identified a complex
containing all six subunits. Using this system we have probed the
protein-protein interactions between the subunits that are necessary
for assembly of the complex. Our results suggest that the HsOrc2,
HsOr3, and HsOrc4 proteins form the core complex to which HsOrc5 and
HsOrc1 bind in an ordered manner. HsOrc6 appears to be the most weakly bound subunit in the complex. The identification of human ORC should
facilitate the identification of replication origins in human cells.
Antibodies--
To generate antibodies, the six HsORC
subunits were expressed individually in bacteria as recombinant fusion
proteins tagged with either the His6 epitope or the
glutathione S-transferase (GST) moiety. For HsORC1, the
1050-bp sequence that encodes the C- terminal 350 amino acids was
amplified using Pfu polymerase (Stratagene),
oligonucleotides 5'-CGCGGATCCCAAGACATCTACAATTTTGTGGAA-3' and
5'-CCGGAATTCCTCGTCTTTCAGCGCATACAGCAC-3' as primers, and the plasmid
pGEX-HsORC12 as template. The
PCR product was digested with BamHI/EcoRI and cloned into the corresponding sites in the plasmid pET28a (Novagen). For HsORC2, the 810-bp sequence that encodes the first 270 amino acids
was amplified using oligonucleotides
5'-GAAGATCTATGAGTAAACCAGAATTAAAGGAAGACAAG-3' and
5'-TCCCCCCGGGTTGCTGATCCAGTTTAGCTCTCTTTAGCTTC-3' as primers and the
plasmid pMHC1H2 (kindly provided by Dr. B. Stillman, Cold Spring Harbor
Laboratory) as template. The PCR product was digested with
BglII/XbaI and cloned into the
BamHI/XbaI sites of the plasmid pGEX-4T-1
(Amersham Pharmacia Biotech). Oligonucleotides
5'-CGCGGATCCATGGACTGCTGTGTAGATATAAAATCC-3' and
5'-ATAAGAATGCGGCCGGAAAAATGAGTATTAGTGGAAATTC-3' were used as primers,
and a HeLa MATCHMAKER cDNA library (CLONTECH)
was used as template to amplify a 284-bp fragment that encodes the
first 96 amino acids of HsORC3. Oligonucleotides
5'-CGCGGATCCTGCCGGCGCCATGGGGTCGGAGCTGATCGGG-3' and
5'-ATAAGAATGCGGCCGCTCACTCTGCTGTAGCCTTTTGAGC-3' were used as primers,
and a HeLa MATCHMAKER cDNA library was used as the template to
amplify a 799-bp fragment that encodes the full-length HsOrc6p. The
HsORC3 and HsORC6 PCR products were digested with
BamHI/NotI and individually cloned into the
corresponding sites in the plasmid pET28a. For HsORC4, a 1290-bp
fragment that encodes the full-length protein was amplified using
oligonucleotides 5'-CGGGATCCATGAGCAGTCGTAAATCAAAGAGTAAC-3' and
5'-TCCCCCCGGGTCATAACCAGCTTAGTGAGGATG-3' as primers and the plasmid
pBluescript II SK+-HsORC4 (a kind gift of Dr. A. Dutta, Harvard Medical
School) as template. The PCR product was digested with
BamHI/SmaI and cloned into the
BglII/PvuII sites of the plasmid pRSETb
(Invitrogen). An 810-bp fragment that encodes the first 270 amino acids
of HsORC5 was amplified using oligonucleotides 5'-CGGGATCCATGCCCCACTTGGAAAACG-3' and
5'-TCCCCCCGGGTCAGAGATAAACAGTCTGCATAG-3' as primers and the plasmid
pBluescript-HsORC5 (kindly provided by Dr. J. Hurwitz, Memorial
Sloan-Kettering Cancer Center) as template. The PCR product was
processed and cloned into the plasmid pRSETb in a similar manner as
that used for HsORC4. All HsORC constructs were confirmed by DNA sequencing.
Recombinant proteins that contained the His6 epitope tag
were expressed in bacteria and purified on a nickel resin column according to instructions provided by the manufacturer (Qiagen, Inc.).
Recombinant fusion proteins that were fused with the GST moiety were
expressed in bacteria and purified on a glutathione column according to
the manufacturer's instructions (Amersham Pharmacia Biotech). The
purified proteins were used as antigens to inject rabbits and obtain
antigen-specific polyclonal antisera (Covance Research Products,
Denver, PA).
Cloning of HsORC Genes into Baculovirus Vectors--
For
expression of HsORC1 in insect cells, the full-length gene was first
amplified by PCR using oligonucleotides
5'-CGGGATCCGCCGGAAGCCATGGCACACTACCC-3' and
5'-GCTCTAGATTACTCGTCTTTCAGCGCATACAG-3' as primers and the plasmid
pGEX-HsORC1 as template. The PCR product was then digested with
BamHI/XbaI and cloned into the corresponding
sites in the plasmid pFastBac1 (Life Technologies, Inc.). The
baculovirus vector containing HsORC2 was constructed by amplifying the
full-length gene by PCR using oligonucleotides
5'-GAAGGCCTGTTGGCAACAATGAGTAAACCA-3' and
5'-GGGGTACCTCAAGCCTCCTCTTCTTCCTTTTC-3' as primers and a HeLa MATCHMAKER
cDNA library as template. The PCR product was digested with
StuI/KpnI and cloned into the corresponding sites
in the plasmid pFastBac1. PCR products containing the HsORC3 and -6 were made by using oligonucleotides
5'-CGGGATCCCAGTAAGACCATGGCTACGTCCTC-3' and
5'-GGGGTACCCTAGCAGCCTCCCCATGTTAGTCT-3' and oligonucleotides 5'-CGGGATCCTGCCGGCGCCATGGGGTCGGAGCT-3' and
5'-GGGGTACCTCACTCTGCTGTAGCCTTTTGAGC-3' as primers, respectively, and a
HeLa MATCHMAKER cDNA library as template. The construct containing
HsORC4 was made by amplifying the full-length gene by PCR using
oligonucleotides 5'-CGGGATCCATTTGTTGAAATGAGCAGTCGTAA-3' and
5'-GGGGTACCTCATAACCAGCTTAGTGAGGATGT-3' as primers and the plasmid
pBluescript II SK+-HsORC4 as template. The vector encoding the HsORC5
gene was made by amplification of the full-length gene by PCR using
oligonucleotides 5'-CGGGATCCGCCTGCCAGAATGCCCCACTTGGA-3' and
5'-GGGGTACCTCACAAGAAATCATACAAGTATTT-3' as primers and the plasmid
pBluescript-HsORC5 as template. After digestion with
BamHI/KpnI, the PCR products containing the
HsORC3, HsORC4, HsORC5, and HsORC6 genes were individually cloned into
the corresponding sites in the plasmid pFastBac1. The constructs were
confirmed by DNA sequencing. All recombinant viruses were
produced and amplified according to the manufacturer's instructions
(Bac-to-Bac TMBaculovirus Expression Systems, Life
Technologies, Inc.).
Preparation of Nuclear Extracts--
Spinner cultures of HeLa
cells were grown at 37 °C in Spinner minimum Eagle's medium
with glutamine (Quality Biological, Inc.) and supplemented with 5%
fetal bovine serum and penicillin (100 units/ml), streptomycin (100 µg/ml). Nuclei were prepared exactly as described by Challberg and
Kelly (38). Nuclear extracts were prepared by first extracting the
nuclei with OPB2 buffer (50 mM Hepes, pH 7.5, 5 mM MgCl2, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM dithiothreitol, 10% glycerol,
0.01% Tween 20) plus 100 mM NaCl on ice for 1 h. After centrifugation at 16,000 × g for 20 min at
4 °C, the resulting pellet was then extracted with OPB2 buffer plus
500 mM NaCl on ice for 1 h. The suspension was
centrifuged at 140,000 × g for 1 h at 4 °C.
The resulting supernatant was used in immunoprecipitation experiments.
Sf9 insect cells were cultured at 27 °C in Grace's medium
(Life Technologies, Inc.) supplemented with 10% fetal bovine serum and
penicillin (50 units/ml), streptomycin (50 µg/ml). For expression of
various HsORC complexes, Sf9 insect cells were grown to
~7 × 106 cells in 10-cm plastic dishes, co-infected
with various combinations of recombinant baculoviruses expressing HsORC
subunits, each at a multiplicity of infection of 5, and incubated at
27 °C for 48 h. Cells were harvested by centrifugation at
2000 × g for 5 min and then washed with cold
phosphate-buffered saline (10 mM
Na2HPO4, 1.8 mM
KH2PO4, pH 7.3, 140 mM NaCl, 2.7 mM KCl). Nuclei were prepared as described by Challberg and
Kelly (38) and lysed by incubation with ISLB (20 mM
Tris-HCl, pH 6.8, 0.4 M sorbitol, 150 mM
potassium acetate, 5 mM MgCl2, 5 mM
MgSO4, 1% Triton X-100) on ice for 5 min. The suspension
was centrifuged at 21,000 × g for 5 min at 4 °C,
and the resulting pellet was extracted with OPB2 plus 500 mM NaCl on ice for 1 h. The supernatant collected
after centrifugation at 21,000 × g at 4 °C for 15 min was used in immunoprecipitation experiments.
Immunoprecipitation and Immunoblotting Procedures--
For
immunoprecipitation experiments, anti-HsORC2 and the anti-HsORC3
antibodies were separately cross-linked to rProtein A-Sepharose (Amersham Pharmacia Biotech) as described in Harlow and Lane (39) with
minor modifications. The solution 0.1 M
Na3BO3, pH 9.0, was used in all steps instead
of 0.2 M Na3BO3, pH 9.0. Cross-linking was achieved with dimethyl suberimidate 2HCl (Pierce) at
a final concentration of 20 mM.
Immunoprecipitation experiments were performed by incubating either
HeLa cell nuclear extract or baculovirus-infected Sf9 insect
cell nuclear extract with cross-linked antibody beads for 2-3 h at
4 °C. The suspensions were then washed 3 times with 10× bead volume
of OPB2 buffer plus 500 mM NaCl. The immunoprecipitated proteins were released from the beads with 1% SDS and analyzed by 10%
SDS/PAGE followed either by transferring separated proteins to
nitrocellulose membranes and Western blotting or staining by silver.
For Western blotting, the following concentrations of antibodies were
used; HsORC1 (1:1000), HsORC2 (1:6000), HsORC3 (1:3000), HsORC4
(1:1000), HsORC5 (1:3000), and HsORC6 (1:3000). Goat anti-rabbit IgG
conjugated with horseradish peroxidase (Pierce) was used as the
secondary antibody at a concentration of 1:6500.
Gel Filtration Analysis--
A fraction of the
baculovirus-infected Sf9 insect cell nuclear extract containing
all six of the HsORC components was applied to a SuperdexTM-200 HR
10/30 gel filtration column (Amersham Pharmacia Biotech) equilibrated
with OPB2 buffer plus 500 mM NaCl. Fractions (500 µl)
were collected and analyzed for HsORC subunits by 10% SDS/PAGE
followed by transferring to nitrocellulose membranes and Western blotting.
Identification of an Endogenous HsORC Composed of Five Subunits in
HeLa Cells--
To begin biochemical characterization of HsORC, we
first raised polyclonal antibodies against recombinant forms of the six subunits that had been individually expressed in bacteria and purified
by affinity chromatography. The specificity of these antibody
preparations was assessed in several ways. Western blotting experiments
demonstrated that each antibody recognized an endogenous HeLa protein
with the molecular weight expected for the corresponding HsORC subunit.
In addition, antigen competition experiments were performed for five of
the six HsORC subunits (HsOrc5 could not be tested in this manner
because of insolubility of the antigen). In every case tested, the
signal detected by Western blotting could be eliminated by
preincubation of the antibodies with an excess of the appropriate
antigen but was not affected by preincubation with an excess of control
antigen (data not shown). Finally, each antibody specifically
recognized the corresponding HsORC subunit when individually
expressed in insect cells using the baculovirus system (see below).
To examine the localization of HsORC subunits in human cells we carried
out subcellular fractionation experiments. HeLa cells were disrupted by
Dounce homogenization at low ionic strength, and the cytoplasmic and
nuclear fractions were separated by centrifugation. The nuclear
fraction was then extracted sequentially with 100 and 500 mM NaCl to elute proteins bound to chromatin with different affinities. Western blot analysis revealed that the HsOrc2-HsOrc6 subunits were found almost exclusively in the 500 mM NaCl
chromatin extract. A significant fraction of HsOrc1 was present in the
same fraction, but HsOrc1 was also detected in the cytoplasmic and 100 mM NaCl chromatin extract (data not shown). This result
suggests that a portion of cellular HsOrc1 is either not associated
with the other HsORC subunits or is more weakly bound.
Preliminary experiments established that our polyclonal HsOrc2
antibody could be used to specifically and quantitatively
immunoprecipitate the HsOrc2 present in the 500 mM
chromatin extract. Fig. 1 shows an
experiment in which we probed such immunoprecipitates for the other
HsORC subunits. Western blot analysis revealed that HsOrc1-HsOrc5 were
co-immunoprecipitated with similar efficiencies by the HsOrc2 antibody
(Fig. 1A). None of the subunits was detected in control experiments with a preimmune antibody. HsOrc6p was not found in the
immunoprecipitated fraction, suggesting that it is not part of the
endogenous complex or is dissociated by washing the immunoprecipitates in 500 mM NaCl (Fig. 1A).
We also examined the complexity of the immunoprecipitated fraction by
SDS/PAGE followed by silver staining. Fig. 1B shows that the
immunoprecipitates obtained with the antibody against HsOrc2 contained
five major proteins not present in control immunoprecipitates. The
mobilities of these proteins were consistent with the calculated molecular weights of HsOrc1-HsOrc5 proteins and were the same as the
corresponding bands observed by Western blotting. As previously observed, HsOrc2 protein appeared as a closely spaced doublet at ~72
kDa. To a first approximation, the bands corresponding to HsOrc2-HsOrc5
stained with similar intensities, suggesting that they were present in
roughly equal amounts. On the other hand, HsOrc1 consistently stained
with lower intensity, suggesting that it may not be as abundant as the
other HsORC subunits in the 500 mM chromatin extract. In
fact, the amount of HsOrc1 protein in the immunoprecipitates varied
somewhat from experiment to experiment, suggesting that the reduced
level of HsOrc1 may be due in part to uncontrolled losses during
fractionation. These conclusions are subject to the caveat that the
relative intensity of silver staining is not always a reliable
indicator of abundance, so further work will have to be done to
establish the stoichiometry of the various subunits. The pattern of
bands in the HsORC immunoprecipitate is strikingly similar to that
observed for X. laevis ORC (xlORC) immunoprecipitated with
antibody against xlORC2 (40). Indeed, the similarity even extends to
the fact that xlOrc3p, like HsOrc3, consistently stains more
intensely than the other subunits. In the case of xlORC it was
demonstrated that xlOrc3 stains aberrantly with silver and is actually
equimolar with the other subunits. Thus, our data indicate that HeLa
cell chromatin contains tightly bound complexes comprised of
HsOrc1-HsOrc5. Our data also suggest that some of these complexes may
lack HsOrc1. Consistent with this possibility, we show below that
complexes containing either HsOrc1-HsOrc5 or HsOrc2-HsOrc5 are stable.
Reconstitution of HsORC Containing Six Subunits in
Baculovirus-infected Insect Cells--
To facilitate the biochemical
characterization of HsORC, we cloned cDNAs encoding each of the six
HsORC subunits into baculovirus expression vectors and isolated the
individual recombinant viruses. Sf9 insect cells were
co-infected with all six recombinant baculoviruses, and after 48 h, 500 mM nuclear extracts were prepared by a method similar to that employed to isolate HsORC from HeLa cells (see "Experimental Procedures"). Recombinant HsORC was
immunoprecipitated with the antibody against HsOrc2 and analyzed by
SDS/PAGE. Examination of the silver-stained gel revealed that all six
HsORC proteins were co-immunoprecipitated with HsOrc2 (Fig.
2A). The bands corresponding to HsOrc2, HsOrc3, HsOrc4, and HsOrc5 proteins stained with similar intensity, indicating that they were present in roughly equivalent amounts. The bands corresponding to HsOrc1 and HsOrc6 proteins were
less well stained, suggesting the possibility that they might be
present in sub-stoichiometric amounts. An unidentified protein with an
apparent molecular mass of 35 kDa was also immunoprecipitated (Fig.
2A). The presence of all of the HsORC subunits was confirmed by Western blot analysis (Fig. 2B). Except for the presence
of HsOrc6, the composition of the recombinant complex was markedly similar to that of the endogenous complex.
We carried out two control experiments to verify the specificity of the
immunoprecipitation reaction. In the first experiment the antibody
against HsOrc2 was incubated with an excess of a specific competitor,
recombinant GST-HsOrc2 (residues 1-270), before immunoprecipitation.
This preincubation completely blocked the co-immunoprecipitation of the
six HsORC subunits. Instead, only the competitor (marked by an
asterisk in Fig. 2C) was recovered in the
immunoprecipitates. In a second control experiment we co-infected Sf9 insect cells with viruses encoding all of the HsORC subunits except HsOrc2. Extracts from the infected cells were incubated with
antibody to HsOrc2, and immunoprecipitates were analyzed by SDS-PAGE.
Silver staining of the resulting gel demonstrated the complete absence
of HsORC subunits in the immunoprecipitates (Fig. 2D). These
results indicate that the observed co-immunoprecipitation of
recombinant HsORC subunits, including HsOrc6, is a result of the
formation of specific multi-subunit complexes and is not due to
nonspecific precipitation or cross-reacting antibodies.
To estimate the size of the recombinant HsORC we carried out gel
filtration chromatography on chromatin extracts of Sf9 cells co-infected with the six subunits (Fig.
3). Western blot analysis revealed that
all six subunits co-eluted from the column in fractions corresponding
to a molecular mass of ~400 kDa (fractions 19-21). This result is
consistent with a holo-complex containing one molecule of each subunit.
The distribution of HsOrc1 was slightly broader than that of the other
subunits and was skewed toward lower molecular mass fractions. We
suggest that this may reflect some dissociation of HsOrc1 from the
complex during chromatography. Although HsOrc6 was present in the same
fractions as HsOrc1-HsOrc5, the peak of HsOrc6 eluted slightly before
the peak of the other subunits. One reasonable explanation for this
behavior is that some of the complexes lack HsOrc6 and that these elute
slightly later than complexes containing the subunit. HsOrc4, HsOrc5,
and HsOrc6 were present in a second peak that eluted from the column in
fractions corresponding to molecular mass of ~50 kDa. This peak
presumably contains free, uncomplexed subunits. The presence of excess
HsOrc4, HsOrc5, and HsOrc6 subunits in the extracts probably reflects the fact that smaller proteins are generally expressed from recombinant baculoviruses with greater efficiencies than larger proteins. Thus, our
gel filtration data together with the co-immunoprecipitation data
described above indicate that a holo-complex containing HsOrc1-HsOrc6 can be efficiently reconstituted from recombinant subunits.
Binary Interactions of Recombinant HsORC Subunits--
We
exploited the baculovirus system to explore in detail the
subunit-subunit interactions that are required for the assembly of
HsORC. For this purpose, HsORC subunits were expressed in various combinations, and chromatin extracts were prepared in the same manner
as that used to isolate the HsORC holo-complex. Interactions among the
subunits were detected by co-immunoprecipitation experiments carried
out with antibodies against HsOrc2 or HsOrc3. In the initial experiments we sought to identify stable binary interactions (Fig. 4). For example, the first panel of Fig.
4A shows the results obtained when Sf9 cells were
co-infected with HsOrc2 and HsOrc3. Western blotting revealed that both
subunits were immunoprecipitated from extracts by antibody against
HsOrc2 but not by control preimmune antibody. Moreover, the two
subunits stained equally with silver, suggesting that they were present
in the immunoprecipitates in similar amounts. We conclude that
HsOrc2 and HsOrc3 form a binary complex that is stable enough to
survive washing with buffers containing 500 mM NaCl. The
data in Fig. 4 demonstrate that similar binary complexes can be formed
between HsOrc2 and HsOrc4 and between HsOrc3 and HsOrc4.
We also detected interactions between recombinant HsOrc6 and either
HsOrc2 or HsOrc3. In the case of the HsOrc2-HsOrc6 combination, only a
small amount of HsOrc6 was recovered in the immunoprecipitates, as
demonstrated by both Western blotting and silver staining. However, the
co-precipitation of HsOrc2 and HsOrc6 was reproducible and was clearly
dependent upon specific antibody. Significantly greater quantities of
HsOrc6 co-immunoprecipitated with HsOrc3 (Fig. 4B),
suggesting that the HsOrc3-HsOrc6 interaction may be stronger than the
HsOrc2-HsOrc6 interaction. The HsOrc6 subunit stained weakly with
silver even in the case of the HsOrc3-HsOrc6 immunoprecipitates,
raising the possibility that not all HsOrc3 subunits were complexed
with HsOrc6 subunits. However, estimates of the relative amounts of the
two subunits from the intensity of staining are unlikely to be accurate
given the large difference in size and composition between HsOrc6 and HsOrc3.
Formation of Complexes Containing Recombinant HsOrc5--
Using
the same general approach we next explored the requirements for
assembly of HsOrc5 into the recombinant HsORC complex. As shown in Fig.
5, A and B, HsOrc5
did not efficiently form binary complexes with either HsOrc2 or HsOrc3.
Thus, we tested various combinations of three subunits for complex
formation. We observed three stable trimeric complexes:
HsOrc2-HsOrc3-HsOrc5, HsOrc2-HsOrc4-HsOrc5, and
HsOrc3- HsOrc4-HsOrc5. In all three instances, considerably more
HsOrc5p was co-immunoprecipitated than when HsOrc5 was expressed with
HsOrc2 or HsOrc3 alone. Complex formation by the combination of
HsOrc2-HsOrc3-HsOrc5 was especially efficient, as the three subunits
appeared to be present in roughly equal amounts in the immunoprecipitates (Fig. 5A). These data indicate that
complex formation by HsOrc5 requires any two of subunits HsOrc2,
HsOrc3, and HsOrc4. There are two possible explanations for these
observations. HsOrc5 may interact weakly with several subunits, and the
formation of stable complexes is a result of the additive effects of
such interactions. Alternatively, binding of HsOrc5 may require
conformational changes brought about by interactions of the other
subunits. It should be noted that we were unable to directly test for a
binary interaction between HsOrc5 and HsOrc4 because the corresponding antibodies are not suitable for immunoprecipitation studies. However, it is clear from the above data that HsOrc4 is not essential for assembly of HsOrc5 into a multi-subunit complex.
Assembly of Recombinant HsOrc1 into a Multi-Subunit Complex
Requires HsOrc2, HsOrc3, HsOrc4, and HsOrc5--
We extended our
studies of HsORC complex formation to determine the requirements for
the interactions of recombinant HsOrc1 with the other subunits. Fig.
6 shows some of the data that we obtained. Our initial experiments indicated that HsOrc1 did not form a
binary complex with HsOrc2 (first panel, Fig. 6). Thus, we
investigated the possibility that HsOrc1 might interact with various
sub-complexes containing other HsORC subunits. Our data demonstrated
that HsOrc1 did not co-immunoprecipitate with the HsOrc2-HsOrc3,
HsOrc2-HsOrc3-HsOrc4, or HsOrc2-HsOrc3-HsOrc5 sub-complexes identified
in the experiments described above (Fig. 6). Similar results were
obtained with the combinations, HsOrc1-HsOrc2-HsOrc4, HsOrc1-HsOrc2-HsOrc5, HsOrc1-HsOrc2-HsOrc3-HsOrc6, and
HsOrc1-HsOrc2-HsOrc4-HsOrc5 (data not shown). We only observed
co-immunoprecipitation of HsOrc1 when HsOrc2, HsOrc3, HsOrc4, and
HsOrc5 were all present (last panel, Fig. 6). Since HsOrc2,
HsOrc3, HsOrc4, and HsOrc5 readily formed a four-subunit complex in the
absence of HsOrc1 (data not shown), the simplest explanation of our
data is that association of HsOrc1 requires the prior assembly of the
four-subunit complex.
Although previous studies have identified individual subunits of
HsORC and probed some of the interactions among them, a multi-subunit ORC from human cells has not been reported (29, 36, 37). We have
identified a HsORC from HeLa cells that contains the five HsORC
subunits HsOrc1-HsOrc5. The complex is tightly associated with
chromatin and requires elevated salt concentrations (~500 mM NaCl) to dissociate it. Our results indicate that the
general architecture of HsORC is probably similar to that of ORCs
described in yeast and other metazoans (10, 17-19). Previous
difficulties in identifying HsORC are probably consequences of the
lack of good immunological reagents and inefficient extraction
conditions. The endogenous HsORC lacks detectable HsOrc6, and a
fraction of the complexes may also lack HsOrc1. This finding may be due
to the partial (HsOrc1) or complete (HsOrc6) dissociation of these subunits during preparation and extraction of chromatin, but there are
other possibilities as well (see below). In addition to the identification of the endogenous complex, we have achieved
reconstitution of HsORC by co-expression of recombinant subunits in the
baculovirus expression system. The recombinant HsORC is a holo-complex
containing all six subunits but otherwise closely resembles the
endogenous complex. We have made use of the baculovirus expression
system to define the complexes that can be formed by subsets of the
recombinant HsORC subunits. These experiments have provided insight
into the organization of HsORC and provided a rationale for the
observed heterogeneity of the complexes extracted from human cells.
Assembly of Recombinant HsORC--
The protein-protein
interactions that we have observed in our studies of sub-complexes of
recombinant HsORC are summarized in Fig.
7. Due to the many combinatorial
possibilities, our analysis, although extensive, is not exhaustive.
Nevertheless, the data illuminate some important features of the
architecture of HsORC. The strong binary interactions among HsOrc2,
HsOrc3, and HsOrc4 suggest that these three subunits represent the
core of the complex. Formation of stable complexes containing HsOrc5
required the presence of at least two of the core subunits, whereas
formation of complexes containing HsOrc1 required the presence of the
four subunits HsOrc2, HsOrc3, HsOrc4, and HsOrc5. These data strongly
suggest that the assembly of HsORC is an ordered process,
i.e. the association of HsOrc5 or HsOrc1 requires the prior
formation of specific sub-complexes. As noted above, there are two
possible mechanisms to account for the dependencies that we have
observed. One possibility is that the association of each subunit
(HsOrc5 or HsOrc1) requires the additive effects of relatively weak
interactions with two or more subunits present in a specific
sub-assembly. The second possibility is that the association of each
subunit is mediated by binding sites that are only uncovered by
conformational changes that occur during formation of a specific
sub-assembly. Further work will be required to distinguish between
these two possibilities.
HsOrc6--
Several observations strongly suggest that HsOrc6 is a
bona fide HsORC subunit even though it was not observed in
the endogenous complex. In the baculovirus expression system we
observed binary interactions of recombinant HsOrc6 with both HsOrc2 and
HsOrc3. Moreover, HsOrc6 was present in a high molecular mass complex containing all of the other HsORC subunits. Our control experiments demonstrated that the presence of HsOrc6 in this holo-complex was due
to specific interactions with the other subunits. Thus, the absence of
HsOrc6 in the endogenous complex was somewhat puzzling. One plausible
explanation is that HsOrc6 binds only weakly to the other subunits and
therefore dissociates from the endogenous complex under our extraction
conditions. We would attribute our ability to detect HsOrc6 in the
recombinant complex to the fact that HsOrc6 is expressed at very high
levels in co-infected insect cells, thus increasing the probability of
complex formation by simple mass action. An alternative, although
perhaps less likely, explanation is that the absence of HsOrc6 from the
endogenous complex is a consequence of post-translational modifications
or specific interactions with other chromatin proteins that do not occur in insect cells. It has been observed that HsOrc6 is
constitutively phosphorylated in human cells, but the possible
biological consequences of the modification are unknown (34). The
functional role of Orc6 is not known in any system. In S. cerevisiae it has been demonstrated that Orc6 is not required for
assembly of the other five ScORC subunits into a complex that binds
specifically to origins of DNA replication (35). However, mutants
lacking ScOrc6 are inviable, indicating that the subunit does mediate
some essential cellular function (12).
HsOrc1--
Our data suggest that HsOrc1 is consistently present
in sub-stoichiometric amounts in endogenous HsORC extracted from HeLa chromatin. Thus, our extracts likely contain two forms of HsORC, a
five-subunit complex containing HsOrc1 and a four-subunit complex lacking HsOrc1. Consistent with this observation, we recovered a
significant fraction of HsOrc1 in the 100 mM NaCl chromatin extract, whereas most of the other HsORC subunits were recovered almost
exclusively in the 500 mM NaCl chromatin extract. These findings indicate that not all of the cellular HsOrc1 is stably bound
to the other subunits of HsORC. The reason for this behavior is not
clear at this point, but one possible explanation is that the
association of HsOrc1 with the other HsORC subunits is regulated during
the cell cycle. It is known that the expression of HsOrc1, but not the
other HsORC subunits, is under the control of the cell cycle-regulated
transcription factor E2F (41). Moreover, two studies provide evidence
that mammalian Orc1 is more easily dissociated from chromatin in M
phase and perhaps early G1 (42, 43). This change in the
properties of HsOrc1 is correlated with the hyperphosphorylation of the
subunit that occurs during M phase, suggesting that the association of
HsOrc1 with the other human ORC subunits may be regulated by
post-translational modification (42). It has also been reported that
human cells have two populations of HsOrc2 that differ in their
association with HsOrc1 (37). One population of HsOrc2 can be extracted
from chromatin at 250 mM NaCl and another at 450 mM NaCl. The majority of the latter population, but not the
former, was shown to be associated with HsOrc1 in immunoprecipitation
experiments. These results are consistent with our suggestion that
HsORC extracted from chromatin at 500 mM NaCl
concentrations consists of two distinct complexes, one that contains
HsOrc1 and the other which does not. It was also reported that the
HsOrc1 dissociates from chromatin during S phase in HeLa cells and that
it may be degraded at this time (37). If this result can be confirmed,
it would provide a potentially attractive mechanism for preventing
re-initiation of DNA replication within genomic segments that have
already been replicated. However, it should be noted that several other
reports have concluded that the total amount of cellular HsOrc1 does
not significantly change during the cell cycle (42, 44). Thus, whereas
it is not yet clear whether regulation of the association of HsOrc1
with the other HsORC subunits plays a role in controlling the
initiation of DNA replication in human cells, several observations,
including our finding that HsORC can exist in at least two types of
complexes, certainly raise this possibility (37, 43).
HsORC--
Our data indicate that HsORC is generally similar in
structure to the previously purified S. cerevisiae,
Drosophila, and Xenopus ORCs (10, 18, 19).
However, there may be some subtle differences, including the propensity
for dissociation of HsOrc1 and HsOrc6. Whether or not these apparent
differences are simply due to differences in methods of extraction and
characterization or whether they reflect some differences in function
or regulation remains to be determined. To date only S. cerevisiae ORC has been shown to exhibit high affinity binding to
specific sequence elements within origins of DNA replication (8, 9). A
small fraction of Drosophila ORC purified from embryos shows
preferential association to a region of DNA implicated in gene
amplification, but the basis for the specificity of this association
has not yet been defined (27). Xenopus ORC appears to be
capable of interacting directly or indirectly with many unrelated DNA
sequences (45). Thus, it will be of great interest to define the
interactions of HsORC with DNA. Such studies will be facilitated by the
identification of HsORC complexes reported here.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Immunoprecipitation of endogenous HeLa
HsORC. A, a 500 mM NaCl chromatin extract
from HeLa cells was incubated with protein A beads coupled to either
control preimmune (P) or anti- HsORC2 antibody
(I). Proteins from the immunoprecipitated fractions were
separated by SDS/PAGE and analyzed by Western immunoblotting with
anti-HsORC1, HsORC2, HsORC3, HsORC4, HsORC5, and HsORC6 polyclonal
antibodies. B, a parallel gel was stained with silver. HsORC
subunits are marked by arrows.
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Fig. 2.
Reconstitution of recombinant HsORC in insect
cells. A, Sf9 cells were co-infected with
baculoviruses encoding the six HsORC subunits. After 48 h, the
cells were processed as described under "Experimental Procedures"
to obtain a 500 mM NaCl nuclear extract. Aliquots of the
extract were incubated with protein A beads coupled to either control
preimmune (P) or anti-HsORC2 antibody (I). The
bound proteins were analyzed by SDS/PAGE and detected by silver
staining. Polypeptides present in the immune precipitate (I)
but not in the control preimmune precipitate (P) are marked
by arrows. B, bound proteins separated on a
parallel gel were analyzed by Western immunoblotting with anti-HsORC1,
HsORC2, HsORC3, HsORC4, HsORC5, and HsORC6 polyclonal antibodies.
C, protein A beads coupled to anti- HsORC2 antibody were
preincubated with either an excess of specific competitor, GST-HsOrc2
(residues 1-270) (specific competition (SC)) or buffer (no
competition (NC)). The beads were then incubated with 500 mM NaCl nuclear extract from insect cells co-infected with
baculoviruses encoding the 6 HsORC subunits. The bound proteins were
separated by SDS/PAGE and analyzed by Western immunoblotting with
anti-HsORC1, HsORC2, HsORC3, HsORC4, HsORC5, and HsORC6 polyclonal
antibodies as in B. The asterisk marks the
location of the GST-HsORC2 competitor. D, Sf9 cells
were co-infected with baculoviruses encoding all of the HsORC subunits
except HsOrc2. After 48 h, a 500 mM nuclear extract
was prepared and incubated with protein A beads coupled to either
control preimmune (P) or anti-HsORC2 antibody
(I). The immunoprecipitated fractions were analyzed by
SDS/PAGE and silver staining.
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Fig. 3.
Gel filtration of recombinant HsORC. A
500 mM nuclear extract from insect cells co-infected with
recombinant baculoviruses encoding the six HsORC subunits was applied
to a Superdex 200 gel filtration column. Proteins from alternate
fractions (fractions 15-39) were separated on SDS/PAGE, transferred to
nitrocellulose membranes, and probed with antibodies against HsOrc1,
anti-HsOrc2, anti-HsOrc3, anti-HsOrc4, anti-HsOrc5, and anti-HsOrc6.
The positions of molecular mass markers thyroglobulin (T;
670 kDa), 
globulin (G; 158 kDa), ovalbumin
(O; 44 kDa), and myoglobulin (M; 17 kDa)
(Bio-Rad) are marked by arrows.
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Fig. 4.
Binary interactions of recombinant HsOrc2 and
HsOrc3 proteins. A, protein A beads coupled to
either control preimmune (P) or anti-HsOrc2 antibody
(I) were incubated with insect cell nuclear extract
containing recombinant HsOrc2 and either HsOrc3, HsOrc4, or HsOrc6. The
bound proteins were separated on SDS/PAGE and analyzed by both Western
immunoblotting (panels above) or silver staining
(panels below). The three lanes in each Western
blot correspond to the input nuclear extract, the immunoprecipitate
with preimmune antibody, and the immunoprecipitate with anti-HsOrc2
antibody. Western blots were probed with the indicated antibodies.
B, protein A beads coupled to either control preimmune
(P) or anti- HsOrc3 antibody (I) were incubated
with insect cell nuclear extract containing recombinant HsOrc3 and
either HsOrc4 or HsOrc6. Proteins in the immune precipitates were
analyzed as in A.
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Fig. 5.
HsOrc5 forms ternary complexes any two of
subunits HsOrc2, HsOrc3, and HsOrc4. Nuclear extracts from insects
cells expressing HsOrc5 and various combinations of other HsORC
proteins were incubated with protein A beads coupled to either control
preimmune (P) or anti-HsOrc2 or anti-HsOrc3 antibodies
(I). Bound proteins were separated on SDS/PAGE and analyzed
as described in the legend to Fig. 4. A,
immunoprecipitations with anti-HsOrc2 antibody. B,
immunoprecipitations with anti-HsOrc3 antibody.
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Fig. 6.
Complex formation by HsOrc1 requires
HsOrc2, HsOrc3, HsOrc4, and HsOrc5. Nuclear extracts from
insect cells expressing HsOrc1 and various combinations of other HsORC
proteins were incubated with protein A beads coupled with either
control preimmune (P) or anti-HsORC2 antibody
(I). Bound proteins were separated on SDS/PAGE and analyzed
as described in the legend to Fig. 4.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 7.
Summary of interactions among HsORC
subunits. Interactions between individual HsORC subunits
identified in co-expression experiments are indicated by
arrows. In the model, HsOrc2, HsOrc3, and HsOrc4 form a
core upon which other subunits build. HsOrc6 forms a specific binary
complex with HsOrc3 and to a lesser extent with HsOrc2. HsOrc5 does not
form strong binary complexes but interacts with any two of the core
subunits. Complex formation by HsOrc1 requires HsOrc2, HsOrc3, HsOrc4,
and HsOrc5.
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ACKNOWLEDGEMENTS |
|---|
We thank Drs. B. Stillman, A. Dutta, and J. Hurwitz for providing plasmids. We also thank Drs. L. Chrétien, M. Davenport, M. Frattini, C. Houchens, and M. Taipale for helpful comments on the manuscript and all of the members of the Kelly lab for thoughtful discussion.
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FOOTNOTES |
|---|
* This work was supported by National Institutes of Health Grant CA40414.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Supported by a postdoctoral fellowship from National Institutes of Health Grant GM19675.
** To whom correspondence should be addressed: Dept. of Molecular Biology and Genetics, 725 N. Wolfe St., Baltimore, MD 21205. Tel.: 410-955-3292; Fax: 410-955-0831; tkelly{at}jhmi.edu.
Published, JBC Papers in Press, April 25, 2001, DOI 10.1074/jbc.M102493200
2 D. Herendeen and T. Kelly, unpublished information.
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ABBREVIATIONS |
|---|
The abbreviations used are: bp, base pair; ACS, ARS consensus sequence; ORC, origin recognition complex; ScORC, S. cerevisiae ORC; xlORC, X. laevis ORC; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; GST, glutathione S-transferase.
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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