|
|
J Biol Chem. 2008 October 31; 283(44): 30216–30224. doi: 10.1074/jbc.M802649200. | PMCID: PMC2573090 |
Copyright © 2008, The American Society for Biochemistry
and Molecular Biology, Inc. Multiple Mechanisms Contribute to Schizosaccharomyces pombe
Origin Recognition Complex-DNA
Interactions *Christopher R. Houchens,‡1 Wenyan Lu,‡ Ray-Yuan Chuang,2 Mark G. Frattini,§ Alex Fuller,‡ Pam Simancek,‡ and Thomas J. Kelly‡ ‡Program in Molecular Biology and §Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, New York 10021 Received April 4, 2008; Revised August 22, 2008. Eukaryotic DNA replication requires the assembly of multiprotein
pre-replication complexes (pre-RCs) at chromosomal origins of DNA replication.
Here we describe the interactions of highly purified Schizosaccharomyces
pombe pre-RC components, SpORC, SpCdc18, and SpCdt1, with each other and
with ars1 origin DNA. We show that SpORC binds DNA in at least two
steps. The first step likely involves electrostatic interactions between the
AT-hook motifs of SpOrc4 and AT tracts in ars1 DNA and results in the
formation of a salt-sensitive complex. In the second step, the salt-sensitive
complex is slowly converted to a salt-stable complex that involves additional
interactions between SpORC and DNA. Binding of SpORC to ars1 DNA is
facilitated by negative supercoiling and is accompanied by changes in DNA
topology, suggesting that SpORC-DNA complexes contain underwound or negatively
writhed DNA. Purified human origin recognition complex (ORC) induces similar
topological changes in origin DNA, indicating that this property of ORC is
conserved in eukaryotic evolution and plays an important role in ORC function.
We also show that SpCdc18 and SpCdt1 form a binary complex that has greater
affinity for DNA than either protein alone. In addition, both proteins
contribute significantly to the stability of the initial SpORC-DNA complex and
enhance the SpORC-dependent topology changes in origin DNA. Thus, the
formation of stable protein-DNA complexes at S. pombe origins of
replication involves binary interactions among all three proteins, as well as
interactions of both SpORC and SpCdt1-SpCdc18 with origin DNA. These findings
demonstrate that SpORC is not the sole determinant of origin recognition. The timely and faithful duplication of eukaryotic genomes involves the
coordinated initiation of DNA replication from hundreds or thousands of
chromosomal replication origins during S phase. The initiation reaction
requires the assembly and activation of pre-replication complexes
(pre-RCs) 3 at
replication origins, leading to the establishment of two replication forks
that move in opposite directions (reviewed in Refs.
1– 4).
Pre-RC formation involves the selection of replication origins by the origin
recognition complex (ORC), followed by ORC-dependent recruitment of Cdc6 and
Cdt1. The resulting complex catalyzes the ATP-dependent loading of the
heterohexameric minichromosome maintenance helicase complex onto DNA to
establish a functional pre-RC. Eukaryotic chromosomal origins were first identified in the budding yeast
Saccharomyces cerevisiae as isolated chromosomal DNA fragments
capable of supporting the autonomous replication of extrachromosomal plasmids
( 5,
6). Budding yeast origins, most
of which are utilized efficiently in their chromosomal context, are relatively
small DNA segments comprised of a highly conserved and essential 11-bp ARS
consensus sequence and auxiliary B elements that enhance origin efficiency
( 7– 12).
S. cerevisiae ORC binds in a sequence-specific and ATP-dependent
manner to the ARS consensus sequence and B1 elements. Notably, mutations in
these elements that reduce binding of S. cerevisiae ORC in
vitro eliminate origin function in vivo
( 13,
14). The properties of S.
cerevisiae origins suggested that the initiation of eukaryotic DNA
replication might conform to the replicon hypothesis posited by Jacob et
al. over 40 years ago
( 15). However, work in the fission yeast Schizosaccharomyces pombe and
metazoan cells suggests that the budding yeast paradigm may not be as
generally applicable to all eukaryotes as first imagined. Most important,
conserved and essential ARS consensus sequence-like sequences have not been
identified in eukaryotic replication origins outside of budding yeast. In
fission yeast, both the size and AT content of origins, but not specific ARS
consensus sequence-like sequences, contribute to origin activity
( 16– 20).
Similarly, in Drosophila and Xenopus embryos, virtually any
DNA fragment is capable of supporting DNA replication
( 21– 25),
and in mammalian cells, DNA replication appears to initiate randomly from
numerous chromosomal sites within large zones of initiation
( 26– 30). Consistent with these observations, both S. pombe and metazoan
ORCs, although lacking sequence-specific DNA-binding activities,
preferentially bind AT-rich DNA sequences, a common feature of both fission
yeast and metazoan replication origins
( 24,
31). The S. pombe ORC
subunit SpOrc4 possesses an N-terminal extension, also present in other
Ascomycota Orc4 homologs, that is not found in budding yeast and metazoan Orc4
homologs. The N-terminal domain of fission yeast SpOrc4 is composed of nine
AT-hook DNA-binding motifs that are necessary and sufficient for the
DNA-binding activities of SpORC in vitro
( 31– 37).
Biochemical and structural studies have shown that AT-hook motifs bind the
minor groove of short AT tracts. Proteins with repeated AT-hook motifs can
bind in a multivalent manner to sequential AT tracts along DNA with nanomolar
binding affinities
( 38– 40).
It is likely that some or all of the SpOrc4 AT-hook motifs target the SpORC
holocomplex to AT-rich fission yeast chromosomal origins and that the relative
DNA-binding affinity of SpOrc4 is a function of the total number and spatial
arrangement of simple AT-core sequences distributed throughout an origin. Because S. pombe and metazoan origins, unlike those of S.
cerevisiae, do not appear to contain highly conserved sequence elements,
it becomes important to define the factors that contribute to origin
recognition in these species. We have examined the interactions of highly
purified S. pombe ORC, Cdc18, and Cdt1 proteins with one another and
with the fission yeast ars1 origin of replication. We have found that
SpORC binds to ars1 DNA in at least two distinct steps. The first
step results in formation of a salt-sensitive SpORC-DNA complex that is likely
mediated by the AT-hook domain of SpOrc4. The second step involves the slow
conversion of the salt-sensitive complex to a salt-resistant complex that
involves additional non-electrostatic interactions between SpORC and DNA. We
also found that negative supercoiling facilitates SpORC-DNA interactions, and
as previously observed, that binding of SpORC induces topological changes in
DNA consistent with untwisting or negative writhing. Finally, we show that
SpCdt1 and SpCdc18 form a binary DNA-binding complex and that each protein
facilitates SpORC-DNA interactions and SpORC-dependent changes in DNA
topology. Our data demonstrate that SpORC is not the sole determinant of
origin recognition, but that DNA structure and interactions with other pre-RC
proteins also make major contributions. Expression and Purification of SpORC, SpCdc18, SpCdt1, and HsORC in
Insect Cells—Recombinant SpORC was purified essentially as
described previously ( 31) with
the following modifications. Sf9 insect cells were co-infected with five
recombinant baculoviruses expressing SpORC subunits 1, 2, 3, 4, and 6 and a
recombinant baculovirus expressing FLAG epitope-tagged SpOrc5. After
clarification of the salt-extracted chromatin-enriched pellet, the collected
supernatant (12 ml) was incubated with 1 ml of anti-FLAG antibody-conjugated
agarose (M2-agarose) (Sigma) at 4 °C for 2 h. The beads were washed twice
with 10 ml of E buffer and once with 10 ml of F buffer
( 31). The M2-agarose-bound
proteins were eluted by incubation with 1.5 ml of F buffer containing 0.4
mg/ml FLAG peptide (Sigma) at 4 °C for 2 h. Recombinant FLAG-SpCdc18
( 31) and recombinant HsORC
( 24) were purified as
described previously. Recombinant SpCdt1-His 6-FLAG was purified as
follows. Sf9 cells (2 × 10 6 cells/ml) were infected with
recombinant baculovirus expressing SpCdt1-His 6-FLAG at a
multiplicity of infection of 5. After 48 h, cells were harvested and washed
once with ice-cold phosphate-buffered saline (10 m m phosphate
buffer, pH 7.3, 140 m m NaCl, 2.7 m m KCl) and collected
by centrifugation. Infected cell pellets from 250-ml cultures were lysed in 18
ml of L buffer ( 31). The
suspension was centrifuged at 1,400 × g for 5 min at 4 °C,
and the resulting supernatant was centrifuged at 100,000 × g
for 30 min at 4 °C. The collected supernatant (18 ml) was incubated with 1
ml of M2-agarose at 4 °C for 2 h. The beads were washed twice with 10 ml
of E buffer and once with 10 ml of F buffer
( 31). The M2-agarose-bound
proteins were eluted by incubation with 1.5 ml of F buffer containing 0.4
mg/ml FLAG peptide at 4 °C for 2 h. DNA Binding Experiments—For DNA-bead binding experiments, a
biotinylated 1153-bp DNA fragment containing the ars1 origin was
synthesized by PCR and coupled to streptavidin-conjugated magnetic beads as
described previously ( 31). The
binding assays were performed by incubating the indicated amounts of purified
SpORC, SpCdc18, and SpCdt1 with beads containing 0.5 pmol of ars1 DNA
in 25 μl of B buffer (50 m m Hepes-NaOH, pH 7.5, 60 m m
or 500 m m NaCl, 1 m m EDTA, 1 m m EGTA, 5
m m magnesium acetate, 10% glycerol, 1 m m dithiothreitol)
with 0.1 mg/ml bovine serum albumin, 1 m m ATP, and 1 ×
protease inhibitor mixture (Roche Applied Bioscience) for 30 min at 30 °C.
Where indicated, 5 pmol of ars1 competitor DNA was included in the
reaction mixtures. Beads were washed twice for 1 min each with 200 μl of B
buffer containing 0.1% Nonidet P-40 (low salt wash) or B buffer containing 500
m m NaCl and 0.1% Nonidet P-40 (high salt wash). The bound proteins
were released by the addition of 1 × SDS loading dye and separated by
10% SDS-PAGE, followed by Western blot analysis using rabbit polyclonal
antibodies against SpOrc3, SpORC5, or SpCdt1. Anti-FLAG antibody (M2, Sigma)
was used for detecting recombinant FLAG-SpCdc18. For competition nitrocellulose filter binding experiments, a 1153-bp DNA
fragment containing the ars1 origin was synthesized and radiolabeled
with [α-32P]dATP by PCR. The binding assays were performed by
incubating 50 fmol (2 nm) of purified SpORC with 12.5 fmol (0.5
nm) of radiolabeled ars1 DNA in 25 μl of B buffer with
0.1 mg/ml bovine serum albumin, 1 mm ATP, and 1 × protease
inhibitor mixture for 30 min at 30 °C in the absence or presence of
supercoiled and relaxed pARS1 plasmid competitor DNA. The reaction mixtures
were passed through nitrocellulose filters (HA, Millipore Corp.), washed with
2 ml of low salt wash buffer, dried, and counted in a scintillation
spectrometer. Topoisomerase I Assay—The 3737-bp pARS1 plasmid containing
the 1153-bp ars1 origin DNA sequence was generated by removing a
2967-bp PflFI-BamHI DNA fragment containing the LEU2 gene from pRC20
( 41) and ligation of the
product. The supercoiled and relaxed pARS1 plasmids used in Figs.
and
were generated by
incubating 5 μg of pARS1 for 2 h at 37 °C in the presence or absence of
60 units of topoisomerase I (Invitrogen). The plasmids were
ethanol-precipitated and dissolved in Tris/EDTA buffer. The topoisomerase I
assays were performed by incubating purified SpORC, SpCdc18, and SpCdt1 with
40 ng of pARS1 in 25 μl of B buffer with 0.1 mg/ml bovine serum albumin, 1
m m ATP, and 1× protease inhibitor mixture for 10 min at 30
°C, followed by addition of 30 units topoisomerase I for 30 min at 37
°C. The reactions were stopped and deproteinized by addition of 5.5 μl
of stop buffer (6% SDS, 30% glycerol, 10 m m EDTA, bromphenol blue,
and xylene cyanol FF) and incubation at 42 °C for 10 min. The samples were
electrophoresed in 0.8% agarose in 0.5× Tris/EDTA buffer lacking or
containing 1 μg/ml chloroquine for 16 h at 45 V. Gels were washed three
times for 20 min in 0.5× Tris borate/EDTA and then stained with SYBR
gold diluted 1:10,000 in 0.5× Tris borate/EDTA for 2 h. The DNA was
detected using a UV transluminator. SpORC Binds DNA in a Biphasic Manner— S. pombe ORC,
Cdc18, and Cdt1 were purified from Sf9 insect cells infected with
baculoviruses expressing FLAG epitope-tagged recombinant proteins
(). The purity
of each protein preparation, as analyzed by silver staining of
SDS-polyacrylamide gels, was determined to be >90%. In initial experiments,
we made use of an ars1 DNA-bead binding assay to examine the
formation and stability of SpORC-DNA complexes at different salt
concentrations. In this assay, SpORC was incubated with an ars1 DNA
fragment coupled to magnetic beads ( ars1-beads) for 30 min in buffer
containing either 60 m m NaCl (low salt) or 500 m m NaCl
(high salt). The beads were collected and washed with either low or high salt
wash buffer, and the bound SpORC was detected by immunoblotting. Consistent
with results from previous studies
( 31), we observed that high
salt prevented the binding of SpORC to ars1 DNA
(, compare
lanes 2 and 3) likely because of reduction of electrostatic
interactions between the AT-hooks of SpOrc4 and AT-rich sequences within
ars1 DNA. Paradoxically, we also observed that SpORC-DNA complexes
formed after 30 min in low salt buffer were highly resistant to washing with
high salt buffer (compare lanes 2 and 4). In control
experiments, SpORC did not bind beads lacking DNA (data not shown), indicating
that the salt-resistant SpORC-DNA complexes are not simply aggregated or
insoluble forms of SpORC. These results suggested that SpORC can form two
distinct complexes with origin DNA. To investigate this possibility further,
we examined the kinetics of formation of salt-sensitive and salt-resistant
complexes ().
For this purpose, SpORC was incubated with ars1-beads for 1–30
min in low salt buffer, followed by washing with buffer containing either a
low or a high salt concentration. A significant fraction of SpORC bound to
ars1 DNA within 1 min, but this complex was almost completely
sensitive to dissociation by high salt (compare lanes 2 and
7). However, a second SpORC-DNA complex, resistant to washing with
high salt, formed gradually and by 30 min accounted for most of the bound
SpORC (compare lanes 6 and 11). Similar results were
obtained when the reactions were carried out in the absence of ATP (data not
shown). Thus, two SpORC-DNA complexes form with different kinetics: a
salt-sensitive complex (complex I) that forms rapidly and presumably involves
significant electrostatic interactions and a second, salt-resistant complex
(complex II) that forms slowly and presumably involves additional
non-electrostatic SpORC-DNA interactions. Complex II is quite stable, as no
appreciable dissociation was observed in the high salt wash buffer during a
30-min incubation. SpORC-DNA Complex I Is a Precursor to Complex II—There are
two possible interpretations of the binding data described above: either the
two SpORC-DNA complexes form independently of one another or complex I is an
obligatory precursor to complex II. To distinguish between these two
possibilities, we incubated SpORC with ars1 DNA-beads for 1 min in
low salt buffer to allow complex I to form and then added competitor
ars1 DNA to prevent further binding of SpORC to ars1
DNA-beads. If complex I is a precursor to complex II, then the presence of
ars1 competitor DNA should not affect formation of the salt-stable
complex II on the ars1 DNA-beads. Alternatively, if the two SpORC-DNA
complexes form independently of one another, then addition of the competitor
DNA should completely prevent formation complex II on the ars1
DNA-beads. We confirmed that the concentration of added ars1
competitor DNA was sufficient to prevent SpORC binding to ars1-beads
when added to the binding reaction prior to SpORC
(, lanes
2 and 5). However, when this concentration of competitor DNA was
added after formation of complex I, the amount of complex II formed in 30 min
was completely unaffected (compare lanes 6 and 7). These
results demonstrate that SpORC binds ars1 DNA to form an initial
SpORC-DNA complex I that does not readily dissociate at low salt
concentrations (compare lanes 3 and 4) and is slowly
converted to the salt-stable complex II. SpORC Preferentially Binds Negatively Supercoiled DNA—It has
been reported that Drosophila melanogaster ORC has an affinity for
negatively supercoiled plasmid DNA that is ~30-fold greater than its
affinity for relaxed plasmid DNA
( 42). To determine whether
this is the case for S. pombe ORC, we used a competition
nitrocellulose filter DNA-binding assay to compare the relative binding
affinities of SpORC for a negatively supercoiled or relaxed plasmid DNA
containing the ars1 origin (pARS1)
(). In this
assay, SpORC was incubated for 30 min with a radiolabeled ars1 DNA
probe in the presence of increasing concentrations of unlabeled negatively
supercoiled or relaxed pARS1, followed by filtration through a nitrocellulose
filter. Under these conditions, the apparent binding affinity of SpORC for
ars1 DNA was determined to be ~2 n m (data not shown),
consistent with previously reported measurements
( 35). Negatively supercoiled
pARS1 was a significantly more effective competitor for SpORC binding to the
ars1 probe (, blue line) than relaxed pARS1 DNA ( red
line). Binding of SpORC to the ars1 probe was reduced by
~50% in the presence of a 4-fold excess of negatively supercoiled pARS1
competitor DNA, whereas the addition of an ~50-fold excess of relaxed
pARS1 competitor DNA was required for a similar reduction in SpORC binding to
the ars1 probe. These results demonstrate that SpORC, like D.
melanogaster ORC, has a significantly higher apparent affinity for
negatively supercoiled plasmid DNA than for relaxed plasmid DNA. Theses
results are highly reproducible and are consistent with the existence of a
SpORC-DNA complex in which the bound DNA is structurally distorted, so as to
induce local untwisting or negative writhe or both. In covalently closed
circular DNA molecules, such a local change in DNA structure upon SpORC
binding would induce compensatory positive writhe in the plasmid DNA,
increasing the free energy change of the binding reaction relative to that of
the binding reaction with topologically unconstrained ( e.g. linear)
DNA. In a negatively supercoiled DNA, the positive writhe cancels negative
writhe, decreasing the relative free energy change for the reaction. SpORC and HsORC Alter the Structure of Bound DNA—To verify
that binding of SpORC can induce changes in the twist or writhe of bound DNA,
we carried out topoisomerase relaxation assays. In these assays, pARS1 was
incubated with increasing concentrations of SpORC to allow complex formation,
and then topoisomerase I was added to relax unconstrained regions of the
plasmid. Following deproteinization, the plasmid DNA was analyzed by native
agarose gel electrophoresis in the absence
(,
left) or presence (, right) of chloroquine. We first confirmed
previous observations ( 33)
that binding of S. pombe ORC can alter the topology of negatively
supercoiled plasmid DNA. Incubation with SpORC resulted in a shift in the
topoisomer distribution toward lower mobilities when gel electrophoresis of
the topoisomerase-treated DNA was carried out in the absence of chloroquine
(,
left, compare lanes 2 and 6). Conversely, the
topoisomer distribution shifted toward higher mobilities when gel
electrophoresis was carried out in the presence of chloroquine
(,
right). Similar results were obtained when the reaction mixtures
lacked ATP (data not shown). Increasing the topoisomerase I concentration in
the assay or the duration of the incubation with topoisomerase I did not alter
the topoisomer distributions (data not shown), indicating that SpORC was not
simply inhibiting the activity of topoisomerase I. The results shown in
, together with
analysis of the distribution of pARS1 topoisomers by agarose gel
electrophoresis in different concentrations of chloroquine (data not shown),
demonstrated that the linking number of pARS1 DNA bound to SpORC was lower
than that of free pARS1 DNA following relaxation with topoisomerase I. This
observation is consistent with the hypothesis that the binding of SpORC can
untwist DNA or stabilize left-handed negative writhe, perhaps via
wrapping. We next used the topoisomerase relaxation assay to examine the effect of
SpORC binding on the topology of relaxed pARS1 plasmid DNA
(). Unlike
supercoiled plasmid DNA (compare lanes 2 and 3), the relaxed
plasmid DNA did not exhibit a change in topoisomer distribution as a result of
incubation with increasing concentrations of SpORC (compare lanes 5
and 6). Thus, under the conditions of these experiments, negative
supercoiling facilitates the change in DNA structure that accompanies SpORC
binding. Our data indicate that the properties of S. pombe ORC are similar
to those of D. melanogaster ORC, which also binds preferentially to
supercoiled DNA and alters the topology of supercoiled, but not relaxed, DNA.
Thus, the ability to untwist or induce negative writhe may be a general
property of ORC. To examine this possibility further, we carried out
topoisomerase relaxation assays with highly purified recombinant human ORC
(HsORC) (). We
used supercoiled pARS1 plasmid DNA in these experiments because we have shown
previously that HsORC has relatively little sequence specificity. The binding
of HsORC to pARS1 DNA resulted in a shift in the topoisomer distribution
(compare lanes 2 and 6), similar in magnitude to that
observed with SpORC at similar concentrations (lane 7). Thus, the
ability of ORC to alter DNA structure and topology is an evolutionarily
conserved property of ORC that can be found in yeast, flies, and humans. SpCdc18 and SpCdt1 Facilitate SpORC-DNA Interactions—We
examined the interactions of purified SpORC, SpCdc18, and SpCdt1 with each
other and with ars1 DNA during the formation of salt-stable
nucleoprotein complexes. For this purpose, we incubated various combinations
of SpORC, SpCdc18, and SpCdt1 with ars1-beads for 10 min and then
washed the beads with high salt (500 mm NaCl) buffer
(). In the
absence of the other proteins, a small amount of SpCdt1 bound to the
ars1 DNA (lane 6). Under the same conditions, no DNA binding
by SpCdc18 was observed (lane 5). When the two proteins were
incubated together, we observed a significant enhancement of the binding of
both proteins to ars1 DNA (compare lanes 5 and 6
with lane 7). This observation strongly suggests that SpCdt1 and
SpCdc18 form a complex that has an intrinsic DNA-binding activity greater than
that of the individual proteins and is independent of SpORC. The presence of SpORC increased the association of SpCdt1 or SpCdc18 with
ars1-beads, indicating that both proteins interact independently with
SpORC (,
compare lanes 5 and 6 to lanes 8 and 9).
Conversely, we observed that the presence of either SpCdt1 (lane 8)
or SpCdc18 (lane 9) enhanced SpORC binding to ars1 DNA to
form the salt-stable complex. SpORC, SpCdc18, and SpCdt1 all bound DNA most
efficiently when all three proteins were present (lane 10). Similar
results were observed when the reactions were carried out in the absence of
ATP (data not shown). We also investigated the effects of SpCdt1 and SpCdc18
on the formation of the salt-sensitive SpORC-DNA complex. For this purpose,
the incubation was shortened to 1 min, and the beads were washed with either
low salt buffer (, left) or high salt buffer
(,
right). Under low salt wash conditions, we observed a similar
enhancement of SpORC binding by SpCdt1 or SpCdc18
(,
left). However, these complexes were not resistant to washing with
high salt buffer (, right). These results suggest that SpCdc18
and SpCdt1 facilitate the initial interaction between SpORC and ars1
DNA that leads the formation of a salt-stable complex. We also carried out a topoisomerase I relaxation assay to examine whether
SpCdc18 and/or SpCdt1 enhances SpORC-dependent structural changes in
negatively supercoiled pARS1 plasmid DNA. In this assay, we used a
concentration of SpORC that we previously determined had a minimal effect on
altering pARS1 topology (, lane 3). Neither SpCdc18 (lane 4)
nor SpCdt1 (lane 5) had a significant affect on pARS1 topology either
alone or in combination (lane 6). However, incubation of SpORC with
either SpCdc18 (lane 8) or SpCdt1 (lane 7) resulted in a
significant shift in the distribution of topoisomers, with the greatest effect
observed when all three proteins were present (lane 9). These changes
in pARS1 topology are similar to those we observed using higher SpORC
concentrations (, lane 6) and thus are consistent with the
observation that SpCdt1 and SpCdc18 independently facilitate the binding of
SpORC to DNA (). The increased binding of SpORC to pARS1 DNA due to
interactions with SpCdt1 and SpCdc18 may be sufficient to explain the observed
enhancement of the topological changes, but we cannot rule out the possibility
that the SpORC-Cdt1-Cdc18 complexes induce greater structural alterations in
the bound DNA than SpORC alone. We have used highly purified recombinant S. pombe ORC, Cdc18, and
Cdt1 proteins to examine the molecular interactions that occur during
pre-replication complex assembly at the fission yeast ars1 origin of
DNA replication. SpORC binds ars1 DNA in a biphasic manner in which
the initial salt-sensitive complex I is slowly converted to the highly stable
salt-resistant complex II (). The formation of complex I likely involves interactions
between the nine AT-hook motifs in the SpOrc4 subunit and AT tracts in
ars1 DNA. Previous studies have shown that the AT-hook motifs are
necessary and sufficient to target SpORC to DNA and that the binding affinity
of SpORC for a segment of DNA is determined largely by its length and AT
content
( 31– 37).
Moreover, thermodynamic studies have demonstrated that salt-sensitive
electrostatic interactions are the dominant forces in the binding of AT-hooks
to DNA ( 43). The nature of
complex II is not yet clear, but its salt resistance indicates that additional
non-electrostatic interactions between SpORC subunits and DNA make a
substantial contribution to its stability. The formation of complex II is a
slow step that only occurs at an appreciable rate when SpORC is tethered to
origin DNA via complex I. Thus, the AT-hooks of SpOrc4 likely function to
properly localize SpORC to AT-rich fission yeast chromosomal origins of DNA
replication, which are preferentially located in intergenic DNA
( 18,
20,
44– 46).
Because AT tracts are highly abundant in the genome, particularly in
intergenic DNA, SpORC does not bind to a highly specific site like S.
cerevisiae ORC, but rather is distributed at multiple sites even within a
single ars element like ars1. Once SpORC is tethered to the origin
DNA via the AT-hooks, additional SpORC-DNA interactions occur that are
presumably required for the subsequent steps in the assembly of the pre-RC. In
addition to simply recruiting SpORC to the vicinity of origin DNA, it is
possible that the AT-hook motifs more directly facilitate the formation of
complex II. For example, a number of proteins containing AT-hooks
( e.g. high mobility group) appear to facilitate the formation of
regulatory protein-DNA complexes at promoters ( e.g. the
interferon-β promoter)
( 47). Depending on the case,
high mobility group can facilitate the binding of other transcriptional
regulatory proteins either by direct protein-protein interactions or by
modulating the structure of the DNA. Further work will be required to
determine whether the AT-hook motifs of SpOrc4 act in a similar way.
Interestingly, it has been reported that the stability of Xenopus ORC
binding to chromatin changes during the cell cycle
( 48). In metaphase, the
Xenopus laevis ORC-DNA complexes are salt-sensitive, but they become
salt-resistant in early interphase. The complexes become salt-sensitive again
after XlMcm2–7 loading completes the formation of active pre-RCs. Several lines of evidence indicate that complex II does not simply
represent an aggregated or insoluble form of SpORC. First, no precipitation of
SpORC was observed in control experiments in which SpORC was incubated with
beads lacking DNA. Second, excess competitor DNA was sufficient to prevent
formation of both complexes I and II, indicating that both species represent
DNA-bound forms. Finally, the SpORC-DNA complexes formed under conditions that
generate the salt-stable complex are associated with a distinct and stable
topological state of the bound DNA (see below). We observed that SpORC in complex II induces a significant change in the
structure of the bound DNA. Consistent with previous studies of D.
melanogaster and S. pombe ORCs, topology assays with closed
circular DNA demonstrated that the DNA is underwound or has negative writhe or
both ( 33,
42). We observed that highly
purified human ORC induced a similar topological change, indicating that this
property of ORC has been conserved during eukaryotic evolution and probably
plays an important role in ORC function. The loading of the Mcm2–7
helicase likely requires local unwinding of the origin DNA, so it is possible
that the ORC-induced structural alteration in the DNA contributes to this
function. We observed that SpORC, like D. melanogaster ORC, has
little effect on the topology of covalently closed, but relaxed, plasmid DNA
in the topoisomerase assay. This observation is consistent with the
expectation that the SpORC-induced changes in topology would be disfavored in
such a DNA molecule because of the energetic penalty resulting from the
accumulation of compensatory positive writhe. On the other hand, it would be
expected that binding and the concomitant changes in DNA structure would be
facilitated by negative supercoiling. These effects explain our observation
that the affinity of SpORC for relaxed plasmid DNA is much less than its
affinity for supercoiled plasmid DNA. The higher affinity of SpORC for supercoiled DNA may be relevant to SpORC
function in vivo. It is interesting that the Escherichia
coli replication initiator DnaA protein, which has structural similarity
to ORC1, -4, and -5 ( 49) in
the AAA+ domains, preferentially binds to negatively supercoiled oriC
replication origin DNA sequences and, upon binding, wraps the DNA and promotes
localized DNA unwinding
( 50– 53).
In E. coli, negative supercoiling is maintained by the activity of
gyrase, but there is no comparable mechanism in eukaryotes. However, in
eukaryotic cells, chromosomal origins are packaged into nucleosomes that must
be cleared during the assembly of pre-replication complexes
( 54– 58).
The removal of nucleosomes by chromatin remodeling factors might result in the
transient formation of negatively supercoiled DNA that could potentially
facilitate ORC binding and the accompanying perturbation of DNA structure. Of
course such a mechanism presumes that the rate of relaxation of the
supercoiled region by topoisomerases is not more rapid than that of ORC
binding. We have observed a number of interactions among S. pombe ORC,
Cdc18, Cdt1, and ars1 DNA. As expected, SpORC greatly enhanced the
binding of SpCdt1 and SpCdc18 to ars1 DNA, consistent with the
current view that ORC recruits the other pre-RC proteins to DNA
( 1– 3).
However, we observed that in the absence of SpORC, SpCdt1 has detectable
affinity for ars1 DNA. This interaction has been described previously
in the Xenopus system, and the DNA-binding activity has been
attributed to the N-terminal region of Cdt1
( 59). Interestingly, we also
observed that SpCdt1 can form a binary complex with SpCdc18 that has
significantly greater affinity for DNA than SpCdt1 or SpCdc18 alone. We
suggest that this SpCdt1-Cdc18-DNA complex may play a role in pre-RC
formation, so SpORC may not be the only determinant of the chromosomal sites
of pre-RC assembly. Consistent with this possibility, it has been reported
that ScCdc6, the orthologue of SpCdc18, can alter origin site selection by
S. cerevisiae ORC
( 21,
60,
61). Finally, we observed that
SpCdt1 and SpCdc18 individually enhance the binding of SpORC to ars1
DNA and that maximal SpORC binding is observed when all three proteins are
present. The effect of SpCdc18 and SpCdt1 is to facilitate the initial
salt-sensitive interaction between SpORC and DNA that leads to the formation
of the salt-stable complex II. Consistent with these results, we found that
SpCdc18 and SpCdt1 also facilitate SpORC-dependent changes in DNA topology in
complex II. Thus, our data indicate that the formation of the pre-RC involves
binary interactions among all three proteins (SpCdt1-Cdc18, SpCdt1-ORC, and
SpCdc18-ORC) as well as interactions of both SpORC and SpCdt1-Cdc18 with
origin DNA. Whereas SpORC is sufficient to induce topological changes in
origin DNA, the stability of the SpORC-DNA complex may likely depend upon all
of these interactions. 1. Bell, S. P., and Dutta, A. (2002. ) Annu.
Rev. Biochem. 71
333-374 [PubMed] 2. Kelly, T. J., and Brown, G. W. (2000. ) Annu.
Rev. Biochem. 69
829-880 [PubMed] 3. Teer, J. K., and Dutta, A. (2006. ) Results
Probl. Cell Differ. 42
31-63 [PubMed] 4. Nishitani, H., and Lygerou, Z. (2002. ) Genes
Cells 7
523-534 [PubMed] 5. Fangman, W. L., Hice, R. H., and Chlebowicz-Sledziewska, E.
(1983. ) Cell
32 831-838 [PubMed] 6. Kearsey, S. (1983. ) EMBO J.
2
1571-1575 [PubMed] 7. Huang, R. Y., and Kowalski, D. (1993. ) EMBO
J. 12
4521-4531 [PubMed] 8. Marahrens, Y., and Stillman, B. (1992. )
Science 255
817-823 [PubMed] 9. Rao, H., Marahrens, Y., and Stillman, B. (1994. )
Mol. Cell. Biol. 14
7643-7651 [PubMed] 10. Theis, J. F., and Newlon, C. S. (1994. ) Mol.
Cell. Biol. 14
7652-7659 [PubMed] 11. Breier, A. M., Chatterji, S., and Cozzarelli, N. R.
(2004. ) Genome Biol.
5 R22. [PubMed] 12. Wyrick, J. J., Aparicio, J. G., Chen, T., Barnett, J. D., Jennings,
E. G., Young, R. A., Bell, S. P., and Aparicio, O. M. (2001. )
Science 294
2357-2360 [PubMed] 13. Bell, S. P., and Stillman, B. (1992. )
Nature 357
128-134 [PubMed] 14. Biswas, S. B., Khopde, S. M., and Biswas-Fiss, E. E.
(2005. ) Cell Cycle
4 494-500 [PubMed] 15. Jacob, F., Brenner, S., and Cuzin, F. (1964. )
Cold Spring Harbor Symp. Quant. Biol.
28 329-348. 16. Dubey, D. D., Kim, S. M., Todorov, I. T., and Huberman, J. A.
(1996. ) Curr. Biol.
6 467-473 [PubMed] 17. Clyne, R. K., and Kelly, T. J. (1997. )
Methods (Orlando) 13
221-233. 18. Dai, J., Chuang, R.-Y., and Kelly, T. J. (2005. )
Proc. Natl. Acad. Sci. U. S. A.
102
337-342 [PubMed] 19. Okuno, Y., Satoh, H., Sekiguchi, M., and Masukata, H.
(1999. ) Mol. Cell. Biol.
19
6699-6709 [PubMed] 20. Segurado, M., de Luis, A., and Antequera, F. (2003. )
EMBO Rep. 4
1048-1053 [PubMed] 21. Harvey, K. J., and Newport, J. (2003. ) J.
Biol. Chem. 278
48524-48528 [PubMed] 22. Mechali, M., and Kearsey, S. (1984. )
Cell 38
55-64 [PubMed] 23. Arias, E. E., and Walter, J. C. (2004. )
Front. Biosci. 9
3029-3045 [PubMed] 24. Vashee, S., Cvetic, C., Lu, W., Simancek, P., Kelly, T. J., and
Walter, J. C. (2003. ) Genes Dev.
17
1894-1908 [PubMed] 25. Chesnokov, I., Gossen, M., Remus, D., and Botchan, M.
(1999. ) Genes Dev.
13
1289-1296 [PubMed] 26. Mesner, L. D., Li, X., Dijkwel, P. A., and Hamlin, J. L.
(2003. ) Mol. Cell. Biol.
23 804-814 [PubMed] 27. Dijkwel, P. A., Wang, S., and Hamlin, J. L. (2002. )
Mol. Cell. Biol. 22
3053-3065 [PubMed] 28. Dijkwel, P. A., Mesner, L. D., Levenson, V. V., d'Anna, J., and
Hamlin, J. L. (2000. ) Exp. Cell Res.
256
150-157 [PubMed] 29. Dijkwel, P. A., Vaughn, J. P., and Hamlin, J. L.
(1994. ) Nucleic Acids Res.
22
4989-4996 [PubMed] 30. Norio, P., Kosiyatrakul, S., Yang, Q., Guan, Z., Brown, N. M.,
Thomas, S., Riblet, R., and Schildkraut, C. L. (2005. )
Mol. Cell 20
575-587 [PubMed] 31. Chuang, R.-Y., Chretien, L., Dai, J., and Kelly, T. J.
(2002. ) J. Biol. Chem.
277
16920-16927 [PubMed] 32. Chuang, R.-Y., and Kelly, T. J. (1999. )
Proc. Natl. Acad. Sci. U. S. A.
96
2656-2661 [PubMed] 33. Gaczynska, M., Osmulski, P. A., Jiang, Y., Lee, J. K., Bermudez,
V., and Hurwitz, J. (2004. ) Proc. Natl. Acad. Sci. U.
S. A. 101
17952-17957 [PubMed] 34. Kong, D., and DePamphilis, M. L. (2001. )
Mol. Cell. Biol. 21
8095-8103 [PubMed] 35. Lee, J. K., Moon, K. Y., Jiang, Y., and Hurwitz, J.
(2001. ) Proc. Natl. Acad. Sci. U. S. A.
98
13589-13594 [PubMed] 36. Takahashi, T., and Masukata, H. (2001. )
Genes Cells 6
837-849 [PubMed] 37. Moon, K. Y., Kong, D., Lee, J. K., Raychaudhuri, S., and Hurwitz,
J. (1999. ) Proc. Natl. Acad. Sci. U. S. A.
96
12367-12372 [PubMed] 38. Bustin, M., and Reeves, R. (1996. ) Prog.
Nucleic Acid Res. Mol. Biol. 54
35-100 [PubMed] 39. Huth, J. R., Bewley, C. A., Nissen, M. S., Evans, J. N., Reeves,
R., Gronenborn, A. M., and Clore, G. M. (1997. ) Nat.
Struct. Biol. 4
657-665 [PubMed] 40. Maher, J. F., and Nathans, D. (1996. ) Proc.
Natl. Acad. Sci. U. S. A. 93
6716-6720 [PubMed] 41. Clyne, R. K., and Kelly, T. J. (1995. ) EMBO
J. 14
6348-6357 [PubMed] 42. Remus, D., Beall, E. L., and Botchan, M. R. (2004. )
EMBO J. 23
897-907 [PubMed] 43. Cui, T., Wei, S., Brew, K., and Leng, F. (2005. )
J. Mol. Biol. 352
629-645 [PubMed] 44. Hayashi, M., Katou, Y., Itoh, T., Tazumi, A., Yamada, Y.,
Takahashi, T., Nakagawa, T., Shirahige, K., and Masukata, H.
(2007. ) EMBO J.
26
1327-1339 [PubMed] 45. Feng, W., Raghuraman, M. K., and Brewer, B. J. (2007. )
Methods (Orlando) 41
151-157. 46. Heichinger, C., Penkett, C. J., Bahler, J., and Nurse, P.
(2006. ) EMBO J.
25
5171-5179 [PubMed] 47. Reeves, R., and Beckerbauer, L. (2001. )
Biochim. Biophys. Acta
1519 13-29 [PubMed] 48. Rowles, A., Tada, S., and Blow, J. J. (1999. )
J. Cell Sci. 112
2011-2018 [PubMed] 49. Clarey, M. G., Erzberger, J. P., Grob, P., Leschziner, A. E.,
Berger, J. M., Nogales, E., and Botchan, M. (2006. )
Nat. Struct. Mol. Biol.
13 684-690 [PubMed] 50. Bramhill, D., and Kornberg, A. (1988. )
Cell 54
915-918 [PubMed] 51. Bramhill, D., and Kornberg, A. (1988. )
Cell 52
743-755 [PubMed] 52. Messer, W. (2002. ) FEMS Microbiol.
Rev. 26
355-374 [PubMed] 53. Sekimizu, K., Bramhill, D., and Kornberg, A. (1988. )
J. Biol. Chem. 263
7124-7130 [PubMed] 54. Gerbi, S. A., and Bielinsky, A. K. (2002. )
Curr. Opin. Genet. Dev.
12 243-248 [PubMed] 55. Aggarwal, B. D., and Calvi, B. R. (2004. )
Nature 430
372-376 [PubMed] 56. Melendy, T., and Li, R. (2001. ) Front.
Biosci. 6
D1048-D1053 [PubMed] 57. Biamonti, G., Paixao, S., Montecucco, A., Peverali, F. A., Riva,
S., and Falaschi, A. (2003. ) Chromosome
Res. 11
403-412 [PubMed] 58. Iizuka, M., Matsui, T., Takisawa, H., and Smith, M. M.
(2006. ) Mol. Cell. Biol.
26
1098-1108 [PubMed] 59. Lee, C., Hong, B., Choi, J. M., Kim, Y., Watanabe, S., Ishimi, Y.,
Enomoto, T., Tada, S., Kim, Y., and Cho, Y. (2004. )
Nature 430
913-917 [PubMed] 60. Speck, C., Chen, Z., Li, H., and Stillman, B. (2005. )
Nat. Struct. Mol. Biol.
12 965-971 [PubMed] 61. Speck, C., and Stillman, B. (2007. ) J. Biol.
Chem. 282
11705-11714 [PubMed]
|
This article has been 
