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Molecular and Cellular Biology, May 2000, p. 3086-3096, Vol. 20, No. 9
Cold Spring Harbor Laboratory, Cold Spring
Harbor, New York 11724,1 and Graduate
Program in Molecular Genetics and Microbiology, School of Medicine,
State University of New York at Stony Brook, Stony Brook, New York
117942
Received 24 September 1999/Returned for modification 12 November
1999/Accepted 10 January 2000
In Saccharomyces cerevisiae, replication origins are
activated with characteristic timing during S phase. S-phase
cyclin-dependent kinases (S-CDKs) and Cdc7p-Dbf4p kinase are required
for origin activation throughout S phase. The activation of S-CDKs
leads to association of Cdc45p with chromatin, raising the possibility that Cdc45p defines the assembly of a new complex at each origin. Here
we show that both Cdc45p and replication protein A (RPA) bind to Mcm2p
at the G1-S transition in an S-CDK-dependent manner. During
S phase, Cdc45p associates with different replication origins at
specific times. The origin associations of Cdc45p and RPA are mutually
dependent, and both S-CDKs and Cdc7p-Dbf4p are required for efficient
binding of Cdc45p to origins. These findings suggest that S-CDKs and
Cdc7p-Dbf4p promote loading of Cdc45p and RPA onto a preformed
prereplication complex at each origin with preprogrammed timing. The
ARS1 association of Mcm2p, but not that of the origin recognition complex, is diminished by disruption of the B2 element of
ARS1, a potential origin DNA-unwinding element. Cdc45p is
required for recruiting DNA polymerase In eukaryotic cells, DNA replication
initiates from multiple origins scattered along chromosomes, making
duplication of large genomes within a single cell cycle possible. Every
replication origin fires once and only once with a characteristic
timing during the S phase. Although the cell-cycle control of DNA
replication has been gradually unveiled, how DNA replication is
initiated at chromosomal origins remains largely unknown (reviewed in
references 16, 21, 46, and 57).
Studies of Saccharomyces cerevisiae and other organisms have
revealed that a prereplication complex (pre-RC) is assembled at each
origin during late mitosis or early G1 phase, the period when cyclin-dependent kinases (CDKs) are inactive (11, 12, 15, 40,
51, 58, 59). The origin recognition complex (ORC), Cdc6p, and the
six minichromosome maintenance (MCM) proteins Mcm2p to -7p are present
in the pre-RC (2, 19, 40, 55, 59, 63). To start DNA
synthesis, S-phase CDKs (S-CDKs) and Cdc7p-Dbf4p kinase need to be
activated at the G1-S transition. Upon activation of these
kinases, the pre-RC undergoes a transformation, and it is eventually
converted to the post-RC and replication forks when initiation of DNA
replication is completed (16, 21, 46, 57). To understand how
DNA replication is initiated at chromosomal origins, we sought to
uncover how the complexes at origins change during initiation and how
they interact with origins. Furthermore, we pursued the mechanisms by
which the activating kinases are involved in this transition.
In the well-characterized simian virus 40 (SV40) system, large T
antigen is the origin recognition protein and also the helicase. Initiation of DNA replication from the SV40 origin begins with local
unwinding of the origin by cooperative action of large T antigen and
replication protein A (RPA; reviewed in references 29 and 62). Subsequently, DNA
polymerase The architecture of the complex at origins remains largely unknown.
With the exception of the ORC, little is known about how the proteins
at origins interact with origin DNA. A single leading-strand start site
was recently identified at the chromosomal autonomously replicating
sequence 1, (ARS1), a well-characterized yeast replication origin (6). This site is located between the binding site of the ORC (the A and B1 elements of ARS1 [5, 52,
54]) and a potential DNA-unwinding element (the B2 element of
ARS1 [41]). It is likely that this site
coincides with the site of initial unwinding at ARS1 and/or
the entry site for the replication machinery. However, the proteins
associating with this region of ARS1 have not been identified.
In this study, we show that Cdc45p, RPA, and Mcm2p start to associate
with each other at the onset of S phase. Cdc45p associates with
different replication origins at specific times during S phase, and the
origin associations of Cdc45p and RPA are mutually dependent. Both
S-CDKs and Cdc7p-Dbf4p are required for efficient binding of Cdc45p to
replication origins. Together, these findings suggest that S-CDKs and
Cdc7p-Dbf4p promote formation of a complex containing Cdc45p, RPA, and
the MCM proteins at each origin with preprogrammed timing. Association
of Mcm2p with ARS1 requires the potential unwinding element
B2. Cdc45p is required for loading Pol Yeast strains and methods.
All the yeast strains used in
this study are listed in Table 1. The
Cdc45HA3p and Cdc45myc3p in the corresponding strains were expressed
from their endogenous promoters at the original chromosomal location
and can support normal cell growth. The integrated DBF4MYC18
in YB0552 was derived from the strain K6388 provided by K. Nasmyth
(60). The GAL1-SIC1
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Assembly of a Complex Containing Cdc45p, Replication Protein
A, and Mcm2p at Replication Origins Controlled by S-Phase
Cyclin-Dependent Kinases and Cdc7p-Dbf4p Kinase
and
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
onto chromatin, and it
associates with Mcm2p, RPA, and DNA polymerase 
only during S phase.
These results suggest that the complex containing Cdc45p, RPA, and MCMs is involved in origin unwinding and assembly of replication forks at
each origin.
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
-primase is recruited to the unwound origin to synthesize
the first Okazaki fragment, and then the DNA polymerase 
complex is
loaded by a polymerase switching mechanism (62). Unlike the
SV40 large T antigen, yeast ORC is not a helicase. Instead, as
indicated by amino acid sequence composition and a study of three of
the six human MCM proteins (32, 38), yeast MCMs might
function as a helicase. The MCM proteins, but not RPA, are present at
origins during G1, and no origin firing can be detected
until S-CDKs and Cdc7p-Dbf4p are activated at the G1-S
transition. Three recent studies have suggested that Cdc45p and RPA
might mediate the activation of origins by the kinases (45, 60,
67). Cdc45p, a protein essential for initiation of DNA
replication (26, 30, 49, 66), binds to MCMs and chromatin
upon the activation of S-CDKs (45, 67). Association of RPA
with origins depends on both S-CDKs and Dbf4p-dependent kinase
(60). Moreover, Cdc45p is required for recruiting DNA polymerase (Pol) onto chromatin in Xenopus
(45), and RPA is needed to recruit primase to origins in
yeast (60).
onto chromatin, and it
associates with DNA polymerase (Pol) during S phase. We propose,
therefore, that Cdc45p, RPA, and the MCM proteins collaborate to unwind
origins and recruit other proteins to assemble replication elongation complexes.
MATERIALS AND METHODS
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
NT in YB0553 was derived
from YCS12 provided by J. Diffley (14). The integrated
POLHA3 in YB0550 and YB0551 was derived from OAy618
provided by S. Bell (2). The integrated ARS1
mutations were derived from the corresponding strains published by
Marahrens and Stillman (44). In all the experiments
involving temperature-sensitive mutant strains, -factor arrest was
performed at 25°C for 3 h. In the experiments using only
non-temperature-sensitive strains (including the cold-sensitive cdc45-1 strain), cells were arrested in -factor at 30°C
for 3 h. Expression of Sic1NTp was either induced in yeast
extract-peptone medium with 2% galactose or remained uninduced in 2%
raffinose.
TABLE 1.
S. cerevisiae strains used in this study
Chromatin immunoprecipitations. Chromatin immunoprecipitations were conducted essentially as previously described (2). Cdc45HA3p, Mcm2p, Orc2p, Orc3p, and RPA p70 were immunoprecipitated by the monoclonal antibodies 12CA5, Mcm2-49, SB3, SB46, and a polyclonal rabbit anti-serum against RPA p70, respectively. All the PCR products were between 200 and 350 bp in size, and were separated on 2.3% agarose gels. The sequences of PCR primers used in this study are available upon request.
Chromatin binding analysis. Preparation and fractionation of cell lysates were performed as described previously (40) with several modifications. The incubation in prespheroplasting buffer was done on ice. Spheroplasts were washed three times with lysis buffer and lysed in the presence of 1% Triton X-100. The quality of fractionation was monitored by the separation of chromatin-bound Orc3p from the soluble protein cross-reacting with the SB3 antibody (anti-Orc3p [40]).
Immunoprecipitation and immunoblotting.
Immunoprecipitations
were carried out essentially as described previously (30)
except that 150 Kunitz units of DNase I (Sigma) was added to the
whole-cell lysates. Cdc45myc3p, Mcm2p, and RPA p70 were
immunoprecipitated with monoclonal antibodies 9E10, Mcm2-49, and rabbit
anti-serum against RPA p70, respectively. The antibodies used for
immunoblotting were monoclonal 12CA5 (for PolHA3p or Cdc45HA3p),
9E10 (for Cdc45myc3p or Dbf4myc3p), 6D2 (for Pol p86), Mcm2-28, SB3
(for Orc3p), and polyclonal antisera against RPA p70 and p34.
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Cdc45p associates with Mcm2p, RPA, and Pol in a cell
cycle-regulated manner.
The binding of Cdc45p to chromatin and the
association of Cdc45p with the MCM proteins indicate the transformation
from the pre-RC to a preinitiation complex (pre-IC
[67]). To test whether Cdc45p and MCMs were part of a
larger complex, we immunoprecipitated Cdc45p and Mcm2p, respectively,
and analyzed the proteins coprecipitated with them (Fig.
1). Cells were treated with hydroxyurea
(HU) to slow down fork movement and prolong the period in which the
potential complex might be present. Because the potential complex was
likely chromatin bound, DNase I was used to release the possible
interacting proteins from chromatin. Immunoprecipitation of either
myc-tagged Cdc45p or intact Mcm2p specifically brought down the
hemagglutinin (HA)-tagged Pol catalytic subunit and intact RPA p70,
the largest subunit of RPA (Fig. 1A and C and data not shown).
Similarly, precipitation of RPA p70 brought down Cdc45p, Mcm2p, and
Pol (Fig. 1B). Furthermore, Cdc45p was detected in the Pol
immunoprecipitate (data not shown). These interactions were also
observed in the absence of HU (Fig. 2 and
data not shown). It is unlikely that these interactions were mediated
by chromatin because several other chromatin-bound proteins were not
detected in the immunoprecipitates (data not shown). The network of
interactions among Cdc45p, Mcm2p, RPA, and Pol suggest that these
proteins are present in a complex or multiple complexes in vivo. It
should be noted that only small fractions of Pol and RPA
coprecipitated with Cdc45p in these experiments. This could be due to
the transient and/or unstable nature of the complex or the inefficiency
of the coimmunoprecipitation procedure. When Mcm2p was precipitated
from an extract derived from mcm2-1 cells, hardly any RPA
p70, Cdc45p, or Pol were brought down even at the permissive
temperature (Fig. 1C and D and data not shown). Thus, the anti-Mcm2p
antibody did not recognize RPA p70, Cdc45p, and Pol directly, and
more importantly, functional Mcm2p was critical for the stability of
the complex. Although an interaction between Cdc45p and Pol was
reported in Xenopus (45), we were unable to
detect Pol in the Cdc45p immunoprecipitate, perhaps because the
interaction was very transient.
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were regulated during the cell cycle, we monitored these interactions
across a complete cell cycle. Cells expressing both Cdc45myc3p and
PolHA3p were synchronously released from an -factor block, and
Cdc45myc3p was immunoprecipitated at 10- or 15-min intervals (Fig. 2A
and B). All the proteins examined in this experiment, including Cdc45p,
Mcm2p, Pol, and both the p70 and p34 subunits of RPA, were present
at roughly constant levels throughout the cell cycle (data not shown).
Cdc45myc3p was precipitated throughout the cell cycle, with a modest
increase in S phase. Mcm2p and both the p70 and p34 subunits of RPA
coprecipitated with Cdc45p during early S phase, whereas Pol was
brought down throughout S phase. Importantly, both the Cdc45p-Mcm2p and
the Cdc45p-RPA interactions were established 20 to 30 min after
release, concomitant with the onset of S phase. A slower-migrating form
of RPA p34 was observed when cells reached early S phase, indicating
the cell cycle-regulated phosphorylation of this protein. Nevertheless,
both forms of RPA p34 were brought down by Cdc45p, suggesting that the
phosphorylation of RPA p34 may not affect its association with Cdc45p.
Mcm2p, Pol, and RPA p70 and p34 reappeared in the Cdc45p
immunoprecipitate 120 min after release. It is likely that some cells
had started the second S phase by that time.
Association of Mcm2p with Cdc45p and RPA requires active
S-CDKs.
Association of Cdc45p with chromatin requires activation
of S-CDKs, indicating that S-CDKs regulate a key step of the assembly of the pre-IC (67). To test directly whether S-CDK activity was required for establishing the complex containing Cdc45p, RPA, and
Mcm2p, we examined the effect of overexpression of Sic1p, an S-phase
CDK inhibitor, on the interactions among these three proteins.
Sic1NTp, a mutant of Sic1p that lacks the N-terminal 50 amino acids,
is more stable than the wild-type Sic1p and is functional in inhibiting
S-phase CDKs (14). Cells overexpressing Sic1NTp from the
GAL1,10 promoter were synchronized in G1 with -factor and were released into either raffinose- or
galactose-containing medium (Fig. 3). In
raffinose-containing medium, Cdc45p and both p70 and p34 of RPA
coprecipitated with Mcm2p as cells entering S phase (Fig. 3). In
contrast, cells were stalled at the G1-S boundary in
galactose, and neither Cdc45p nor RPA coprecipitated with Mcm2p. Thus,
Cdc45p and RPA could not interact with Mcm2p when S-CDKs were
inactivated by overexpression of Sic1NTp.
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Cdc45p and Mcm2p associate differently with replication origins. To analyze the distribution of the pre-IC on chromatin, we carried out a chromatin immunoprecipitation (CHIP) assay on Cdc45p. In this assay, proteins were cross-linked in vivo to chromatin with formaldehyde, the chromatin was then sheared, and the bound Cdc45p was immunoprecipitated. To detect DNA sequences that were associated with Cdc45p, specific primer pairs were used to analyze coprecipitated DNA by PCR.
A recent study using the CHIP assay suggested that Cdc45p was a component of the pre-RC in G1 (2). However, we were unable to detect association of Cdc45p with chromatin or with MCMs at Start in G1 (67). To determine whether Cdc45p was indeed a component of the pre-RC, we first compared the origin association of Cdc45p with that of Mcm2p, a known pre-RC component, by using CHIP assays. CHIP analyses of Cdc45HA3p and Mcm2p were performed in parallel with cells collected either at the-factor block in
G1 or at the beginning of S phase (30 min after release
from the -factor block). Coprecipitated DNA fragments were amplified
for ARS1, ARS501, ARS301, and a
nonorigin control region (Fig. 4A). Both ARS1 and ARS501 are active origins, but
ARS1 fires early whereas ARS501 fires late in S
phase (24). ARS301 only fires at low frequency
(<15%) at its chromosomal location (20). No clear enrichment of Cdc45p was detected at these three origins in
G1 (Fig. 4A). In early S phase, Cdc45p was enriched at
ARS1 but not ARS301 or ARS501. On the
other hand, Mcm2p was found to associate with ARS1,
ARS301, and ARS501 in both G1 and
early S phase (Fig. 4A). In addition, Mcm2p associated with all three
origins equally well. Thus, the association of Mcm2p with origins was
temporally different from that of Cdc45p. Unlike Cdc45p, Mcm2p binding
did not distinguish early and late origins, nor did it differentiate between active and inactive origins. Nevertheless, Cdc45p and Mcm2p
coexisted on ARS1 in early S phase, suggesting that they might be present in a complex at this origin during initiation.
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8
kb, regions on both sides of ARS1 that are 4 or 8 kb away.
(C) DNA content of the time point samples used in panels B and D. (D)
Association of Cdc45p with ARS1 is delayed in the absence of
CLB5 and CLB6. YB0477 (clb5,6Cdc45p associates with different replication origins in a
temporally controlled manner.
To investigate the cell-cycle
regulation of the association of Cdc45p with different origins, we
carried out CHIP assays on Cdc45HA3p across the cell cycle. Wild-type
cells were synchronized in G1 with -factor and then
released into the cell cycle (Fig. 4C). The DNA fragments
coprecipitated with Cdc45p were analyzed by PCRs using four pairs of
primers that amplified either ARS1 or one of the three
flanking regions (Fig. 4B). In the -factor-arrested cells, none of
the four chromosomal loci was significantly enriched in the Cdc45p
immunoprecipitate. Twenty minutes after release, Cdc45p began to
associate with ARS1 but not the surrounding regions. The
specific enrichment of Cdc45p at ARS1 was most evident 30 min after release, when the cells were clearly in early S phase. As the
cells proceeded through S phase, the enrichment of Cdc45p at
ARS1 declined. Simultaneously, Cdc45p became associated with the regions flanking ARS1. When S phase was complete in most
cells, Cdc45p could no longer be detected at ARS1 and its
flanking regions. Similar observations were also made in the cells
synchronously released from a dbf2 block in mitosis (data
not shown). These results show that Cdc45p specifically associates with
ARS1 in early S phase rather than in G1 as
suggested by the earlier study (2). However, our data agree
with the observation that at least some Cdc45p may move away from
origins after initiation (2).
-factor. Only 10 min after release, weak association of Cdc45p
with ARS305 and ARS607 was detected. By 20 min,
Cdc45p was clearly bound to the two early origins. Cdc45p bound to
these two early origins approximately 5 to 10 min before it bound to
ARS1, which correlated well with the timing of initiation at
these origins (25, 53, 65). Similar analyses were also performed with two late origins, ARS501 and
ARS603 (Fig. 4B). Cdc45p began to associate with these late
origins 40 min after release, more than 10 min after it bound to
ARS1. This is also in agreement with the timing of
initiation at these loci (24, 25, 65). Persistent
association of Cdc45p with ARS1 and late origins were
observed even in late S phase. This is possibly due to the rapid loss
of cell cycle synchrony after cells have entered S phase. Together,
these results show that Cdc45p binds to early-firing origins early and
late-firing origins late in S phase, suggesting that binding of Cdc45p
to each origin is concomitant with initiation at that origin.
Association of Cdc45p with ARS1 requires S-CDK
activity.
Binding of Cdc45p to chromatin is delayed in
clb5,6 mutant cells due to the lack of S-phase cyclins
(67). To test whether the specific association of Cdc45p
with ARS1 was also postponed in the absence of
CLB5 and CLB6, we performed CHIP analysis on Cdc45p in the clb5,6 cells synchronously released from an
-factor block in G1 (Fig. 4D). As indicated by the
fluorescence-activated cell-sorting analyses, clb5,6
mutant cells entered S phase approximately 50 min after release, which
was delayed by about 30 min relative to the wild type (Fig. 4C).
Specific Cdc45p-ARS1 association was first detected 50 min
after release, concomitant with the apparent G1-S
transition (Fig. 4C and D). Compared with that in the wild-type cells,
binding of Cdc45p to ARS1 in the clb5,6 cells
was delayed for approximately 30 min. This is in good agreement with
the delay of ARS1 firing in the same mutant (18).
Because only the cell cycle events relying on S-CDKs are delayed in the
clb5,6 mutant, this result shows that the specific
association of Cdc45p with ARS1 requires S-CDK activity.
Association of Cdc45p with chromatin, Mcm2p, and ARS1
is affected in cdc7 and dbf4 mutants.
Significant amounts of Cdc45p were detected on chromatin in the
cdc7-1 cells that were arrested by shifting asynchronously growing cultures to 37°C (67). However, two recent studies
showed that Cdc7p-Dbf4p was important not only for entry into S phase but also for progression through S phase (7, 17). Therefore, inactivation of Cdc7p-Dbf4p in an asynchronous population might trap
early S-phase cells in S phase, and the origins that had fired in these
cells could be responsible for the detection of chromatin-bound Cdc45p.
To assess this possibility, we examined the chromatin association of
Cdc45p in the arrested cdc7-1, cdc7-4, and
dbf4-1 cells that had been synchronized in G1
prior to the temperature shift (Fig. 5A).
The mutants and the congenic wild-type cells were released from an
-factor block at either 25 or 35°C and were given 30 min to enter
S phase or reach the arrest point at the G1-S boundary
(data not shown). At 25°C, cdc7-4 cells entered S phase
more slowly than wild-type, cdc7-1, and dbf4-1
cells. The amount of Cdc45p on chromatin in cdc7-4 cells was
also smaller than that in the other three strains (Fig. 5A and data not
shown). At 35°C, all three mutant strains were stalled at the
G1-S boundary 30 min after release from -factor (data
not shown). Chromatin-bound Cdc45p was hardly detectable in
cdc7-4 cells and was significantly reduced in
cdc7-1 cells, but the reduction of chromatin-bound Cdc45p in
dbf4-1 cells was very modest (Fig. 5A).
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-factor and
then released at 37°C so that all mutant cells were arrested at the
G1-S boundary (Fig. 5B). Compared to that in the wild-type
cells, the Cdc45p-Mcm2p interaction was reduced to various extents in
the three mutants (Fig. 5B). cdc7-4, which entered S phase
more slowly than the other strains even at 25°C, exhibited the most
dramatic reduction of Cdc45p-Mcm2p interaction at 37°C. Overall, the
reduction of Cdc45p-Mcm2p interaction in the cdc7 and
dbf4 mutants correlated well with the reduction of
chromatin-bound Cdc45p in these mutants (Fig. 5A and B and data not
shown). These results suggest that Cdc7p-Dbf4p is important, if not
essential, for the association of Cdc45p with Mcm2p and chromatin.
To investigate directly the effect of inactivation of Cdc7p-Dbf4p on
origin association of Cdc45p, we performed CHIP assays on Cdc45p in
cdc7-1, cdc7-4, and dbf4-1 mutant
cells. Wild-type and mutant cells were synchronized with -factor and
released at 35°C (Fig. 5C and D). Cdc45p specifically bound to
ARS1 when the wild-type cells reached early S phase. In
contrast, no enrichment of Cdc45p could be detected at ARS1
in all three mutants arrested at the G1-S boundary (Fig.
5C). Mcm2p, on the other hand, remained bound to ARS1 in the
arrested cdc7 and dbf4 cells (data not shown), indicating that ARS1 did not fire efficiently. Thus,
Cdc7p-Dbf4p appeared to be essential for the Cdc45p-ARS1 interaction.
ARS1 associations of Cdc45p and RPA are mutually
dependent.
Both Cdc45p and RPA require S-CDKs and Cdc7p-Dbf4p
kinase to associate with replication origins (Fig. 4 and 5)
(60). We therefore investigated whether there was an
interdependence between Cdc45p and RPA for their binding to origins. A
cold-sensitive cdc45-1 mutant and the congenic wild-type
cells were synchronized with -factor and released at 13°C, at
which temperature cdc45-1 cells were stalled at the
G1-S boundary (Fig. 6B). To
detect origin association of RPA in these cells, we carried out CHIP
analysis with an antibody against RPA p70. Within 2 h after
release at 13°C, wild-type cells had entered S phase and specific
association of RPA p70 with ARS1 was detected (Fig. 6A). In
contrast, RPA p70 did not bind to ARS1 in the
cdc45-1 cells stalled at the G1-S boundary. To
determine whether RPA was required for loading Cdc45p onto origins, we
synchronized wild-type and rfa2-2 (a temperature-sensitive mutant of RPA p34) cells with -factor and then released them at
35°C (Fig. 6D). Cdc45p bound to ARS1 in the wild-type
cells 20 min after release, but it did not do so in the
rfa2-2 cells stalled at the G1-S boundary (Fig.
6C). Hence, the ARS1 association of Cdc45p and RPA were
interdependent, indicating that Cdc45p and RPA were recruited to this
origin at roughly the same time. Since RPA is required for origin
unwinding in the SV40 system, these results raise the possibility that
the complex containing Cdc45p, RPA, and the MCM proteins is involved in
unwinding the origins.
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Cdc45p is required for loading Pol p86 but not Dbf4p onto
chromatin.
Consistent with the idea that the pre-IC is involved in
origin unwinding, it was shown in Xenopus that Cdc45p was
required for association of Pol with chromatin (45). To
investigate whether such a mechanism was conserved in budding yeast, we
analyzed chromatin association of Pol in the cold-sensitive
cdc45-1 mutant and wild-type cells released from an
-factor block at 13°C (Fig. 7B). As
previously described, cell lysates were fractionated into supernatants
and chromatin-enriched sediments (Fig. 7A). In the -factor-arrested
cells, Pol p86 was virtually undetectable on chromatin. As wild-type
cells entered S phase, the level of chromatin-bound Pol p86
increased. However, such an increase was hardly detectable in the
cdc45-1 cells stalled at the G1-S boundary.
Thus, association of Pol with chromatin requires Cdc45p function in
budding yeast.
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-factor block at 13°C, Dbf4p bound to chromatin in both wild-type
and cdc45-1 cells with similar kinetics (Fig. 7A).
Therefore, the cdc45-1 allele is not defective for loading
Dbf4p onto chromatin, even though it is incompetent to recruit RPA and
Pol.
The B2 element of ARS1 is involved in association with
Mcm2p.
The association of the ORC with ARS1 requires
the A and B1 but not the B2 element of the origin (5, 52,
54). Disruptions of either A or all three B elements abolished
association of Mcm7p with ARS1 (59). However,
none of these studies identified the proteins that associate with B2,
the potential DNA-unwinding element. If the complex containing Cdc45p,
RPA, and Mcm2p were involved in origin unwinding, then one would
predict that it should be present in close proximity to B2. To test
this possibility, we performed CHIP analyses on Mcm2p with cells in
which the chromosomal ARS1 was mutated at either A or one of
the three B elements (44). The association of Mcm2p with
ARS1 was abolished in the A
mutant, just like
the association of the ORC (Fig. 8). In
the B1 mutant, although no ORC was detected at the mutant
origin, a weak Mcm2p-ARS1 association was observed by CHIP
(Fig. 8). Unlike mutations of the essential A element, disruption of
the B1 element only weakened but did not abolish the origin activity of
ARS1 (43). It is likely that the ORC could
transiently associate with the B1 ARS1 and
recruit some MCM proteins to this mutant origin. Interestingly, disruption of the B2 element weakened the association of Mcm2p with
ARS1 but not the association of the ORC with this mutant origin (Fig. 8). Therefore, although the B2 element of ARS1
is not required for ORC binding, it is involved in the stable
association with Mcm2p and perhaps Cdc45p and RPA.
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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In budding yeast, initiation of DNA replication is accomplished by a number of sequential transitions at origins. The first transition is to establish the pre-RC at replication origins in late mitosis or early G1, when CDKs are inactive (11, 12, 19, 40, 51, 58, 59). During late G1 and S phases, a series of less-understood transitions occur at individual origins, and DNA replication is launched from different origins at specific times. Two of the critical events that take place during the transitions at each origin are the unwinding of origin DNA and the assembly of replication elongation complexes. Accompanying these events, the pre-RC at each origin undergoes a transformation and is eventually converted to the post-RC and replication forks. Here we have uncovered a number of events that are part of this transformation, thereby shedding light on the mechanisms of origin unwinding and replication fork assembly at eukaryotic chromosomal origins.
The transformation of the pre-RC was first detected through the disappearance of Cdc6p from chromatin and origins at the G1-S transition (11, 31, 59, 63). At about the same time, Cdc45p, RPA, and Mcm2p start to associate with each other in vivo (Fig. 1 to 3) (13, 30, 67). CHIP experiments show that both Cdc45p and RPA begin to associate with early-firing origins at this moment of the cell cycle (Fig. 4 and 5) (60). Mcm2p, a component of the pre-RC and one subunit of the MCM complex, is also present at origins at this time (Fig. 4) (2, 59). It was shown that Cdc45p required Mcm2p to associate with chromatin and that RPA depended on Mcm5p to bind to origins (60, 67). We show here that the origin associations of Cdc45p and RPA are mutually dependent. Together, these findings suggest that Cdc45p and RPA bind to the MCM proteins at origins at the onset of S phase and that together they form the pre-IC. Interestingly, we find that binding of Cdc45p to replication origins is temporally controlled. Cdc45p binds to early-firing origins early and late-firing origins late in S phase, and the timing of Cdc45p binding at each origin correlates well with the timing of initiation at that origin. Since RPA associated with early- but not late-firing origins in the presence of HU, it was postulated that the timing of RPA binding differed between early- and late-firing origins (60). Given that Cdc45p and RPA are both essential for initiation and that their binding to each origin coincides with firing of the origin, the formation of the pre-IC is likely a key step for origin activation.
Unlike the formation of the pre-RC in late mitosis or early G1, the later transitions at origins require activation by CDKs and Cdc7p-Dbf4p. The opposite requirement for CDKs ensures that these transitions always occur after the pre-RC is formed. Several recent studies suggested that S-CDKs and Cdc7p-Dbf4p might function at origins (7, 17, 18). Although a number of proteins present in the pre-RC appear to be substrates of CDKs or Cdc7p in yeast or other organisms (8, 22, 34, 35, 39, 42, 50, 56, 64), how these phosphorylations trigger the transitions remains to be addressed. We have previously shown that Cdc45p was associated with chromatin upon activation of S-CDKs, suggesting that Cdc45p could play an important role in mediating the origin activation by S-CDKs (67). Here we show that S-CDKs are indeed required for the association of Cdc45p with ARS1 and most likely other origins in a temporally specific manner. Furthermore, S-CDK activity is needed for establishing the interactions among Cdc45p, RPA, and Mcm2p. In addition to S-CDKs, Cdc7p-Dbf4p is also required for efficient binding of Cdc45p to ARS1. Likewise, binding of RPA to origins also requires S-CDKs and Dbf4p-dependent kinase (60). Therefore, formation of the pre-IC at individual origins may be the key event during origin activation that is controlled by S-CDKs and Cdc7p-Dbf4p. Because inactivation of Cdc7p-Dbf4p in early S phase leads to silencing of late-firing origins, Cdc7p-Dbf4p probably executes its function at individual origins in a temporally controlled manner (7, 17). The requirement for Cdc7p-Dbf4p in loading Cdc45p and RPA onto origins perhaps contributes to formation of the pre-IC at each origin, with a characteristic timing during S phase.
We noticed that although the Cdc45p-ARS1 association was not detected by CHIP in the arrested cdc7 and dbf4 cells, binding of Cdc45p to Mcm2p and chromatin was only weakened but not abolished (Fig. 5). Unlike chromatin fractionation and Mcm2p coimmunoprecipitation, CHIP analysis examines the enrichment of Cdc45p at a single replication origin. It is possible that in the arrested cdc7 and dbf4 cells, Cdc45p associated with origins other than ARS1 and/or even nonorigin sequences. Alternatively, when Cdc7p-Dbf4p activity was limited, the enrichment of Cdc45p at each origin might not be sufficient to be detected by CHIP. When combined, even the inefficient binding of Cdc45p to a number of origins could give detectable amounts of chromatin-bound Cdc45p and Cdc45p-Mcm2p interaction. Since we have shown that Cdc45p could interact weakly with Mcm2p off the chromatin (67), Cdc7p-Dbf4p might be required for recruiting Cdc45p to origins and establishing a more stable Cdc45p-MCM interaction.
The conclusion of Aparicio et al. (2) that Cdc45p is a
component of the pre-RC must be reassessed. We did not find Cdc45p on
chromatin until the S-CDKs were activated (67), and here we
show that Cdc45p associates with individual origins in a temporally regulated manner during S phase. Indeed, Aparicio et al. (3) agree with this result for most origins, except for the very early origins. We find that, even at the earliest origins, Cdc45p is loaded
after the MCM proteins are loaded, the latter step being the one that
best defines pre-RC formation and which even occurs upon exit from
mitosis (15). Furthermore, since we have used CHIP and
direct chromatin binding, as well as coimmunoprecipitation with MCM
proteins, we suggest that the differences are unlikely to be due to
different methods (as suggested by Aparicio et al. [3]). Nevertheless, we agree that Cdc45p is required
for Pol loading and that it might move away from origins after initiation.
How does the pre-IC interact with origins? Disruption of the B2 element
of ARS1 diminished its origin activity but did not affect
ORC-ARS1 binding (Fig. 8) (43, 52). In contrast,
we found that the Mcm2p-ARS1 association was weakened in the
B2 mutant, suggesting that the B2 element might be
specifically involved in the association with the MCMs. Since only the
8-bp linker substitution in the B2 element, but not the
single-base-pair changes in this region, affected the origin activity
of ARS1 (43), the B2 element is likely a
structural element rather than a sequence-specific binding site for
proteins. Indeed, it has been suggested that the B2 element is a
DNA-unwinding element (41), and it is very close to the site
of initiation of leading-strand DNA replication (6).
Therefore, we suggest that the MCMs preferentially associate with the
easily unwound sequences (such as the B2 element of ARS1) adjacent to the ORC binding sites. The low helical stability and the
position relative to the ORC binding site, but not the actual DNA
sequence, might be important for this association. Furthermore, Cdc45p
and RPA may be recruited to these regions by the MCMs to form the
pre-ICs.
What, then, is the function of the pre-IC at origins? In the SV40
system, RPA binds to the origin that is locally "melted" by T
antigen, and then it stimulates the helicase activity of T antigen and
stabilizes the unwound origin (29). Subsequently, T antigen,
RPA, and the unwound origin recruit Pol-primase to form a complex
called the primosome (62). At chromosomal origins, the MCM
proteins might function as a T-antigen-like helicase. A helicase
activity was reported to be associated with a complex of human Mcm4p,
Mcm6p, and Mcm7p (32). Six fission yeast MCM proteins form a
globular complex in vivo (1). Our data suggest that the MCM
complex might associate with the B2 element of ARS1, a
potential DNA-unwinding element (41). However, unlike SV40 T
antigen, which can "melt" the origin by itself, it is unlikely that
MCM proteins can unwind origins in G1 when they are present in the pre-RC. If the MCM complex functions as a helicase, then its
activity might need to be stimulated by S-CDKs and Cdc7p-Dbf4p at the
G1-S transition. Together with the findings by Tanaka and Nasmyth (60), our results show that S-CDKs and Cdc7p-Dbf4p
promote the binding of Cdc45p and RPA to the MCM proteins at origins. From genetic evidence, both Cdc7p-Dbf4p and Cdc45p have been implicated in the activation of MCMs. A mutant allele of MCM5 called
bob1 can suppress the lethality caused by deletions of
either CDC7 or DBF4 (27, 33). The loss
of Cdc45p function in the cdc45-1 mutant can be bypassed by
cdc46-1 and cdc47-1, two mutant alleles of
MCM5 and MCM7 (28). Furthermore, RPA
can stimulate the helicase activity of T antigen in the SV40 system,
indicating that it might also be involved in the activation of MCMs in
yeast (29). Therefore, it is plausible that Cdc45p and RPA
collaborate to activate the MCM helicase and/or to increase its
processivity. Consistent with their potential roles in origin
unwinding, Cdc45p is required for loading Pol onto chromatin, and
RPA is needed for recruiting primase to origins (Fig. 7) (45,
60).
The function of Cdc45p may not be restricted to origin unwinding and
assembly of replication forks. We find that Cdc45p associates with
Mcm2p, RPA, and Pol during S phase. Unlike Pol and Pol, Pol
is not required for in vitro replication of SV40 DNA (62). Consistent with this, it was recently shown that Pol polymerase activity was not essential in yeast (37). Nevertheless, the carboxyl terminus of Pol is essential for viability, and it may function as a sensor of stalled replication forks (47). Two recent studies using CHIP analysis suggested that Cdc45p, RPA, MCMs,
and Pol might move away from origins after initiation (2, 60). Moreover, Xenopus Cdc45p was colocalized with
Pol throughout S phase (45). Together, these findings
argue that Cdc45p, RPA, and MCMs move with replication forks. Cdc45p,
together with RPA, may continue to stimulate MCM helicase activity
during replication elongation, or it may play an important structural
role at replication forks. In addition to chromosomal replication,
Pol and RPA are also implicated in the repair of UV-induced DNA
damage (9, 61). Furthermore, DPB11, a gene that
interacts genetically with both POL2 (encoding Pol) and
CDC45, is involved in the S-phase-induced checkpoint arrest
of entry into mitosis (4, 36). It will be interesting to
test whether Cdc45p plays a role in nucleotide excision repair and
replication-induced checkpoints.
Our results suggest that S-CDKs and Cdc7p-Dbf4p control the formation of the pre-IC at origins. However, it is still unclear how phosphorylation is involved in this process. Identification of the key substrates of the kinases will be crucial for solving the puzzle. Our findings also present us with new challenges. Are Cdc45p, RPA, and MCMs sufficient for origin unwinding? Does the formation of the pre-IC require an initial melting of origins? If so, then what melts the origins? Extensive biochemical investigations will be essential to address these questions.
| |
ACKNOWLEDGMENTS |
|---|
We thank K. Nasmyth, J. Diffley, S. Bell, O. Aparicio, and P. Plevani for yeast strains, M. Akiyama and P. Plevani for antibodies, O. Aparicio for a chromatin immunoprecipitation protocol, and X. H. Zou-Yang for critical reading of the manuscript.
This work was supported by a grant from the National Institutes of Health (GM45436 to B.S.).
| |
FOOTNOTES |
|---|
* Corresponding author. Mailing address: Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724. Phone: (516) 367-8383. Fax: (516) 367-8879. E-mail: stillman{at}cshl.org.
Present address: Department of Biochemistry, Baylor College of
Medicine, Houston, TX 77030.
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REFERENCES |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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