From the Department of Molecular and Developmental
Biology, Institute for Medical Science, The University of Tokyo, 4-6-1 Shirokanedai Minato-ku, Tokyo 108-8639, Japan, ¶ Imperial
Cancer Research Fund, 44 Lincolns Inn Fields, London WC2A 3PX,
United Kingdom, and
Core Research for Evolutional Science and
Technology (CREST), Tokyo 108-8639, Japan
Received for publication, January 2, 2001
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ABSTRACT |
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Assembly of replication complexes at the
replication origins is strictly regulated. Cdc45p is known to be a part
of the active replication complexes. In Xenopus egg
extracts, Cdc45p was shown to be required for loading of DNA polymerase
Chromosomal DNA replication requires a series of complex events
including recognition of origins, firing of replication origins, loading of DNA polymerases onto origins, and elongation of newly synthesized DNA. Initiation of DNA replication takes place only at
specific loci on the chromosomal DNA, replication origins (1-3). Origin recognition complexes, which have been shown to be
associated specifically with replication origins throughout the cell
cycle in budding yeast, may serve as hallmarks of the origins. Newly synthesized CDC6/Cdc18p temporally associates with origin recognition complexes at the G1-S boundary (4, 5). This association is
followed by the loading of minichromosome maintenance
(MCM)1 protein complexes onto
the replication origins, leading to the formation of prereplicative
complexes (preRCs) (6). After the MCM loading, Cdc6/Cdc18p is
phosphorylated by S-phase cyclin-dependent kinase (Cdk),
and this phosphorylation leads to the rapid degradation of CDC6/Cdc18p
to prevent reinitiation in yeasts (5, 7). Preceding the firing of
origins, MCM proteins in preRCs are phosphorylated by CDC7-DBF4 kinase
complexes, and CDC45 is loaded onto preRCs at the replication origins
(9-11). Finally, replication proteins, such as replication protein A
(single-stranded DNA-binding protein) and DNA polymerases, are
loaded onto replication complexes (12). After DNA synthesis is
initiated, some of the MCM components may leave the complexes,
resulting in postreplicative complexes.
The model above is based mostly on findings in budding yeast and in
Xenopus egg extracts. Although only limited analyses have been performed in other organisms, much of the data from these analyses
are consistent with it. However, considering that the origin structure
of budding yeast diverges from that of other eukaryotes, it would be
important to study the mechanisms of the initiation in other model
systems to clarify the precise mode of initiation in higher eukaryotes.
Fission yeast, in which the origin structures may share some similarity
with those of higher eukaryotes (13), may serve as another useful
model. In fission yeast, the genes encoding six MCM protein homologues
(14-18) and origin recognition complexes components have been
identified (19-21). In addition, homologues for cdc7,
dbf4, and cdc45 have also been identified
(22-25).
Although the formation of preRCs is relatively well understood in
budding yeast, how replication proteins are assembled into active
replication complexes remains mostly unclear in other organisms. DNA
polymerase In this study, we addressed these issues through the characterizations
of Sna41p in fission yeast. Fission yeast Pol Strains
The strains used were all derived from strain SP812
h Media
Edinburgh minimal medium and appropriate supplements were used
for all biochemical analyses.
Molecular Biology Techniques
Unless stated otherwise, all molecular biology techniques were
performed as described previously (45). Transformation of yeast strains
was performed essentially by the lithium acetate procedure (33).
Constructions of Strains Expressing Tagged Proteins
sna41HA3 tagged strain was constructed using a
sna41 Preparations of Cell Extracts
Whole Cell Extracts--
Harvested cells (1 volume) were washed
with 3 volumes of cold water three times and then washed by 3 volumes
of EB buffer (25 mM Hepes, pH 7.6, 50 mM NaCl,
5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 2 mM dithiothreitol, 0.25% Triton
X-100, and proteinase inhibitors) three times. The cells were then
resuspended in 1 volume of EB buffer containing NaCl at a concentration
indicated. After the addition of the same volume of acid-washed glass
beads, cells were broken by a bead beater. After removal of glass beads by filtration, the suspensions were further lysed by sonication and
then centrifuged in a microfuge at 15,000 rpm for 25 min. The
supernatants were harvested as whole cell extracts. Denatured whole
cell extracts were prepared by using DEB buffer (25 mM
Hepes, pH 7.6, 6 M urea, and 1% SDS) in place of EB buffer
and without the sonication step.
Chromatin-enriched Fractions--
Harvested cells were washed
with 3 volumes of cold water three times and with 3 volumes of SB
buffer (25 mM Hepes, pH 7.6, 1.2 M sorbitol, 50 or 150 mM NaCl, 5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, and 2 mM
dithiothreitol and proteinase inhibitors) three times. The cells were
then resuspended in the same volume of SB buffer containing Zymoryase
T100 (Seikagaku-Kogyo) and spheroplasted by a 20-min incubation at
30 °C. After cells were washed with 3 volumes of SB buffer three
times, Triton-soluble fractions were extracted by a 30-min incubation
at 4 °C in 2 volumes of EBT buffer (25 mM Hepes, pH7.6,
50 or 150 mM NaCl, 5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, and 2 mM dithiothreitol and proteinase inhibitors) followed by
centrifugation at 3,000 rpm for 5 min. The pellets were resuspended in
2 volumes of EBT, and Triton-insoluble fractions (chromatin-enriched
fractions) were extracted by sonication, followed by centrifugation at
15,000 rpm for 25 min to ensure the removal of any insoluble
aggregates. Immunoprecipitation experiments were performed basically as
described previously (32), with some modification. For all processes,
EB buffer (with 50 or 150 mM NaCl) was used as washing
buffer, and the 1:1 mixture of protein A- and protein G-Sepharose beads
was used for precipitation. Extracts were precleared by incubation with
Sepharose beads without antibody to suppress nonspecific absorption of
proteins to beads. To prepare DNase I-treated extracts, cell
suspensions were treated with 0.5 mg/ml DNase I for 15 min after glass
bead lysis and filtration. The breakage of DNA was confirmed by agarose
gel electrophoresis. The samples were centrifuged at 15,000 rpm for 25 min to obtain supernatants.
Immunofluorescence Studies
An spp1-GFPKan strain expressing Spp1p tagged at the
C terminus with the GFP was constructed by the PCR-based method
recently described in Ref. 47. The spp1-GFPKan PCR product
was transformed into h+/h Sna41p Physically Interacts with Pol Sna41p and Pol Sna41p Interacts with Mis5p Only in the Chromatin-enriched
Fractions in Early S Phase--
The physiological interactions between
CDC45 and MCM complex have been suggested in Saccharomyces
cerevisiae (8, 10). Therefore, we have examined the interaction
between Sna41HA3p and MCM complex. We examined one of the MCM proteins,
Mis5p/SpMcm6p (15, 38). First, we performed immunoprecipitation
experiments in exponentially growing cells. We observed
co-immunoprecipitation of Sna41HA3p and Mis5p with either anti-Mis5p
antibody (38) (data not shown) or anti-HA antibody (Fig.
1D). Because DNase I treatment did not affect the amount of
Mis5p immunoprecipitated by anti-HA antibody, the interaction between
Mis5p and Sna41HA3p is not mediated by DNA. Next, we analyzed the
interaction in cultures synchronized by HU. The interaction between
Sna41HA3p and Mis5p was cell cycle-regulated and correlated with the
septation index (Fig. 3). Because Schizosaccharomyces pombe
undergoes septation in G1-S phase, we concluded that the
interaction took place in S phase. To further examine this regulation,
we next tested the interaction in cell cycle-arrested cultures.
Sna41HA3 strain was arrested by various cell cycle mutants,
cdc10-129, cdc20-M10, cdc22-M45,
cdc23-M36 (35), pol sna41goa1 Mutant Has No Defect in Chromatin Loading of
Pol sna41goa1 Is Defective in Association of Pol
Recently, Griffiths et al. (39) isolated and
characterized S. pombe DNA primase 1, spp1,
encoding a subunit of DNA polymerase
We have already shown that Sna41HA3p interacts with Pol It was previously reported that DNA polymerase The Interaction between Sna41p and Mis5p is Cell Cycle- and
Localization-dependent--
In budding yeast, physical and
genetic interactions of CDC45 with MCMs have been reported (8, 10).
Although the interaction is known to take place at the G1-S
boundary, what exactly triggers this interaction has remained unclear.
We have found that Sna41p and Mis5p interact exclusively in the
chromatin-enriched fractions in S. pombe. At present, we
cannot completely rule out the possibility that this interaction is
mediated indirectly by DNA linking the two proteins. However, we think
this possibility is rather unlikely because the average sizes of DNA
are less than 300 base pairs after sonication of the Triton-insoluble
fractions and also because we detected the interaction in DNase
I-treated extracts (Fig. 1D).
It is probable that the interaction between these two proteins is
regulated by a third component such as Hsk1p-Dfp1/Him1 (22-24, 40),
S-phase Cdk, or unknown mediator(s). Two different bands of Mis5p were
detected on SDS-PAGE when extracts were prepared by the spheroplast
method. In addition, the upper band was maximized in the
sna41goa1-arrested fraction (Fig. 5B) and
minimized in cdc25 or cdc10 (data not shown).
Although we cannot conclude at this moment that this mobility shift is
due to phosphorylation, this possibility needs to be examined in the
future experiments.
Pol
The model can explain the recent finding in S. cerevisiae
that CDC45 and DNA polymerase onto chromatin. The fission yeast cdc45 homologue was identified as
a suppressor for nda4 and named sna41. Nevertheless, it is
not known how Cdc45p facilitates loading of DNA polymerase
onto
chromatin, particularly to prereplicative complexes. To gain novel
insight into the function of this protein in fission yeast, we
characterized the fission yeast Cdc45 homologue, Sna41p. We have
constructed C-terminally epitope-tagged Sna41p and Pol
p and replaced
the endogenous genes with the corresponding tagged genes. Analyses of
protein-protein interactions in vivo by the use of these
tagged strains revealed the following: Sna41p interacts with Pol
p
throughout the cell cycle, whereas it interacts with Mis5p/Mcm6p in
the chromatin fractions at the G1-S boundary through S
phase. In an initiation-defective sna41 mutant,
sna41goa1, interaction of Pol
p with Mis5p is not
observed, although Pol
p loading onto the chromatin that occurs
before G1 START is not affected. These results show
that fission yeast Sna41p facilitates the loading of Pol
p onto
minichromosome maintenance proteins. Our results are consistent
with a model in which loading of Pol
p onto replication origins
occurs through two steps, namely, loading onto chromatin at
preSTART and association with prereplicative complexes at
G1-S through Sna41p, which interacts with minichromosome maintenance proteins in a cell cycle-dependent manner.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(pol
), the only replicative polymerase equipped with
primase activity, plays essential roles in initiation at the origins
(26). The second largest subunit of DNA polymerase
, b subunit,
which undergoes cell cycle-regulated phosphorylation (27, 28), has been
suggested to have a regulatory role (29). In budding yeast,
unphosphorylated forms of b subunit accumulate only in
G1-arrested cells, and this phosphorylation appears to be
correlated with CDC6-independent Pol
loading onto the chromatin fraction (30), but the loading of Pol
onto preRCs depends on Cdc45p.
In Xenopus egg extracts, XCdc45, the Xenopus
CDC45 homologue, was shown to be required for Pol
loading onto the
chromatin under the control of S-phase Cdk kinases (31). These findings
in two different organisms are apparently inconsistent with each other with regard to the timing and dependence of the Pol
loading onto the chromatin.
p appears to undergo
Cdc18p-independent loading onto chromatin, and this step does not
require the function of Sna41p. Nevertheless, Sna41p is still required
for interaction of Pol
p with Mis5p/Mcm6p at the G1-S
boundary. These results provide evidence for a model of replication
complex assembly in which Pol
p is loaded onto preRCs in two steps.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
ade6-M210 ura4-D18 leu1-32.
strain, which will be described elsewhere
(49). Three HA epitopes, linked by a single glycine, were inserted just
before the termination codon of the sna41 open reading frame
by consecutive PCR method. The PCR fragment was directly
introduced into sna41
strain rescued by pREP3X-Sna41. The
transformed cells were plated on yeast extract with supplements
containing 5'-fluoroacetic acid. The 5'-fluoroacetic acid-resistant colonies, which could grow without pREP3X-Sna41 plasmid, were tested by PCR, genetic cross, and Western blot. Tandem
FLAG tags, linked by a single glycine, were introduced before the
termination codon of the pol
open reading frame as described
previously (18), except that pUC18 carrying a ura4 nutritional marker instead of a leu1 marker was used.
ade6-M216/ade6-M210 wild-type diploid strain, and
transformants were selected for kanamycin resistance. The transformant
was confirmed by Western blotting. For immunostaining with anti-GFP
antibody (a gift from K. Sawin), cells were grown in rich media, and
~2.5 × 108 cells were harvested, either by
centrifugation or filtration. Cells were fixed by the addition of 10 ml
of
80 °C methanol and processed for immunofluorescence essentially
as described previously (48). Secondary antibodies (Alexa; Molecular
Probes) were used at a 1:1,000 dilution, and cells were visualized
using a cooled charge-coupled device camera (Hamamatsu).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
p in Vivo--
It was
reported that, in Xenopus egg extracts, DNA Pol
p is
loaded onto chromatin through interactions with Cdc45p (31). To examine
this possibility, we first constructed strains in which triple
HA-tagged sna41 and tandem FLAG-tagged pol
replaced the endogenous genes, as described under "Experimental
Procedures" (the tags were at the C terminus in both cases). The
presence of the tags did not cause any growth defect in either the
sna41HA3 or pol
FLAG2 strains. The
tagged proteins could be detected by anti-HA and anti-FLAG antibodies
in the whole cell extracts (Fig. 1A). Sna41HA3p migrated at
around 70 kDa on SDS-PAGE, whereas Pol
FLAG2p appeared as a doublet
of 180- and 165-kDa proteins, as described previously (36). To confirm
that these bands actually represent the tagged proteins,
immunoprecipitates by antibodies against the HA or FLAG epitope were
immunoblotted by the antisera against Sna41p or Pol
p as well as by
the epitope antibodies. The same bands reacted with both antibodies
(data not shown). Utilizing these strains, we performed
immunoprecipitation experiments. When Sna41HA3p was precipitated by
anti-HA antibody, Pol
FLAG2p was co-immunoprecipitated (Fig.
1B). Only the upper band representing the intact p180
Pol
FLAG2p interacted with Sna41HA3p. Conversely, in the anti-FLAG
antibody immunoprecipitate, Sna41HA3p was present (Fig. 1C).
Because we sonicated the cells to solubilize chromatin-enriched fractions, it is possible that this interaction is mediated by DNA. To
eliminate this possibility, we also examined the interaction after
DNase I treatment. As shown in Fig. 1D, even more
Pol
FLAG2p was recovered in the anti-HA immunoprecipitate in DNase
I-treated extracts. Therefore, we have concluded that the interaction
between Sna41p and Pol
p is not mediated by DNA.
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Fig. 1.
Sna41p interacts with
Pol p in vivo.
A, expression of the tagged proteins. The integrated tagged
strains, pol
FLAG2 and sna41HA3,
were constructed as described under "Experimental Procedures."
Exponentially growing pol
FLAG2 or
sna41HA3 cells were harvested, and whole cell extracts were
prepared with EB buffer containing 500 mM NaCl or DEB
buffer, respectively. Extracts were run on SDS-PAGE and blotted with
anti-FLAG2 antibody (top panel) or with anti-HA antibody
(bottom panel). Left lane, tagged strain;
right lane, untagged wild-type strain. B,
Pol
FLAG2p co-immunoprecipitated with Sna41HA3p. Whole cell extract
of sna41HA3 polaFLAG2 strain was used for
immunoprecipitation with anti-FLAG antibody (lane 1) or with
anti-HA antibody (lane 2). Gel was blotted with anti-FLAG
antibody. C, Sna41HA3p co-immunoprecipitated with
Pol
FLAG2p. Cells were arrested for 3.5 h at 36 °C, whole
cell extracts were prepared, and immunoprecipitation was conducted with
anti-HA antibody (lanes 1 and 2) or with
anti-FLAG antibody (lanes 3 and 4). Western
blotting was conducted by anti-HA antibody. Tagged,
sna41HA3 pol
FLAG2 cdc20; Untagged,
sna41goa1 pol
FLAG2.
D, Sna41HA3p immunoprecipitation after DNase I treatment.
Whole cell extracts were treated by sonication (lanes 1 and
3) or with DNase I (lanes 2 and 4).
Lanes 1 and 2, 10% of the starting cell extract;
lanes 3 and 4, immunoprecipitates with anti-HA
antibody. The gels were blotted with anti-FLAG (top panel),
anti-Mis5 (middle panel), and anti-HA (bottom
panel) antibodies.
p Are Present at a Constant Level and Interact
with Each Other throughout the Cell Cycle--
In the synchronized
cultures of the sna41HA3 pol
FLAG2 strain
released from HU arrest, the levels of Pol
FLAG2p (32) and Mis5p (33) remained unchanged. The amount of Sna41HA3p also stayed constant regardless of cell cycle stage (Fig.
2A). We also examined the
amounts of Pol
FLAG2p, Mis5p, and Sna41HA3p in various cdc
mutants arrested at different cell cycle stages. We chose cdc10, cdc20, cdc22, cdc23,
pol
(cdc6), cdc17, and
cdc25 to obtain cells arrested at specific cell cycle
stages. cdc10 encodes a component of the transcription
factor essential for cdc18 expression and arrests the cell
cycle at G1 START. cdc20 encodes the DNA polymerase
catalytic subunit and arrests the cell cycle at the G1-S boundary just before the HU arrest point.
cdc22 encodes the large subunit of ribonucleotide reductase
and arrests the cell cycle at the point same as the HU arrest.
cdc23 is the homologue of budding yeast MCM10 and
arrests the cell cycle at S phase after the HU arresting point.
pol
encodes the large subunit of DNA polymerase
and
similarly arrests the cell cycle at S phase. cdc17 encodes
DNA ligase and arrests the cell cycle at the S-G2 boundary.
cdc25, encoding a protein phosphatase that activates Cdc2p,
arrests the cell cycle at G2-M. As shown in Fig.
2B, the amounts of Pol
FLAG2p, Mis5p, and Sna41HA3p were
similar in these extracts arrested at various cell cycle stages. We
also examined the interaction between Pol
p and Sna41p in cultures
synchronously released from HU block. We found that this interaction
could be observed regardless of cell cycle stage (Fig.
3).
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Fig. 2.
Expressions of Pol p,
Mis5p, and Sna41p during the cell cycle. A, the
sna41HA3 pol
FLAG2 cells were arrested in HU
for 3.5 h and released into the cell cycle. After a 30-min
recovery, the samples were collected every 15 min. The septation and
mitotic indices were measured under a microscope. Whole cell extracts
were prepared from all the samples. Top graph, the
vertical axis indicates the septation index (
) or mitotic
index (
). Bottom panels show Pol
FLAG2p, Mis5p, and
Sna41HA3p in the whole cell extracts at each time after release from HU
block. The gels (from top to bottom) were blotted
by anti-FLAG, anti-Mis5, and anti-HA antibodies, respectively.
B, the exponentially growing sna41HA3 strain
under cdc10, cdc20, cdc22,
cdc23, pol
, cdc17, or
cdc25 background was arrested for 4 h at 36 °C, and
the whole cell extract was prepared from each strain. The gels were
blotted with anti-FLAG, anti-Mis5, and anti-HA antibodies (top
three panels). The bottom panel shows the CBB
staining of the same gel to show the loading of approximately equal
amount of proteins in each lane.
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Fig. 3.
Sna41p interacts with Mis5p in S phase.
sna41HA3 pol FLAG2 strain was synchronized by
HU arrest for 3.5 h and subsequent release from the block. After a
30-min recovery, the samples were collected every 15 min. Whole cell
extracts were prepared from all of the samples, and Sna41HA3p was
immunoprecipitated by anti-HA antibody. Left and right
vertical axes of the top graph indicate, respectively,
the percentage of septation index (
) or mitotic index (
) and the
relative extent of Mis5p interaction with Sna41HA3p (
). To generate
the latter values, the band intensities of the second and
third panels were quantified, and the ratio of Mis5p to
Sna41HA3p was calculated with the value at 1.5 h taken as 1. After
electrophoresis on SDS-PAGE, the gel was blotted with anti-FLAG,
anti-Mis5p, and anti-HA antibodies (top three panels). The
panel indicated as CBB stain shows the CBB
staining of the same gel to show the presence of roughly equal amount
of proteins in each lane. In the bottom panel,
immunoprecipitates containing an equal amount of Sna41HA3p were blotted
with anti-Mis5p antibody.
ts01 (36),
cdc17-K42, and cdc25-22 (37). Sna41HA3p
interacted with Mis5p in cdc20-, cdc22-, and cdc23-arrested extracts (Fig.
4A). Interestingly, although
both cdc23 and pol
mutants arrest the cell
cycle with late S-phase DNA contents, only weak interaction was
observed only in pol
. In addition, we tested whether
these interactions were affected by the protein localization. The
Triton-soluble and -insoluble (the chromatin-enriched) fractions were
prepared from the arrested cdc mutant cells. Interestingly,
the interaction was only observed in Triton-insoluble fractions.
Despite the presence of significant amount of Mis5p and Sna41p,
virtually no interaction was observed in triton soluble fractions (Fig.
4B). Because Sna41p appears to be exclusively localized in
nucleus throughout the cell cycle (data not shown), the interaction
between Sna41HA3p and Mis5p is likely to take place exclusively on the
chromatin. Taken together with the results in the previous section that
Sna41HA3p interacts with Pol
p throughout the cell cycle, the role of
CDC45-related proteins in assembly of replication machinery, as
proposed previously (31), may be accomplished by the physical
interaction between Cdc45p and MCM at the G1-S
boundary.
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Fig. 4.
Sna41p interacts with Mis5p exclusively in
chromatin-enriched fractions. Exponentially growing
sna41HA3 strains under cdc10, cdc20,
cdc22, cdc23, pol ,
cdc17, and cdc25 backgrounds were arrested for
3.5 h at 36 °C. A, Sna41HA3p and Mis5p interact in
cdc20-, cdc22-, and cdc23-arrested
extracts. The whole cell extracts were prepared with EB buffer
containing 50 mM NaCl for each strain. The
immunoprecipitates were separated by SDS-PAGE and blotted by anti-FLAG
(top panel), anti-Mis5p (middle panel), and
anti-HA (bottom panel) antibodies. B, interaction
between Sna41HA3p and Mis5p is detected in Triton-insoluble fractions
but not in Triton-soluble fractions. The cells were harvested and
spheroplasted. The Triton-soluble and -insoluble fractions were
prepared as described under "Experimental Procedures." The
immunoprecipitated samples (top and middle
panels) and untreated extracts (bottom panel) were
separated by SDS-PAGE, and the filter was blotted with either
anti-Mis5p (top and bottom panels) or anti-HA
(middle panel) antibody. Left six lanes,
Triton-soluble fractions; right six lanes, Triton-insoluble
(chromatin-enriched) fractions. In both A and B,
immunoprecipitation was performed by anti-HA antibody after the protein
concentrations of the extracts were adjusted.
p--
In Xenopus egg extracts, XCdc45 was shown to
be required for chromatin loading of Pol
p. Independently, in budding
yeast, Pol
complexes were found to undergo a so-called "mitotic
resetting" event and to be loaded onto chromatin fractions before
CDC6 loading in budding yeast (30). These two results suggest that
Sna41p may play essential roles in a similar mitotic resetting event of
Pol
p in fission yeast. To address this question, we examined the
chromatin loading of Sna41HA3p, Mis5p, and Pol
FLAG2p in HU synchronized extracts. The Sna41HA3 pol
FLAG2
strain was arrested by HU for 4 h and released into cell cycle
from the block. Then, chromatin-enriched fractions were prepared at
various times after release. As reported previously, the amount of
Mis5p on the chromatin was cell cycle-regulated and correlated with the
septation index (33). The amount of Sna41HA3p and Pol
FLAG2p in the
same fractions also fluctuated, as did Mis5p, and generally increased
during S phase (Fig. 5A).
However, it was difficult from this experiment to conclude on the order
of the loading of these three proteins onto the chromatin. To clarify
the timing of chromatin loading, we examined the chromatin loading in
cell cycle-arrested extracts. Sna41HA3
pol
FLAG2 strains in combination with the cell cycle mutants cdc10-129, cdc20-M10,
cdc22-M45, cdc23-M36,
pol
ts01, and cdc25-22 were used to
arrest the cell cycle. The chromatin-enriched fractions were analyzed
by Western blotting. Mis5p was recovered in chromatin-enriched
fractions from cdc20-, cdc22-, cdc23-,
and pol
-arrested cells (Fig. 5B), in agreement
with the recent report (33). Sna41HA3p was recovered mainly in
cdc10 and cdc20 as well as in cdc22
and in cdc23 in reduced amount. Pol
FLAG2p was detected in
cdc10-, cdc20-, and cdc22-arrested
fractions. Because no Mis5p was detected in cdc10, Sna41HA3p
and Pol
FLAG2p loading onto chromatin might be controlled by a
mechanism different from that for MCMs. Because cdc10 mutant
fails to progress into S phase due to the lack of Cdc18p expression
(38), we could conclude that, unlike Mis5p, Sna41p and Pol
p loading
onto the chromatins is independent of Cdc18p. We also examined the
chromatin loading of Pol
FLAG2p and Mis5p in
sna41goa1 mutant, which we recently isolated as an
initiation-defective temperature-sensitive mutant and identified as an
allele of sna41 (49). The defect in Sna41p did not
affect the chromatin loading of Pol
FLAG2p and Mis5p (Fig.
5B). Interestingly, Pol
FLAG2p in the chromatin-enriched
fractions was most abundant in the sna41goa1 mutant.
This is unexpected, but we can clearly conclude that Sna41p is not
required for Pol
p loading onto chromatin.
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Fig. 5.
Pol FLAG2p is loaded onto the
chromatin-enriched fractions earlier than Mis5p in a manner independent
of sna41 function. A, Sna41HA3
pol
FLAG2 strain was arrested by HU for 4 h and
released. Chromatin-enriched fractions were prepared and run on
SDS-PAGE. Left vertical axis of the top graph
indicates the percentage of the septation index (
) or mitotic index
(
). B, Sna41HA3 pol
FLAG2 strains
containing a cell cycle mutation indicated at the top (except for
sna41goa1) were analyzed.
sna41goa1 contains only pol
FLAG2p. In both
A and B, EB buffer containing 150 mM
NaCl was used to prepare the extracts, and the gels were blotted with
anti-FLAG (top panels), anti-Mis5p (upper middle
panels), or anti-HA (lower middle panels) antibody. The
bottom panels show CBB staining of the same gel.
p with
MCMs--
Although we showed that chromatin loading of Pol
was not
affected by sna41goa1 mutation, the physical and
genetic interactions among Sna41p, Pol
p, and Mis5p must be the key
to reveal sna41 functions in G1-S transition.
The arresting point of sna41goa1 is close to that of
cdc20 or cdc22 (data not shown), and interaction of Sna41HA3p with MCMs takes place mainly at this cell cycle point. Taking these facts into account, we hypothesized that Sna41p is not
required for chromatin loading of Pol
p but is still required for
association of Pol
p with replicative complexes at the origins of DNA replication.
enzyme complexes. The amount
of this protein remains constant throughout the cell cycle, and the
protein is localized in the nucleus at all cell cycle stages. However,
when Spp1GFPp is stained with anti-GFP antibody by indirect
immunofluorescence, the nuclear staining of Spp1GFPp disappears at the
G1-S boundary or at the HU-arrested stage. We found that
the staining pattern of Spp1GFPp in sna41goa1 stayed
basically the same as the G2 status even after 5 h at the nonpermissive temperature, despite the fact that
sna41goa1 arrests the cell cycle at the
G1-S boundary very close to cdc22 or the HU
arresting point (Fig. 6). This
observation suggests that some G1-S-specific alterations of
Pol
complexes may not occur in sna41goa1,
consistent with our proposal that sna41p is required for interaction of
Pol
p with preRC.
View larger version (22K):
[in a new window]
Fig. 6.
G1-S specific
regulation of Pol FLAG2p is absent in
sna41goa1. A, exponentially
growing spp1-GFPKan or spp1-GFPKan
sna41goa1 cells were arrested either by 10 mM HU or by temperature shift to 36 °C, respectively.
The cells were fixed by methanol and then processed for
immunofluorescence with anti-GFP antibody and staining by
4',6-diamidino-2-phenylindole. First row, exponentially
growing spp1-GFPKan sna41goa1at 25 °C;
second and third rows, spp1-GFPKan
sna41goa1 arrested at 36 °C for 3 h; fourth
row, spp1-GFPKan sna41goa1 arrested at 36 °C
for 5 h; fifth and sixth rows,
spp1-GFPKan (sna41+) arrested by HU
for 2.5 h. B, quantitation of GFP staining. In the
merged image, the cells containing nuclei with yellow
staining were counted as positive, and those containing nuclei
with red staining were counted as negative, and fractions of
the cells with positive staining are presented. Approximately 200 cells
were counted.
FLAG2p
regardless of cell cycle stages and with Mis5p at the G1-S boundary in chromatin-enriched fractions. Therefore, it is possible that Pol
complexes are loaded onto replicative complexes with the
aid of Sna41p, which may function as a bridge. To further test this
possibility, we first examined the interaction of Pol
FLAG2p with
Mis5p in the HU-synchronized culture. Pol
FLAG2p was precipitated by
anti-FLAG antibody, and the interactions were analyzed by Western blotting. Consistent with the results in Fig. 2, Pol
FLAG2p
interacted with Sna41p regardless of the cell cycle stage. In
contrast, Mis5p interaction fluctuated during the cell cycle (Fig.
7A). The extent of the
interaction correlated with septation index. This result clearly shows
that Pol
FLAG2p interacted with Mis5p only during S phase. These
results strongly suggest the possibility that Sna41p mediates the
loading of Pol
complexes onto the replication complexes. This
possibility could be examined by analyzing the interaction of Pol
p
with MCM in the sna41goa1 mutant because the mutant
contains a very low level of Sna41p at the nonpermissive temperature
due to its instability (data not shown). Because
sna41goa1 and cdc20-M10 arrest the cell
cycle at a very similar point, we examined the interaction
between Pol
FLAG2p and Mis5p in sna41goa1- and
cdc20-arrested extracts. In the cdc20-arrested
extract, Mis5p was coprecipitated with pol
FLAG2p. In contrast,
Mis5p was not co-precipitated in the
sna41goa1-arrested extract (Fig. 7B). In
conclusion, we show here that Sna41p function is essential for
interaction of Pol
p with Mis5p at the initiation of DNA
replication.
View larger version (28K):
[in a new window]
Fig. 7.
Interaction of
Pol FLAG2p with Mis5p occurs during S phase and
is dependent on Sna41p. A, sna41HA3
pol
FLAG2 strain was arrested by 10 mM HU
for 4 h and released. EB buffer containing 150 mM NaCl
was used to prepare the whole cell extracts. Left vertical
axes of the top graph indicate the percentage of
septation index (
) or mitotic index (
). Immunoprecipitates with
anti-FLAG antibody were blotted with anti-FLAG (top panel),
anti-Mis5 (middle panel), and anti-HA (bottom
panel) antibodies. B, pol
FLAG2
cdc20-M10 (left lanes) and pol
FLAG2
sna41goa1 (right lanes) strains were shifted to
36 °C for 3.5 h, and then the whole cell extracts were prepared
using EB buffer containing 150 mM NaCl. The whole cell
extracts (left panels) and immunoprecipitates by anti-FLAG
antibody (right panels) were blotted with anti-FLAG
(top panel) or anti-Mis5p (second panel)
antibody. The bottom left panel is CBB staining.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
is loaded onto
chromatin through Cdc45 protein in Xenopus egg extracts.
Furthermore, through characterization of a new G1 arrest
mutant, sna41goa1, we found genetic interactions
between sna41 and pol
(49). These findings
lead us to examine physical interactions between Sna41p and Pol
p. We
found that Sna41p and Pol
p interacted throughout the cell cycle.
Sna41p also interacted with Mis5p, the fission yeast homologue of MCM6,
during S phase. Sna41p and Pol
p were loaded onto the
chromatin-enriched fractions in cdc10-arrested extract.
Sna41p is not required for chromatin loading of Pol
p but is still
required for its interaction with Mis5p in S phase.
p Loading onto DNA Replication Origin May Require Two
Independent Steps--
We have found that sna41goa1
suppresses the growth defect of pol
ts13 (49).
Thus, it is most likely that this genetic interaction is attributed to
the physical interaction between Sna41p and Pol
p. In accordance with
this prediction, we found that these two proteins interact with each
other. This is consistent with the result in Xenopus and the
recent in vitro observation in human (41). In Xenopus egg extracts, DNA polymerase
is loaded onto
chromatin at the G1-S boundary in a manner dependent on
XCdc45 and Cdk activity (31). In contrast, budding yeast DNA polymerase
is loaded at the end of mitosis independent of CDC6 (30), although
another report claims that the Pol
b subunit is loaded onto
chromatin at G1-S (42). These somewhat contradictory
results may be due to the different cell cycle operations in yeast and
Xenopus eggs. DNA replication and mitosis are alternating
practically without gap periods in Xenopus egg extracts,
whereas yeast cell cycles are under strict G1 regulations.
Diversity in the methods for preparation of chromatin fractions may
also account in part for these discrepancies. However, our data may
provide a more unified view for the loading of DNA polymerase
. We
found that Pol
p loading on the chromatin-enriched fractions is
observed even in cdc10- or
sna41goa1-arrested extract (Fig. 5B).
This clearly shows that this step is not dependent on either Cdc18p or
Sna41p. This result is consistent with the observation in budding
yeast. We also showed that interaction of Pol
p with MCMs at the
G1-S boundary depends on functional Sna41p (Fig. 7).
Recently, Mis5p/Mcm6p was shown to be loaded at or near autonomously
replicating sequences (33). Although further confirmation is
needed, it is most likely that association of Pol
p with MCMs
facilitates relocation of the former protein to replication
origins. This result is more consistent with the finding in
Xenopus egg extracts. Taken together, our data support the
following model (Fig. 8) of
loading of DNA polymerase
onto replication complexes in the
proliferating cell cycle of somatic cells. The first step may depend on
Pol
b subunit and inactivation of M-phase Cdk. The fact that Pol
p
and Sna41p recovered in the chromatin-enriched fractions are relatively
resistant to extraction with DNase I digestion (data not shown)
suggests that the first step of loading may deliver these proteins not
simply onto the chromatin but onto a particular nuclear structure or
nuclear matrix. Because Sna41p and Pol
p can interact with each other
throughout the cell cycle, the two proteins may be loaded onto
chromatin as a complex. However, because Pol
p is loaded onto
chromatin even in the sna41goa1 mutant, the
association of Sna41p and Pol
p is unlikely to be essential for
Pol
p loading. This step may be required for accumulation of the
proteins involved in DNA synthesis at the specific nuclear structures.
Then, interaction of Sna41p with MCMs brings DNA polymerase
onto
replication complexes at the origins in the second step. This step may
be regulated by S-phase Cdk and/or by Cdc7 kinase. It should be noted
that our results do not exclude the possibility that the observed
interactions are mediated by other proteins. In fact, both Mis5p and
Pol
p are parts of protein complexes (six-protein MCM complex and
four-protein DNA polymerase
complex, respectively) that are
essential components for a larger replication machinery, and therefore
it may be more likely that Sna41p interacts with these complexes as
well as other components in the replication apparatus.
View larger version (16K):
[in a new window]
Fig. 8.
Two-step model for loading of the DNA
polymerase /primase complex onto replication
origins. DNA polymerase
/primase and Sna41p form a complex
throughout the cell cycle. In early G1, both proteins are
loaded onto chromatin or some kind of nuclear structure (Nuclear
Matrix?). This step may depend on DNA polymerase
b subunit, as
was shown in budding yeast. At the G1-S boundary, Pol
complex associates with replication origins through Sna41p-mediated
interaction with MCMs.
are loaded onto late-firing origins later than early ones (43, 44) by postulating that origin-specific phosphorylation events activate association of Cdc45 with MCM. The data
presented in this work indicate that physical interactions between
MCM-Cdc45p-Pol
p constitute an important step for assembly of active
replication machinery. More biochemical studies are needed to
understand the nature of these interactions and the roles of these
complexes in initiation of DNA replication.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Susan Forsburg for the
generous gift of anti-Mis5 antibody. The anti-pol antibody and
strains were kindly provided by Teresa Wang. We thank all members of
our laboratory for useful discussion.
![]() |
FOOTNOTES |
---|
* This work was supported in part by grants-in-aid for scientific research on priority areas from the Ministry of Education, Science, Sports and Culture of Japan.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 Japan Society for the Promotion of Science Research Fellowships for Young Scientists.
** To whom correspondence should be addressed: Dept. of Cell Biology, Tokyo Metropolitan Inst. of Medical Science, 3-18-22 Honkomagome, Bunkyo-Ku, Tokyo 113-8613, Japan. Tel.: 81-3-5685-2264; Fax: 81-3-5685-2932; E-mail: hmasai@rinshoken.or.jp.
Published, JBC Papers in Press, May 8, 2001, DOI 10.1074/jbc.M100007200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
MCM, minichromosome maintenance proteins;
preRC, prereplicative complex;
Cdk, cyclin-dependent kinase;
pol, DNA polymerase
;
HA, hemagglutinin;
PCR, polymerase chain reaction;
GFP, green
fluorescent protein;
PAGE, polyacrylamide gel electrophoresis;
HU, hydroxyurea;
CBB, Coomasie Brilliant Blue R-250.
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