(Received for publication, August 2, 1995; and in revised form, January 31, 1996)
From the
The B subunit of the DNA polymerase (pol) -primase complex
executes an essential role at the initial stage of DNA replication in Saccharomyces cerevisiae and is phosphorylated in a cell
cycle-dependent manner. In this report, we show that the four subunits
of the yeast DNA polymerase
-primase complex are assembled
throughout the cell cycle, and physical association between newly
synthesized pol
(p180) and unphosphorylated B subunit (p86)
occurs very rapidly. Therefore, B subunit phosphorylation does not
appear to modulate p180
p86 interaction. Conversely, by depletion
experiments and by using a yeast mutant strain, which produces a low
and constitutive level of the p180 polypeptide, we found that formation
of the p180
p86 subcomplex is required for B subunit
phosphorylation.
The development of cell-free systems capable to replicate viral DNA molecules has been essential in understanding the fundamental enzymology of eukaryotic DNA replication and in identifying most of the required cellular factors (Challberg and Kelly, 1989; Stillman, 1989; Hurwitz et al., 1990; Waga and Stillman, 1994). The structure of these proteins appears to be conserved in all the eukaryotic organisms analyzed so far, and the genetic amenability of the yeast system has allowed testing of their function in DNA replication in vivo (Campbell, 1993).
Despite considerable advances in understanding the mechanistic properties of the replicative process, very little is known about the mechanisms that couple initiation of DNA replication to cell cycle progression (Coverley and Laskey, 1994; Heichman and Roberts, 1994). Entry into the S phase is likely to be controlled by the combined action of positive and negative regulators. In fact, current evidence is consistent with the notion that the onset of S phase requires the accumulation of cyclin-dependent kinase(s) and the concomitant destruction of cyclin-dependent kinase inhibitors (Hayles et al., 1994; Schwob et al., 1994). The substrates of the cyclin-dependent kinase promoting S phase entry are presently unknown, although it is legitimate to speculate that at least some proteins of the replicative apparatus itself might represent the physiological target. Indeed, specific subunits of critical replication factors are phosphorylated in a cell cycle-dependent manner (Din et al., 1990; Dutta and Stillman, 1992; Foiani et al., 1995; Nasheuer et al., 1991).
Biochemical and genetic evidence
have established that the DNA polymerase -primase complex (pol (
)
-primase) plays an essential role in eukaryotic DNA
replication, because of its unique ability to initiate DNA synthesis de novo (Wang, 1991); as a consequence, it is required for
discontinuous lagging strand synthesis, as well as for initiation of
DNA replication at an origin. This dual role of the pol
-primase
complex makes it a potential target of the regulatory mechanisms
controlling entry into S phase. By using the yeast Saccharomyces
cerevisiae as a model system, we have combined in vitro and in vivo experimental approaches to test whether the
function of the yeast pol
-primase complex is indeed regulated
during the cell cycle. The yeast pol
-primase complex contains
four polypeptides with apparent molecular masses of 180, 86, 58, and 48
kDa (Plevani et al., 1985; Brooke et al., 1991), and
the structure and catalytic properties of this protein complex are
conserved in a wide range of eukaryotic organisms (Wang, 1991). The
p180 polypeptide is the pol
subunit, while DNA primase is a
heterodimer of the 58- and 48- kDa polypeptides (Plevani et
al., 1985; Brooke and Dumas, 1991). The p48 subunit is sufficient
for RNA primer synthesis in vitro (Santocanale et
al., 1993), and the p58 polypeptide stabilizes primase activity
and mediates the interaction between pol
and p48 (Santocanale et al., 1993; Longhese et al., 1993). The p86 protein
species (also called B subunit) directly interacts with the p180
polypeptide, but does not influence any of the enzymatic activity of
the complex (Plevani et al., 1985; Brooke et al.,
1991), suggesting that it may play a regulatory function. Accordingly,
we have recently shown that the yeast B subunit specifically executes
its essential function at the initial stage of DNA replication (Foiani et al., 1994), and it is phosphorylated and dephosphorylated
in a cell cycle-dependent manner (Foiani et al., 1995). These
observations led us to suggest that p86 might be involved in loading
the pol
-primase complex at the origins of replication during the
M/G
transition of the cell cycle. This loading may require
specific protein-protein interactions with some initiation factors and
may be modulated by post-translational modifications. The finding that
the human B subunit physically interacts with the SV40 T-antigen and is
phosphorylated in a cell cycle-dependent manner is in agreement with
this model (Collins et al., 1993).
The yeast genes encoding
the pol -primase subunits are transiently transcribed at the
G
/S boundary (Johnston et al., 1987; Foiani et
al., 1989; Johnston et al., 1990; Foiani et al.,
1995), and this transcriptional program is mediated by cis- and
trans-acting elements (Johnston and Lowndes, 1992; Koch and Nasmyth,
1994). We have recently shown that the pol
-primase subunits are
stable proteins, which are present in large excess within the cell, and
their de novo synthesis, resulting from the late G
transcriptional burst, is not essential to enter S phase (Muzi
Falconi et al., 1993; Foiani et al., 1995).
Therefore, the significance of the transcriptional control of the
corresponding genes remains obscure. By considering that the four
subunits of the pol
-primase complex are always present during the
different steps of the cell cycle and their level is not rate-limiting
for entry into S phase, we wanted to test whether the assembly of the
complex was regulated during the cell cycle and whether it was
influenced by the phosphorylation state of the B subunit.
In this
report we provide evidence that formation of the pol -primase
complex is not restricted to S phase, since the 4-subunit complex is
assembled throughout the cell cycle. Phosphorylation of the B subunit
of the complex (p86) is not required for productive interaction with
the pol
polypeptide (p180), while previous assembly of the
p86
p180 subcomplex is required for B subunit phosphorylation.
Figure 1:
Immunoprecipitation of the pol
-primase complex from synchronously dividing cells. Top,
strain CG378 was synchronized by
-factor treatment and the degree
of synchrony was measured by microscopic analysis of cell budding. Bottom, aliquots of the cell culture (50 ml) were taken at the
indicated times and protein extracts were prepared using non-denaturing
conditions (see ``Experimental Procedures''). For each
sample, 2 mg of total protein were immunoprecipitated and analyzed by
SDS-PAGE electrophoresis and Western blotting with monoclonal or
affinity-purified polyclonal antibodies against the individual pol
-primase subunits (Plevani et al., 1985; Santocanale et al., 1992; Foiani et al.,
1994).
Figure 2:
Kinetics of interaction between newly
synthesized p180 and B subunit. Top, budding profile of strain
CG378 synchronized by -factor treatment. After
-factor
release, cells were labeled in vivo with a 12-min pulse of
[
S]methionine, performed at the time indicated
by bracket, and chased with an excess of cold methionine. Bottom, at the times indicated by the numbered arrows on the top of the figure, aliquots of the culture (2 ml) were
taken and protein extracts were prepared under non-denaturing
conditions. The p180 and B subunit polypeptides were immunoprecipitated
with the two-step method described under ``Experimental
Procedures.''
We previously found that overexpression of the POL12 gene,
encoding the B subunit, resulted in accumulation of the p86
unphosphorylated form (Foiani et al., 1995). To investigate
whether a correct stoichiometry of the pol -primase polypeptides
was required for proper p86 modification, we analyzed its
phosphorylation state in yeast strains carrying the POL1, PRI1, or PRI2 genes, encoding, respectively, the
p180, p48, and p58 pol
-primase polypeptides under the control of
the repressible GAL1 promoter.
Strain CG378POL1 (pMA2)
carrying the GAL1-POL1 fusion gene can grow for several
generations under repressed conditions in glucose-containing media (Fig. 3, panel A) (Muzi Falconi et al., 1993),
even when the level of the p180 polypeptide drops far below its
physiological level (Fig. 3, panel B) (Muzi Falconi et al., 1993). Therefore, it was possible to test whether
phosphorylation of B subunit was correlated to the progressive
depletion of the pol
polypeptide. As shown in Fig. 3(panel B), repression of GAL1-POL1 expression did not change the ratio between the p86 and p91 forms
of B subunit, until the level of p180 was lower than that of the
wild-type control strain. Further decrease in the amount of p180
resulted in a proportional decrease of p91, indicating that the amount
of p180 influences the extent of B subunit phosphorylation.
Figure 3:
Effect of p180 depletion on B subunit
phosphorylation. Panel A, cells of strain CG378POL1
(pMA2) were grown at 28 °C under selective conditions in synthetic
medium (Muzi Falconi et al., 1993) containing 2% galactose to
a concentration of 2
1O
cells/ml. Cells were
filtered, washed, and resuspended in synthetic medium containing 2%
glucose (time 0). Cell number was monitored by microscopic counting.
Aliquots of 3
10
cells were taken at the indicated
times (numbered arrows) for protein extract preparation and
Western blot analysis. Panel B, 25 µg of total protein
extracts were prepared by the trichloroacetic acid method (see
``Experimental Procedures'') and analyzed by SDS-PAGE
electrophoresis followed by Western blotting with the appropriate
antibodies. Control lane is a sample of protein extract (25 µg)
prepared from exponentially growing CG378 isogenic
cells.
Analogous experiments performed on yeast strains carrying,
respectively, the PRI1 and PRI2 genes under the
control of the GAL1 promoter have shown that progressive
depletion of either the p48 or p58 polypeptides did not interfere with
B subunit phosphorylation (data not shown). Therefore, the proper
stoichiometry of all four subunits of the pol -primase complex is
not a prerequisite for B subunit phosphorylation, which is exclusively
dependent on the level of p180. This finding is consistent with the
long standing observation that the pol
-primase complex consists
of two subcomplexes: heterodimeric DNA primase, containing the p48 and
p58 polypeptides; and the p180
p86 subcomplex, which only shows
DNA polymerase activity (Plevani et al., 1985).
As an
alternative approach to correlate the level of p180 with that of
phosphorylated B subunit, we used a yeast strain constitutively
producing a very low amount of the p180 polypeptide. Such a strain was
constructed by replacing the chromosomal copy of the POL1 gene
with the constitutively repressed pol1-6 allele, which
carries a deletion of the POL1 cell cycle regulatory element
(Pizzagalli et al., 1992), and tested the effect of this
deletion on cell growth and B subunit phosphorylation. As shown in Fig. 4A, this mutation in the POL1 promoter
did not affect growth rate, since the kinetic of growth of the pol1-
6 strain was undistinguishable from that of the
isogenic wild-type. However, FACS analysis showed that deletion of the POL1 cell cycle regulatory element resulted in a partial delay
of S phase progression, as visualized by the accumulation of cells with
an intermediate DNA content between 1C and 2C (Fig. 4B). Microscopic analysis of wild-type and pol1-
6 cells indicated that the percentage of unbudded
cells decreased in the mutant strain. This finding was expected, since
it is known that a longer S phase leads to larger daughter cells at the
time of cell separation, reducing the requirement for growth and time
in G
in the subsequent cell cycle (Johnston and Singer,
1983).
Figure 4:
Growth rate and FACS analysis of the pol1-6 strain. Panel A, growth rates of CG378
(
) and pol1-
6 (
) isogenic strains were
monitored by cell counting. Panel B, samples of exponentially
growing CG378 (POL1) and pol1-
6 cells were taken
and the DNA content was measured by FACS analysis. Percentage of small
budded, large budded, and unbudded cells (inset) was monitored
by microscopic examination.
As shown in Fig. 5A, after release from the
-factor block, pol1-
6 cells were able to divide
synchronously with a budding profile that was essentially
undistinguishable from that of isogenic wild-type cells. Moreover,
periodic synthesis of H2A mRNA in S phase was coincident in wild-type
and mutant cells (Fig. 5, B and C). However,
the steady-state level of POL1 mRNA in the pol1-
6 strain was barely detectable and periodic transcription of the POL1 gene in G
/S, which was evident in the
isogenic wild-type strain, was completely abolished. Accordingly, the
amount of the POL1 gene product (p180 polypeptide) was
strongly decreased, compared to that found in wild-type cells. These
data support the notion that periodic transcription of the POL1 gene is not required to drive entry into S phase, and that the
p180 polypeptide is present in excess amount within the cell (Muzi
Falconi et al., 1993). When we tested the level of B subunit
phosphorylation in synchronously dividing pol1-
6 cells,
only the p86 form was detectable on Western blots of total protein
extracts (Fig. 5C), while both p86 and p91 were found
in protein extracts prepared from wild-type cells (Fig. 5B).
Figure 5:
B subunit phosphorylation in synchronized pol1-6 cells. Panel A, budding profile of CG378
(
) and pol1-
6 (
) strains synchronized by
-factor treatment. Panel B, at the indicated times,
samples of the CG378 synchronized culture were taken, protein extracts
were prepared by the trichloroacetic acid method (Experimental
Procedures), and 25 µg of total proteins were analyzed by SDS-PAGE
electrophoresis and Western blotting with the appropriate antibodies.
Total RNA was extracted at the same times, and 5 µg of RNA was
loaded in each lane. The level of POL1, PR1, and H2A
mRNAs was measured by Northern blot analysis as indicated under
``Experimental Procedures.'' Panel C, samples taken
from the synchronous pol1-
6 culture were processed for
Western and Northern blot analysis as described in panel B.
The Northern blot performed to monitor the level of POL1 mRNA
in pol1-
6 cells was developed after 3 weeks, while the
other Northern blots shown in panels B and C were
developed after 3 days.
The correlation between B subunit
phosphorylation and the level of p180 can again be explained by
assuming that physical interaction between p86 and p180 is required for
p86 phosphorylation. In fact, the finding that only p86 is detectable
on Western blots of protein extracts prepared from synchronously
dividing (Fig. 5C) or logarithmically growing pol1-6 cells (Fig. 6A) can be ascribed to
the low amount of B subunit that can associate with p180 due to the
reduced level of the pol
polypeptide in the mutant strain. This
assumption was corroborated by the finding that phosphorylated B
subunit can instead be detected in pol1-
6 cells if a
sufficient amount of protein extract was immunoprecipitated with
anti-p180 monoclonal antibodies (Fig. 6B). This result
indicates that B subunit phosphorylation requires the physical
interaction with p180 and, in turn, this association is modulated by
the level of the pol
polypeptide.
Figure 6:
Phosphorylation state of pol
-associated B subunit in pol1-
6 cells. Panel
A, Western blot analysis of 25 µg of total protein extracts
prepared from exponentially growing CG378 (wt) and pol1-
6 cells. Panel B, 5 mg of total protein
from exponentially growing CG378 (wt) and pol1-
6 cells were immunoprecipitated under non-denaturing conditions,
followed by Western blot analysis with specific antibodies. Panel
C, total protein extracts were prepared from exponentially growing
CG378 (wt) and pol1-
6 cells, or from cells of
the same strains which have been arrested in S phase by treatment with
0.08 M HU for 3 h. 25 µg of total proteins for each sample
were separated by SDS-PAGE electrophoresis, followed by Western
blotting.
Both p86 and p91 were
present in the immunoprecipitate prepared from logarithmically growing pol1-6 cells (Fig. 6B), probably as a
consequence of the cell cycle-dependent phosphorylation of B subunit.
However, only the amount of p86 that is physically associated with p180
can be phosphorylated by the yet unidentified protein kinase
responsible for such post-translational modification. In fact,
phosphorylated p91 accumulated in protein extracts prepared from
wild-type cells arrested by hydroxyurea (HU) treatment (Fig. 6C) (Foiani et al., 1995). Conversely,
only p86 was detected in protein extracts prepared from pol1-
6 cells arrested at the HU-dependent step, indicating that free p86
cannot be phosphorylated, even when cells are blocked for several hours
in S phase, a stage of the cell cycle when a protein kinase capable to
phosphorylate p86 is fully active.
The level of p86 phosphorylation
could be exclusively dependent on the amount of the p86p180
subcomplex or, alternatively, could be limited by one or more
rate-limiting factors. If the first hypothesis is correct, we would
predict that the amount of phosphorylated B subunit will increase in a
yeast strain overproducing the p180 and p86 polypeptides. Indeed, as
shown in Fig. 7, the total level of the p180
B subunit
subcomplex was found to be higher in immunoprecipitates from protein
extracts prepared from a yeast strain transformed with two high copy
number plasmids carrying either the POL1 or the POL12 genes. This overproducing strain did not show any detectable cell
cycle defect (data not shown), and the ratio between unphosphorylated
p86 and phosphorylated p91 was similar to that found in the
untransformed wild-type strain. When we analyzed the level of
associated p48 and p58 in the immunoprecipitates shown in Fig. 7, we found that the amount of these polypeptides was
slightly higher in the co-overproducing strain compared to the
wild-type although their level, measured by Western blotting on total
extracts, was identical in the two strains. We have shown previously
that only 50% of DNA primase is associated in the four subunit pol
-primase complex (Santocanale et al., 1993), and an
increase in the level of the p180
B subunit heterodimer might
favor association of free primase subunits.
Figure 7: Phosphorylation state of B subunit in cells co-overexpressing the POL12 and POL1 genes. 5 mg of total protein prepared from wild-type cells (lane 1) or from cells overexpressing the POL12 and POL1 genes (lane 2) were immunoprecipitated under non-denaturing conditions, followed by Western blot analysis with specific antibodies. Lane 3 is the same sample analyzed in lane 2, except that the Western blot was developed for a few seconds instead than 5 min (lanes 1 and 2).
As expected, single
overexpression of the POL12 gene causes the accumulation of
unphosphorylated p86 (Foiani et al., 1995), while POL1 overexpression does not increase the level of p91 found in
wild-type cells and the ratio between p86 and p91 (see Fig. 3).
These findings indicate that p180p86 association is necessary and
sufficient for proper B subunit phosphorylation.
Replication of the eukaryotic genome is restricted to the S
phase of the cell cycle, but preparation for S phase appears to occur
while cells are exiting from mitosis. Recently it has been shown that
the chromatin structure of yeast origins of replication changes from a
post-replicative to a prereplicative state late in mitosis (Diffley et al., 1994). In this respect, it is quite intriguing that
dephosphorylation of the yeast pol -primase B subunit is
coincident with the appearance of the prereplicative complexes late in
mitosis (Foiani et al., 1995). By considering that the B
subunit becomes phosphorylated in G
/S and executes its
function at the initial stage of DNA replication, before the
HU-sensitive step (Foiani et al., 1994), it is reasonable to
speculate that this protein might be the target of regulatory events
controlling the onset of DNA replication. This hypothesis is
strengthened by the unique ability of the pol
-primase complex in
initiating DNA synthesis de novo (Wang, 1991). The successful
production of a RNA-DNA chain by pol
-primase represents the first
polymerization event occurring at an origin of replication. Moreover,
the switch of the pol
-primase complex to the lagging strand
template (Tsurimoto et al., 1990; Waga and Stillman, 1994)
might signal the establishment of a productive multiprotein complex
competent for further DNA replication.
We are interested in
understanding: (i) the in vivo function of the four pol
-primase subunits, (ii) the physiological role of B subunit
phosphorylation, and (iii) the rules controlling the physical
interaction of the four subunits with each other and with other
components of the replication apparatus. In yeast, the answer to these
questions is further complicated by the finding that the genes encoding
the pol
-primase polypeptides are periodically transcribed during
the cell cycle, while the corresponding gene products are stable
proteins (Muzi Falconi et al., 1993; Foiani et al.,
1995). As a consequence, the cell contains two pools of pol
-primase polypeptides: the first one, which is inherited from the
previous cell cycle (maternal pool); and the second one, which is de novo synthesized in S phase (newly synthesized pool). It is
presently unclear whether these newly synthesized proteins play any
role, since we have shown that proteins of maternal origins are
sufficient to drive entry into S phase and to replicate chromosomal DNA
(Muzi Falconi et al., 1993; Foiani et al., 1995).
A possible way to restrict the function of the pol -primase to
S phase would have been to modulate the assembly of the whole complex
during the cell cycle. This question is particularly relevant in the
yeast S. cerevisiae, because of the presence of maternal and
newly synthesized proteins in normally cycling cells. The
immunoprecipitation experiments described in this report indicate that
the four subunits of the complex are firmly associated with each other
throughout the cell cycle, similarly to what it has been found in human
cells (Nasheuer et al., 1991). Furthermore, association of
newly synthesized subunits is extremely rapid. Therefore, transient
association of one subunit does not appear to regulate the function of
the whole complex.
Moreover, B subunit phosphorylation does not
modulate the assembly of the complex. In fact, not only the B subunit
is associated to the other polypeptides at every stage of the cell
cycle but two complexes, containing either phosphorylated (p91) or
unphosphorylated (p86) B subunit, can co-exist during most of the cell
cycle starting from early S phase until mitosis. The presence of two
complexes differing in the phosphorylation state of B subunit is likely
due to its dual timing of phosphorylation. In fact, we have shown
previously that maternal p86 is phosphorylated early in S phase, while
newly synthesized p86, produced as a consequence of periodic
transcription of the POL12 gene in late G, becomes
phosphorylated 70 min after its synthesis (Foiani et al.,
1995).
Several mechanisms, which are not mutually exclusive, can be
involved in preventing phosphorylation of newly synthesized p86 during
this time interval. For example, it is possible that phosphorylation of
p86 requires the previous assembly of the pol -primase complex,
and productive interaction among the four subunits may be a function of
a threshold level of the de novo synthesized polypeptides.
Alternatively, B subunit phosphorylation might occur only after this
polypeptide, or the whole pol
-primase complex, are transported
within the nucleus and, eventually, after they interact with DNA or
with other protein components. Finally, B subunit phosphorylation might
be controlled by a timing mechanism, and, under unperturbed conditions,
two cyclin-dependent kinases might be responsible for phosphorylation
of maternal or newly synthesized B subunit. According to this last
hypothesis, it is interesting that the phosphorylation timings of
maternal and newly synthesized p86 essentially parallel, respectively,
the timings of Clb5/Clb6 and of Clb1-4 synthesis (Schwob and
Nasmyth, 1993; Schwob et al., 1994).
Our finding that newly
synthesized p86 rapidly associates with the p180/pol polypeptide,
while remaining unphosphorylated for 70 min, indicates that p86
phosphorylation is not a prerequisite for p180
p86 interaction and
suggests that assembly of the complex occurs almost immediately after
synthesis of its subunits. Therefore, assembly of the pol
-primase
complex does not seem to be the rate-limiting step preventing B subunit
phosphorylation under unperturbed conditions. However, p86
phosphorylation requires the formation of the p180
p86 subcomplex,
while it does not appear to be influenced by a correct stoichiometry of
the four pol
-primase subunits. In fact, progressive depletion of
the p48 and p58 primase polypeptides does not change the ratio between
phosphorylated and unphosphorylated B subunit (data not shown).
Conversely, depletion of the p180 polypeptide causes an evident
increase in the amount of unphosphorylated p86.
The correlation
between the level of p180 and B subunit phosphorylation was also
studied by using the pol1-6 strain, which carries an
integrated copy of the POL1 gene lacking its cell cycle
regulatory element and producing a very low amount of p180 polypeptide.
The finding that wild-type and pol1-
6 cells have
identical growth rates further confirms that periodic transcription of
the POL1 gene does not have an essential function under normal
conditions (Muzi Falconi et al., 1993). In the pol1-
6 mutant strain, the level of phosphorylated p91 is much lower than
that found in the isogenic wild-type and correlates with the limited
amount of p180 found in protein extracts prepared from the mutant
strain. Moreover, three sets of evidence indicate that unassembled B
subunit cannot be phosphorylated: first, overexpression of the POL12 gene leads to accumulation of unphosphorylated p86
(Foiani et al., 1995); second, when pol1-
6 cells
are blocked in S phase by HU treatment, the phosphorylation of B
subunit does not increase, while phosphorylated p91 accumulates in
wild-type cells arrested by the same treatment; third, phosphorylated
p91 in pol1-
6 extracts can only be found in
immunoprecipitates obtained with anti-pol
antibodies. Altogether,
these data indicate that formation of the p180
p86 subcomplex is a
prerequisite for B subunit phosphorylation.
The finding that
co-overproduction of p180 and B subunit results in an increased level
of p91, compared to that found in wild-type cells, indicates that the
interaction between these polypeptides is sufficient for B subunit
phosphorylation. Although other explanations can be envisaged, it is
possible that p180p86 association leads to a conformational
change which allows proper B subunit phosphorylation. A major issue
remains the discovery of the kinase(s) responsible for B-subunit
phosphorylation and the definition of the biological significance of
this event. Our finding that p180
p86 association is a
prerequisite for B-subunit phosphorylation further complicates these
studies. In fact, proper p180
p86 interaction will have to be
considered in studying any mutant altered in B-subunit phosphorylation,
and the possibility that specific protein-protein interaction will
affect phosphorylation could be extended to other DNA replication
proteins.