Tài liệu Báo cáo khoa học: Dissociation of DNA polymerase a-primase complex during meiosis in Coprinus cinereus pptx

Dissociation of DNA polymerase a-primase complex during meiosis
in
Coprinus cinereus
Satoshi Namekawa, Fumika Hamada, Tomoyuki Sawado†, Satomi Ishii, Takayuki Nara‡, Takashi Ishizaki,
Takashi Ohuchi, Takao Arai and Kengo Sakaguchi
Department of Applied Biological Science, Faculty of Science and Technology, Tokyo University of Science, Japan
Previously, the activity of DNA polymerase a was found in
the meiotic prophase I including non-S phase stages, in the
basidiomycetes, Coprinus cinereus. To study DNA poly-
merase a during meiosis, we cloned cDNAs for the
C. cinereus DNA polymerase a catalytic subunit (p140) and
C. cinereus primase small subunit (p48). Northern analysis
indicated that both p140 and p48 are expressed not only
at S phase but also during the leptotene/zygotene stages of
meiotic prophase I. Insituimmuno-staining of cells at
meiotic prophase I revealed a sub population of p48 that
does not colocalize with p140 in nuclei. We also purified
the pol a-primase complex from meiotic cells by column
chromatography and characterized its biochemical proper-
ties. We found a subpopulation of primase that was separ-
ated from the pol a-primase complex by phosphocellulose
column chromatography. Glycerol gradient density sedi-
mentation results indicated that the amount of intact pol
a-primase complex in crude extract is reduced, and that a
smaller complex appears upon meiotic development. These
results suggest that the form of the DNA polymerase
a-primase complex is altered during meiotic development.
Keywords: meiotic prophase I; zygotene; pachytene; pol a
catalytic subunit (p140); primase small subunit (p48).
The DNA polymerase a-pol (a)-primase complex plays an
essential role in eukaryotic DNA replication and the

structural and biochemical properties of pol a are conserved
across a wide range of eukaryotes [1,2]. According to the
current model, replicative DNA synthesis initiates
from a short stretch of RNA primer synthesized by the
pol a-associated primase. After generating approximately
20 base pairs of DNA, the pol a-primase complex is
released from the DNA template, and then pol d,and
perhaps also pol e, complete DNA replication [2]. The
pol a-primase complex is composed of four subunits with
distinctive functions [1,3–5]. The largest p180 subunit is a
catalytic core for DNA polymerase activity [6]. Primase
consists of the p49 subunit, where primase activity resides
[7,8], and the p54 subunit, which contains a nuclear
localization signal that is capable of directing both the p54
monomer and the p49-p54 dimer to the nucleus [9]. The p68
subunit binds tightly to the p180 subunit, but not to the
primase subunits, and contributes both to protein synthesis
of p180 and to its translocation into the nucleus [10]. The
p68 subunit is also essential for initial DNA synthesis, and is
phosphorylated and dephosphorylated in a cell cycle-
dependant manner [11,12]. When quiescent cells begin
to proliferate, mRNA levels of all subunits of pol a are
elevated [13], as are consequent translation rates and
enzyme activities [14].
Upon entry into the leptotene stage in meiotic prophase I,
chromosomes that are initially diffused in nuclei form a
thread-like structure and each chromosome acquires an
axial core at which the two sister chromatids attach. During
the next zygotene stage, homologous chromosomes align,
and form the synaptonemal complex. We have previously

reported meiosis-related DNA polymerases and their func-
tions in chromosome pairing and meiotic recombination in
various organisms including the lily, Lilium longiflorum
[15], and a basidiomycete, Coprinus cinereus [16–19]. Several
reports have provided evidence that DNA synthesis takes
place during meiotic prophase I. In C. cinereus,DNArepair
synthesis occurs at the pachytene stage [20] when the a-type
DNA polymerase is present [16,21]. In lily, during meiotic
prophase I, at least two sequential DNA syntheses are
known to play a role in progression of meiosis. A small
amount of DNA is synthesized in meiotic prophase I at the
zygotene and pachytene stages when homologous chromo-
some pairing and recombination occur [22–24]. Further-
more, in yeast, several DNA syntheses relating to meiotic
recombination have been reported. Meiotic recombination
in yeast starts from meiosis-specific double-strand breaks
(DSBs) followed by formation of single-stranded DNA by
exonuclease digestion. The single-strand portion invades
the regions having homologous sequences in the other
Correspondence to Kengo Sakaguchi, Department of Applied
Biological Science, Tokyo University of Science, 2641 Yamazaki,
Noda-shi, Chiba-ken 278–8510, Japan.
Fax: + 81 471 24 1501, Tel.: + 81 471 23 9767 (ext. 3409),
E-mail:
Abbreviations: pol a, DNA polymerase a; DSBs, double strand
breaks; DAPI, 4¢,6-diamidino-2-phenylindole dihydrochloride;
BCAT, bovine catalase; YADH, yeast alcohol dehydrogenase.
Enzymes: DNA-directed DNA polymerase (EC 2.7.7.7).
Present address: Division of Basic Sciences, Fred Hutchinson Cancer
Research Center, Seattle, Washington, 98109–4433.

àPresent address: Department of Food Science and Human Nutrition,
University of Illinois, 62801.
(Received 16 January 2003, revised 5 March 2003,
accepted 12 March 2003)
Eur. J. Biochem. 270, 2137–2146 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03565.x
allele, resulting in formation of the Holiday structure. After
single-ended strand invasion and initial repair synthesis,
crossover and noncrossover pathways diverge. Both cross-
over and noncrossover pathways accompany DNA synthe-
sis [25,26]. Despite the importance of DNA synthesis during
meiosis, the molecular basis is poorly understood.
In C. cinereus meiotic cell cycle, premeiotic S-phase and
meiotic prophase I are distinguished by the karyogamy
stage using light microscopy [20]. This allows us to address
the question of whether DNA polymerase is present and
how it is regulated during meiotic prophase I. We cloned
cDNAs of the C. cinereus pol a catalytic subunit (p140) and
the C. cinereus primase small subunit (p48). We observed
expression of p140 and p48 not only at S phase but also
during the leptotene/zygotene stages of meiotic prophase I.
Immunostaining of meiotic cells with anti-p140 and anti-
p48 Igs revealed that these two subunits do not always
colocalize in the nuclei. Consistent with this, biochemical
experiments suggest that a subpopulation of the p48 subunit
dissociates from the pol a-complex in meiotic cells. Glycerol
gradient density sedimentation results indicated the popu-
lation of intact pol a-primase complex (11S) in crude extract
is reduced, and that a smaller-sized complex appears upon
meiotic development. These results suggest that formation
of the pol a-primase complex is altered or affected during

meiotic development. This may be a novel feature of pol
a-primase regulation, and also may be related to specific
events during meiosis, such as genetic recombination or
chromosome paring.
Materials and methods
Culturing of
C. cinereus
and collection of the fruiting
bodies
The basidiomycete C. cinereus (American Type Culture
Collection no. 56838) was used in this study. The culture
method used here was described previously [21]. These
cultures are incubated from day 0 to day 7 at 37 °Cintotal
darkness and from day 7 onward at 25 °C under a 16-h
light : 8-h dark cycle to allow photoinduction of fruiting
body formation. A series of meiotic events occur synchro-
nously in all the fruiting bodies under the proper light cycles
as described previously [27,28]. Typical procedures of
photoinduction of meiosis is as follows: Karyogamy, which
is defined as the time at which 5% of all basidia had fused
nuclei, begins at 04.00 h (K + 0), 1 h before the light was
turned on. Photoinduction starts at 05.00 h (Karyo-
gamy + 1 h, K + 1). Fruiting caps containing meiotic
cells at the leptotene to the zygotene stages are observed
between 04.00 h (K + 0) and 09.00 h (K + 5). Cells at the
pachytene stage are observed between 10.00 h (K + 6) and
11.00 h (K + 7). Meiosis II cells are observed between
12.00 h (K + 8) and 14.00 h (K + 10).
cDNA cloning of p140 and p48
In order to isolate cDNA clones of p140, two primers

were synthesized corresponding to the amino acid motifs
conserved among species: sense primer (5¢-CATCAT
CCAGGAGTACAACATCTGYTTYACNAC-3¢)and
antisense primer (5¢-CCGAGGCAGCCGTACATNSW
RRTT-3¢)(N¼ A, C, G or T, S ¼ CorG,W¼ AorT,
R ¼ AorG,Y¼ CorT,H¼ A, T or C). These primers
were used for PCR of cDNA generated from total RNA
isolated from meiotic tissues of C. cinereus.ThePCR
product was used to screen the C. cinereus kZAP II cDNA
library as described previously [27]. 5¢RLM-RACE
(Ambion) was performed according to the manufacturer’s
protocol. In the case of p48, two primers were synthesized
corresponding to amino acid motifs conserved among
species: sense primer (5¢-CAGAAGGAGCTCGTCTT
CGAYATHGAYHT-3¢) and antisense primer (5¢-GGAT
GCAGAAGGGGGACTTNARNARRTG-3¢). Identical
methods were used as with p140 except that reagents for
5¢-RACE and 3¢-RACE. Assays were performed according
to the manufacturer’s protocol (Invitrogen). The DDBJ/
EMBL/GenBANK accession numbers of the nucleotide
sequences reported in this paper are AB072453 and
AB072454 for the p140 and the p48 subunits, respectively.
Polyclonal antibodies for p140 and p48
The truncated cDNA corresponding to amino acid residues
from 299 to 718 of p140 was cloned into the expression
vector pET21a (Novagen) in the NdeIandHindIII sites.
The coding region of p48 was cloned into the expression
vector pET21a (Novagen) in the NdeIandXhoIsites.
Recombinant his-tagged proteins were expressed in Escheri-
chia coli BL21 (DE3) (Novagen) and purified using a

Ni-nitrilotriacetic acid column (Amersham).
The polyclonal antiserum against the His-tagged p140
protein was raised in rabbit. To remove the antibody
fraction that reacts with the His
6
protein from the
antiserum, 2 mL of the anti-p140 serum was incubated
with 200 lL of the crude extracts of the E. coli BL21 (DE3)
expressing the His
6
protein. After centrifugation 39 000 g
1
,
the anti-p140 Ig was obtained using N-hydroxysuccinimide
(NHS)
2
-activated Sepharose beads (Amersham) that were
prebound to His-tagged p140 proteins. The polyclonal
antiserum against the p48 subunit was raised in rat. The
purification of p48 polyclonal antibody was perfomed by
the same methods as those described for p140 except that
the NHS-activated Sepharose beads were prebound to the
His-tagged p48 proteins.
Immunostaining of meiotic
C. cinereus
nuclei
Immunostaining of meiotic C. cinereus nuclei was perfomed
as described in previous reports [29–31] with minor modi-
fications. C. cinereus gills were fixed in 4% (v/v) formal-
dehyde, 50 m

M
NaH
2
PO
4
-HCl pH 6.5, 5 m
M
MgCl
2
,5%
(w/v) polyethylene glycol 8000 and 5 m
M
EGTA at room
temperature for 20 min. The gills were applied to glass
slides, cover slips were affixed, and slides were placed in
liquid N
2
for10s.Thecoverslipswerethenremovedand
slides were dried for 2 h. The slides were washed three times
in NaCl/P
i
pH 7.4 for 10 min. The cell walls were then
digested with 0.4% (w/v) Novozyme 234 (Novo Nordisk) in
50 m
M
NaH
2
PO
4
-HCl pH 6.5, 5 m

M
MgCl
2
for 3 min
each, washed three times in NaCl/P
i
pH 7.4 for 10 min, and
then were soaked in a detergent solution (1% Triton X-100,
5m
M
EGTA, 1 m
M
phenylmethanesulfonyl-fluoride, in
NaCl/P
i
(pH 7.4) at room temperature for 20 min. The
2138 S. Namekawa et al. (Eur. J. Biochem. 270) Ó FEBS 2003
slides were then washed three times in NaCl/P
i
(pH 7.4) for
10 min each and incubated overnight at 4 °Cwitha1:100
dilution of either the anti-p140 Ig or the anti-p48 Ig. The
next day, slides were washed three times with NaCl/P
i
(pH 7.4) containing 1% (w/v) BSA for 10 min and treated
at 37 °C for 8 h with either anti-(rabbit IgG) Ig conjugated
with Alexa fluoro 488 (Molecular Probes) for anti-p140
or anti-(rat IgG) Ig conjugated with Alexa fluoro 568
(Molecular Probes) for anti-p48. Both secondary antibodies
were diluted 1 : 1000. Slides were then washed three times

with NaCl/P
i
(pH 8.4) for 10 min. Slides were stained with a
solution of 20 gÆL
)1
DAPI. Specimens were examined under
a fluorescence microscope (Olympus BH2).
Purification of DNA polymerase a from the fruiting
bodies at the meiotic prophase I of
C. cinereus
TEMG buffer contains the following reagents: 50 m
M
Tris/
HCl (pH 7.5), 1 m
M
EDTA, 5 m
M
2-mercaptoethanol,
15% glycerol, plus protease inhibitors (1 lgÆmL
)1
leupeptin
and pepstatin, and 1 m
M
phenylmethanesulfonyl fluoride).
DNA polymerase a was purified using the protocol
described below. All procedures were carried out at 4 °C.
Approximately 20 g of frozen cap tissue at the zygotene
to the pachytene stages were suspended in 40 mL of Tris/
HCl, EDTA, 2-mercaptoethanol, glycerol (TEMG)
3

buffer
containing 600 m
M
NaCl, ground through a French press
and sonicated (20 kHz, 10 s). The supernatant was collected
after centrifugation at 39 000 g for 10 min, and saturated
with 30–55% ammonium sulfate. The ammonium sulfate
precipitate was collected by centrifugation and the pellet
was resuspended in 20 mL of TEMG buffer containing
300 m
M
NaCl and dialyzed against TEMG buffer contain-
ing 300 m
M
NaCl. This fraction was passed through a
DEAE–Sepharose column equilibrated with the same buffer
containing 300 m
M
NaCl. The fraction was diluted
three-fold with the same buffer containing no salt. The
fraction was loaded onto a phospho–cellulose column
(2.5 cm · 5 cm) equilibrated with TEMG containing
100 m
M
NaCl, and eluted with a 200-mL NaCl gradient
from 100 m
M
to 700 m
M
in TEMG buffer. An active DNA

polymerase peak was eluted at 450 m
M
NaCl. The active
fractions were dialyzed against TEMG buffer and were
loaded onto a DEAE–Sepharose column (2.5 · 5cm)
equilibrated with TEMG buffer containing 50 m
M
NaCl.
Then proteins were eluted with a 90-mL NaCl gradient from
50 m
M
to 600 m
M
in TEMG buffer. The DNA polymerase
activity was detected at 150 m
M
NaCl in a single peak. The
active fractions were dialyzed against TEMG buffer and
loaded onto a Heparin agarose column (FPLC system,
5 mL) that had been equilibrated with TEMG buffer
containing 200 m
M
NaCl. The proteins were eluted with a
30-mL NaCl gradient from zero to 1
M
in TEMG buffer.
The active fractions were eluted at 600 m
M
and dialyzed
against TEMG buffer. Then the samples were loaded onto a

single strand DNA cellulose column (1.5 · 4cm)thathad
been equilibrated with TEMG buffer. The proteins were
eluted with a 30-mL NaCl gradient from zero to 600 m
M
in
TEMG buffer. The active fraction was eluted at 150 m
M
of
NaCl and then was dialyzed against TEMG buffer. The
combined active fraction obtained from the ssDNA cellu-
lose column was loaded onto a MonoQ column (FPLC)
that had been equilibrated with TEMG buffer containing
100 m
M
NaCl. The fractions were eluted with a 40-mL
NaCl gradient from zero to 400 m
M
in TEMG buffer. In
the MonoQ column, DNA pol a-primase complex was
eluted at 250 m
M
NaCl as a single peak. The purified
proteins were desalted, concentrated, and stored at )20 °C
in a solution containing 50 m
M
Tris/HCl (pH 7.5), 1 m
M
EDTA, 5 m
M
b-mercaptoethanol, 50% glycerol, 0.01%

Nonidet P-40, and 20% sucrose.
DNA primase assay
TheDNAprimaseassayusingaDE81filterwasthesameas
the DNA polymerase assay except that the RNA priming
activity was monitored by Klenow enzyme (Fig. 5). The
assay mixture (20 mL) contained the following: 50 m
M
Tris/
HCl (pH 7.5) containing 5 m
M
MgCl
2
,5m
M
dithiothreitol,
2m
M
ATP, 0.02
M
dATP, 0.04 U Klenow fragment, 20 l
M
of [
3
H]dATP (4800 c.p.m.Æpmol
)1
), 40 gÆmL
)1
of poly(dT),
and 15% glycerol. Incubation was carried out at 37 °Cfor
30 min.

The primase activity was also tested as follows (Fig. 5B).
The reaction mixture (20 mL) contains 50 m
M
Tris/HCl
(pH 7.5), 10 m
M
MgCl
2
,5m
M
dithiothreitol, 2 m
M
ATP,
80 gÆmL
)1
of poly(dT), 20 l
M
dATP, 4 lCi of [a-
32
P]dATP
(6000 CiÆmmol
)1
), and 4 lL of purified fraction. Incubation
was perfomed at 37 °C for 60 min, and terminated by
ethanol precipitation. The samples were resuspended in
30 lL of formamide dye [90% formamide (v/v) with
bromophenol blue and xylene cyanol], and heated to
95 °C for 5 min. After separation on a 10% polyacryl-
amide/7
M

urea denaturing gel, products were detected by
autoradiography.
Glycerol density gradient sedimentation
Glycerol density gradient sedimentation was performed as
described by Mizuno et al. [10] with some modifications.
Proteins were extracted from C. cinereus meiotic tissues in a
buffer containing 50 m
M
Tris/HCl (pH 7.5), 300 m
M
NaCl,
10% glycerol, 1 m
M
EDTA, 5 m
M
2-mercaptoethanol, and
proteinase inhibitors [1 m
M
phenylmethylsulfonyl fluoride,
1 lgÆmL
)1
leupeptin, 1 lgÆmL
)1
pepstatin A, and Protease
Inhibitor Cocktail (Roche)]. Aliquots of 100 lL containing
1 mg of crude extract protein were layered onto 1900 lLof
a linear 15–35% glycerol gradient in a buffer containing
50 m
M
Tris/HCl (pH 7.5), 300 m

M
KCl, 1 m
M
EDTA and
0.1% Triton X-100. Protein markers [bovine serum albumin
(BSA: 4.4 S), yeast alcohol dehydrogenase (YADH: 7.4 S),
and bovine catalase (BCAT: 11.3 S)] were loaded simulta-
neously with crude extract as an internal control. Centri-
fugation was perfomed at 55 000 r.p.m.
4
for 16 h at 4 °C
(Beckman TLS-55). Fractions were collected from the top
of the gradient. Elution of each subunit was detected by
Western analysis using antibodies specific for each subunit.
Other methods
Southern, Northern, and Western blotting analyses were
performed as described previously [27,28]. Probes were
made using the cDNAs corresponding to the amino acids
1154–1211 of the p140 or 118–314 for the p48 protein.
Ó FEBS 2003 Meiotic expression of Coprinus DNA polymerase a (Eur. J. Biochem. 270) 2139
Immunostaining of meiotic C. cinereus tissues was
performed as described previously [28]. The DNA poly-
merase assay was performed as described previously [21].
Active gel electrophoresis was performed as described
previously [32].
Results
Isolation of homologues of the pol a catalytic and the
primase small subunits in
C. cinereus
meiotic tissues

To study the role of DNA pol a in the meiotic cell cycle, we
first cloned the cDNA encoding the pol a catalytic subunit
and the primase small subunit in C. cinereus. Two degen-
erate PCR primers (see Materials and methods) were used
with cDNA template from C. cinereus meiotic tissues. The
PCR products were used as probes to obtain cDNA clones
encoding the pol a catalytic and the primase small subunits
by hybridization screening of a kZAPII cDNA library of
C. cinereus coupled with 5¢-and3¢-RACE methods.
The cDNA clones containing 4260 bp and 1248 bp were
isolated and found to encode the C. cinereus orthologs of
the pol a catalytic subunit and the primase small subunit,
respectively. The cDNA for the pol a catalytic subunit
encodes a 1420 amino acid long protein, the predicted
molecularmassofwhichis161kDa.ThecDNAforthe
primase small subunit encodes a 416 amino acid protein,
the predicted molecular mass of which is 47.9 kDa. As
described below, the cDNA for the pol a catalytic subunit
and the primase small subunit encode proteins having
140 kDa and 48 kDa, respectively (see below). Thus we
named them p140 and p48. As shown in Fig. 1A and 1B,
both polypeptides contain the regions conserved among
their eukaryotic counterparts. Identity of the amino acid
sequence of p140 with other eukaryotic counterparts is as
follows: Schizosaccharomyces pombe: 38.9%, Saccharomyces
cerevisiae:34.9%,Homo sapiens: 31.8%, Mus musculus:
30.6%, Drosophila melanogaster: 26.7%, Oryza sativa:
27.7%. The amino acid sequence identity of p140 with
corresponding regions of S. pombe pol a are 27.0% for the
nonconserved region (1–449aa) and 44.6% for the con-

served region (450–1420aa). Amino acid sequence identity
of p48 with other eukaryotic counterparts is as follows:
S. pombe: 40.8%, S. cerevisiae: 38.4%, H. sapiens:35.6%,
M. musculus: 35.4%, D. melanogaster: 32.0%. Southern
hybridization analysis revealed that each gene exists as a
single copy in the C. cinereus genome (data not shown).
Northern hybridization analyses of p140 and p48
from meiotic cells
The expression profile of each subunit of DNA polymerase
a-primase has been shown in mammalian somatic cells [13]
and yeast [33,34]. The transcripts of both DNA polymerase
a-primase are strongly induced early in meiosis [33,34]. To
Fig. 1. Schematic representation of Coprinus cinereus DNA polymerase
a and its counterparts. (A) Comparison of C. cinereus DNA polymerase
a catalytic subunit (p140) with its eukaryotic counterparts. The seven
black boxes represent the highly conserved regions (I to VII) among
eukaryotic and prokaryotic DNA polymerases. The five grey boxes
(A–E) represent the conserved regions among DNA polymerase a
catalytic subunits. The hatched box near the C-terminus represents a
zinc finger motif (Zn). (B) Comparison of C. cinereus primase small
subunit (p48) with itseukaryotic counterparts. The five grey boxes (I–V)
represent the conserved regions among DNA primase small subunits.
Fig. 2. Increase of p140 and p48 transcript in leptotene to zygotene during
meiotic prophase I stages. Northern analysis of p140 and p48 expression
at various stages of meiosis. Each lane contained 20 lgoftotalRNA
isolated from fruiting caps of C. cinereus at premeiotic S phase (lane 1),
karyogamy (K + 0), the leptotene/zygotene (K + 2 and K + 5), and
the pachytene (K + 7) stages. The blot was hybridized with either p140
cDNA (upper panel), p48 cDNA (middle panel), or glyceraldehyde
3-phosphate dehydrogenase (G3PDH) cDNA (lower panel).

2140 S. Namekawa et al. (Eur. J. Biochem. 270) Ó FEBS 2003
investigate the expression profile of DNA polymerase a in
C. cinereus where each stage of meiotic cell division is
separable (see Materials and methods section), we obtained
total RNA from a synchronous culture extracted at various
periods after the induction of meiosis, and then analyzed by
Northern hybridization for p140 and p48 (Fig. 2). Tran-
scripts of p140 and p48 accumulated in the premeiotic
S-phase as expected (Fig. 2). Interestingly, despite the lack
of bulk DNA synthesis, we observed significant levels of
expression of p140 and p48 in both the leptotene and
zygotene stages (Fig. 2). In both cases, the highest level of
expression was observed 2 h after karyogamy (K + 2 in
Fig. 2), and baseline levels were restored 5 h after karyo-
gamy (K + 5 in Fig. 2). These results indicate that p140
and p48 were primarily expressed during the leptotene and
zygotene stages when bulk DNA replication is already
completed. We also investigated the distribution of p140
and p48 transcripts using in situ hybridization. Both
transcripts were found exclusively in the meiotic tissues of
the fruiting bodies (data not shown).
Distribution of p140 and p48 during meiotic
cell division
Anti-p140 and anti-p48 were generated as described in
Materials and methods and their specificities were tested
Fig. 3. Localization of p140 and p48 in meiotic tissues. (A) Anti-p140 (left panel) and anti-p48 (right panel) Igs were generated and tested for their
specificity using western analysis of crude extracts of meiotic tissues. Numbers indicate the positions and sizes of the protein standards. (B) Meiotic
tissues from K +0, K + 2, K + 5, K + 7, and K + 9 were sectioned. Sections of the fruiting body were stained with anti-p140 polyclonal Ig (green)
and anti-p48 polyclonal Ig (red). The nuclei were counterstained with DAPI. As a negative control for primary antibodies, preimmune serum of rat
or rabbit (1 : 100 dilution) were tested. The slides were then treated for 4 h with anti-(rabbit IgG) Ig conjugated with Alexa fluoro 488 (Molecular

Probes) or with anti-(rat IgG) Ig conjugated with Alexa fluoro 568 (Molecular Probes), diluted 1 : 1000 as the secondary antibody. (C) Schematic of
synchronous meiotic progression is illustrated to the right. In C. cinereus meiosis begins with karyogamy (K). Fruiting caps containing meiotic cells
at the leptotene to the zygotene stages are observed between 04.00 h (K + 0) and 09.00 h (K + 5). Cells at the pachytene stage are observed between
10.00 h (K + 6) and 11.00 h (K + 7). Meiotic recombination occurs in meiotic prophase I. Meiosis I is reductional division, in which the
chromosome number is reduced in half. Meiosis II cells are observed between 12.00 h (K + 8) and 14.00 h (K + 10). Meiosis II is equational
division in which four nuclei are produced and sporulate.
Ó FEBS 2003 Meiotic expression of Coprinus DNA polymerase a (Eur. J. Biochem. 270) 2141
using Western analysis of crude extract of meiotic tissues
(Fig. 3A). The distributions of p140 and p48 in meiotic
tissues were examined by in situ immunofluorescence
staining using these antibodies (Fig. 3B). Intense signals
for p140 and p48 were detected exclusively in tissues at
meiotic prophase I stages (K + 0 to K + 7 in Fig. 3B),
and at meiosis II stage (K + 9 in Fig. 3B). Notably, both
proteins were colocalized in the same compartment of
tissues where meiotic cells are abundant (yellow) (Fig. 3B).
We also stained nuclei with anti-p140 and p48 Igs to
determine their nuclear localization in the cells at various
stages ranging from premeiotic S phase to meiosis II
(Fig. 4). Both proteins were found in the nuclei throughout
the meiotic stages we tested (Fig. 4). Interestingly, the
signals of p140 and p48 did not always colocalized, while
overlapping signals were abundant in meiotic nuclei
(Fig. 4). During the pachytene stage, there was a noticeable
separation of p48 and p140 signals (white arrows in Fig. 4).
This suggests that p48 and p140 do not always form a
complex during the meiotic cell cycle.
The biochemical profiles of DNA polymerase a from
crude extract of
C. cinereus

meiotic prophase I tissues
To study the mode of pol a-primase complex formation at
meiotic prophase I and its biochemical features, we
isolated the pol a-primase complex from meiotic pro-
phase I tissues in the zygotene and pachytene stages. All
purification procedures are summarized in the Materials
and methods section. Figure 5A shows the elution profile
Fig. 4. Nuclear localization of p140 and p48 in meiotic cells. Nuclei from the basidia were stained with anti-p140 polyclonal Ig (green) and anti-p48
polyclonal Ig (red) as described in the Materials and methods section. The nuclei were counterstained with DAPI. Meiotic stages of these cells are
indicated on the left.
2142 S. Namekawa et al. (Eur. J. Biochem. 270) Ó FEBS 2003
of the DNA polymerase and primase activities from the
phospho-cellulose column. The ammonium sulfate preci-
pitation fraction was passed through a DEAE–Sepharose
column and separated into two primase activity peaks by
the phospho-cellulose column, one of which is not
associated with polymerase activity (fraction I) and other
containing polymerase activity (fraction II) (Fig. 5A).
Western analysis indicates that fraction I contains p48,
while fraction II contains both p140 and p48. Using an
indirect primase assay where even quite low levels of the
RNA priming reaction are detected by either intrinsic or
extrinsic DNA polymerase activity, we also confirmed that
fraction I contains primase activity, while fraction II
contains both DNA polymerase and primase activities
(Fig. 5B). It should be noted that intrinsic DNA
polymerase activity in fraction II has quite low processivity
for DNA synthesis which is a typical feature for replicative
DNA polymerases [1]. Taken together with the immuno-
staining data, this result suggests that there is a population

of p48 that is not complexed with p140 during meiosis.
Alternatively, the p48 subunit may be unstably associated
with the intact complex in vivo.
In order to determine the biochemical features of the
pol a-primase complex, we further purified fraction II using
five different columns as described in the Materials and
methods section. The active fraction from the ssDNA
cellulose column chromatography was purified 307,000-fold
(Table 1). The protein concentration in the fractions after
the MonoQ column was too low to measure (Table 1). The
elution point from the Sephacryl S-300 (Hiprep) gel
filtration column indicates that the native molecular weight
of the complex is approximately 330 kDa (data not shown).
The purified complex displays the features of a typical
pol a-primase complex as reported in other species [1,4]. As
shown in Fig. 6A and B, the enzyme in the active fraction
from the MonoQ column was recognized by anti-p140 and
anti-p48. Active gel analyses, in which the protein complex
from the MonoQ column was further separated by SDS/
PAGE and incubated with DNA substrate during a
renaturation process, indicates that the catalytic core of
DNA polymerase activity resides in the 140 kDa protein
molecule (Fig. 6C). The purified complex also contains
primase activity (data not shown). We found that the DNA
polymerase activity in the purified complex is sensitive to
aphidicolin, and insensitive to ddTTP (data not shown) as
seen in pol a family members in other species. We also
observed that DNA synthesis by the purified complex
occurs in a low processive or distributive manner (data not
shown), and that DNA polymerase activity is inhibited by

high ionic strength (data not shown) as seen in typical
replicative DNA polymerases [1,4].
During a series of purification procedures, we found
that the bulk of primase activity is always associated
with DNA polymerase activity after phospho–cellulose
column fractionation (data not shown). This suggests
that the primase activity separated from the intact complex
may be caused by dissociation of the two subunits that
occurs in in vivo, rather than as a result of instability of
the complex. Taken together with the immunostaining
data, these results suggest that some proportion of primase
dissociates from the p140-containing complex in vivo
during meiotic prophase I.
Fig. 5. Separation of primase activity from pol a-primase complex by
phospho–cellulose column chromatography. (A) DNA polymerase
(circles) and DNA primase activities (squares) were measured in the
elution fractions from phospho-cellulose chromatography. NaCl
concentration in each fractions are shown (triangles). Western analysis
with anti-p140 polyclonal Ig and anti-p48 polyclonal Ig are shown
below the graph. The primase activity was separated into two peaks,
one of which is not associated with polymerase activity (fraction I),
while the other is (fraction II). (B) Primase activity in the fraction I and
II. Synthetic primer-independent DNA polymerization occurs by
internal primase and DNA polymerase activities (see the Materials and
methods section). The reactions in the presense of klenow fragment
contained 1 U of klenow fragment. Lanes 1, 2 represent control lanes
with no protein.
Ó FEBS 2003 Meiotic expression of Coprinus DNA polymerase a (Eur. J. Biochem. 270) 2143
Mode of complex formation of pol a-primase complex
during meiotic stages

We monitored the complex formation of pol a-primase
during meiosis in detail by applying crude extracts from
various stages of meiotic cells to a glycerol density gradient
sedimentation. Various protein markers to crude extract
were used as internal controls (Fig. 7C and data not
shown). Eluted samples were analysed by Western blotting
using anti-p140 (Fig. 7A) and anti-p48 (Fig. 7B). We found
that p140 is eluted in fractions 27–32 when extract from
tissues at premeiotic S phase or K + 3 were used. The peak
p140 signal appeared in fractions 30–32 and its sedimenta-
tion coefficient was 11S. On the other hands, p140 was
eluted at a point corresponding to the lower sedimentation
coefficient, when extract from K + 6 or K + 9 was used
(Fig. 7A, K + 9, fractions 26–32). This suggests that the
mode of pol a–primase complex formation is altered upon
progression of the meiotic cell cycle. Unlike p140, we found
no significant differences in the p48 elution profile: the
signals of p48 were observed throughout fractions 17–32
regardless of the stage in meiosis (Fig. 7B). These results
suggest that the amount of intact pol a-primase complex
(11S) declined gradually during meiotic development.
Furthermore, it appears that pol a–primase complex for-
mation is altered during meiotic prophase I.
Discussion
Biochemical features of pol a-primase complex
during meiotic stages
In this examination of the DNA polymerase a-primase
complex, we determined the molecular mass of the
pol a catalytic subunit and the primase small subunit from
a basidiomycete, C. cinereus. The predicted molecular mass

based on the amino acid sequences from cloned genes is
160 kDa for the pol a catalytic subunit and 48 kDa for the
primase small subunit, respectively. Western analysis of
both crude extract and purified fractions using an antibody
to each subunit indicated that the molecular masses of the
pol a catalytic subunit and primase small subunit are
140 kDa and 48 kDa, respectively. It is possible that the
p140 we detected in Western analysis is a degraded form of
the pol a catalytic subunit, which is often observed in other
species such as Drosophila [35]. Alternatively, a protein
modification may affect the migration of the pol a catalytic
subunit in SDS/PAGE, although we have found that pol a
catalytic subunit purified from somatic cells also shows
140 kDa in SDS/PAGE analysis (data not shown).
Table 1. Purification step of C. cinereus DNA polymerase a. One unit
(1 U) of DNA polymerase was defined as the amount needs to catalyze
the incorporation of 1 pmol of [
3
H]-d TTP into a DNA polymer in
30 min. Protein concentrations were determined using the Coomassie
Brilliant Blue binding technique. ND, not detected.
Purification step
Total
activity
(mU)
Total
Protein
(mg)
Specific
activity

(mUÆmg
)1
)
Purification
(fold)
Crude extract 557
Ammonium sulfate 0.36 547 0.000658 1
Phospho-cellulose 14.8 84.0 0.176 267
DEAE–Sepharose 12.5 4.12 3.03 4 600
Heparin agarose 20.4 0.40 51.0 77 500
ssDNA cellulose 28.3 0.14 202 307 000
Mono Q 41.5 ND
Fig. 6. Characterization of C. cinereus DNA polymerase a. (AandB)
Western analysis of the active fraction from the MonoQ column using
anti-p140 (A) and anti-p48 Igs. (C) Analysis of the active fraction from
the monoQ column by active gel electrophoresis.
Fig. 7. Fractionation of the endogenous C. cinereus DNA polymerase a
during meiotic development by glycerol density gradient sedimentation.
(A and B) Crude extracts of C. cinereus meiotic tissues (Premeiotic S,
K + 3, K + 6, and K + 9) were fractionated by 15–35% glycerol
gradient sedimentation. The fractions were subjected to Western
blotting. Complex formation was monitored by Western analysis using
anti-p140 (A) and anti-p48 Igs (B). The following protein markers were
simultaneously loaded with the extract onto the gradient solution:
Bovine serum albumin (BSA: 4.4 S), yeast alcohol dehydrogenase
(YADH: 7.4S), and bovine catalase (BCAT, 11.3 S). SDS/PAGE was
perfomed and gel was stained with Coomassie Brilliant Blue (C). Each
elution sample was analysed by SDS/PAGE gel and gels were stained
with Coomassie Brilliant Blue. As there is no significant difference in
elution profile of protein markers, only protein markers that are eluted

with the K + 9 extract is shown.
2144 S. Namekawa et al. (Eur. J. Biochem. 270) Ó FEBS 2003
The pol a-primase complex is generally regarded as a
stable protein complex. In both yeast and mammals, the
pol a-primase complex can always be isolated as an intact
complex. Separation of DNA primase activity from the
intact complex usually requires reagents that alter protein
complex conformation such as urea [36] or ethylene glycol
[37,38]. In this study, we found that pol a-primase complex
formation is altered upon meiotic differentiation. Both in situ
immunofluorescence staining of meiotic nuclei and purifi-
cation on a phospho-cellulose column suggest that a sub
population of p48 can be dissociated from the complex
containing p140. Glycerol density gradient sedimentation
revealed reduced levels of intact pol a-primase complex, and
a gradual shift of p140 signals toward a lower sedimentation
coefficient during meiotic development. One explanation for
this shift upon progression of meiosis is that a p140
monomer [21], or a subcomplex such as p140 with the
mediator subunit, forms during meiotic development. In
contrast to the p140 elution profile in glycerol density
gradient sedimentation, p48 was more broadly distributed
across fractions regardless of the meiotic stage. These results
also may indicate the presence of a subcomplex containing
p48 during meiotic development. Recently, Mizuno et al.
[9] showed that various components of the pol a-primase
complex formation exist in NIH3T3 cells. They detected the
coexistence of the intact pol a-primase complex with both
a free p68 monomer and a free p54-p46 dimer [9]. Taken
together with our observations, these results suggest that

complex formation may be an important regulator of
optimal pol a activity. Alternatively, each subcomplex
could have distinct biological functions in the cells.
Expression of DNA polymerase a in meiotic cells
In Lilium cells at the late leptotene to the zygotene stages, it
has been shown that DNA synthesis occurs at long DNA
gaps that are not replicated during premeiotic S phase [22].
Also, DNA repair synthesis was observed at the pachytene
stage during meiotic prophase I [22]. In C. cinereus,we
showed that the p140 and p48 transcripts are present not
only at the premeiotic S phase, but also at the meiotic
prophase I stages. Interestingly, p140 and p48 transcripts
were increased at the leptotene through the zygotene stages
when chromosome paring occurs. In mammals, during the
transition from quiescent to proliferating, steady state pol a
mRNA levels, translation rate, and enzyme activity are all
increased [13,14]. Furthermore, in growing mouse cells the
transcripts of all four pol a subunits have been observed
throughout the cell cycle and slightly increase in number
prior to S phase [13]. Taking these observations into
consideration, the slight increase of p140 transcripts we
found may be associated with DNA synthesis that occurs
during meiotic prophase I, although there is not any direct
evidence of this. A conditional mutant for p140 and p48
would directly address the question of pol a ¢s role in meiotic
chromosome paring and homologous recombination.
Acknowledgements
We would like to thank Dr Jessica Halow and Ms. Joan Hamilton
(Fred Hutchinson Cancer Research Center) and Dr Norikazu Aoyagi
(Tokyo University of Science) for critical reading of the manuscript. We

thank Dr Takeshi Mizuno (RIKEN) for technical advice on glycerol
density gradient sedimentation. We thank Dr M. E. Zolan and
Dr M. Celerin (Indiana University) and Dr Takashi Kamada
(Okayama University) for technical advice on immunostaining. We
thank Dr Seisuke Kimura, Dr Masahiko Oshige, Dr Yoshiyuki
Mizushina, Ms Yuri Tsuya, Mr Narumichi Aoshima, Mr Kei
Watanabe and Mr Kazuki Iwabata (Tokyo University of Science) for
technical assistance.
References
1. Kornberg, A. & Baker, T.A. (1992) DNA Replication.W.H.
Freeman and Company, New York.
2. Baker, T.A. & Bell, S.P. (1998) Polymerases and the replisome:
machines within machines. Cell 92, 295–305.
3. Tsurimoto, T., Melendy, T. & Stillman, B. (1990) Sequential
initiation of lagging and leading strand synthesis by two different
polymerase complexes at the SV40 DNA replication origin.
Nature 346, 534–539.
4. Wang, T.S. (1991) Eukaryotic DNA polymerases. Annu. Rev.
Biochem. 60, 513–552.
5. Ferrari, M., Lucchini, G., Plevani, P. & Foiani, M. (1996) Phos-
phorylation of the DNA polymerase a-primase B subunit is
dependent on its association with the p180 polypeptide. J. Biol.
Chem. 271, 8661–8666.
6. Copeland, W.C. & Wang, T.S. (1991) Catalytic subunit of human
DNA polymerase a overproduced from baculovirus-infected
insect cells. Structural and enzymological characterization. J. Biol.
Chem. 266, 22739–22748.
7. Copeland, W.C. & Wang, T.S. (1993) Enzymatic characterization
of the individual mammalian primase subunits reveals a biphasic
mechanism for initiation of DNA replication. J. Biol. Chem. 268,

26179–26189.
8. Schneider, A., Smith, R.W., Kautz, A.R., Weisshart, K., Grosse,
F. & Nasheuer, H.P. (1998) Primase activity of human DNA
polymerase a-primase. Divalent cations stabilize the enzyme
activity of the p48 subunit. J. Biol. Chem. 273, 21608–21615.
9. Mizuno, T., Yamagishi, K., Miyazawa, H. & Hanaoka, F. (1999)
Molecular architecture of the mouse DNA polymerase a-primase
complex. Mol. Cell. Biol. 19, 7886–7896.
10. Mizuno, T., Ito, N., Yokoi, M., Kobayashi, A., Tamai, K.,
Miyazawa, H. & Hanaoka, F. (1998) The second-largest subunit
of the mouse DNA polymerase a-primase complex facilitates both
production and nuclear translocation of the catalytic subunit of
DNA polymerase alpha. Mol. Cell. Biol. 18, 3552–3562.
11. Foiani,M.,Marini,F.,Gamba,D.,Lucchini,G.&Plevani,P.(1994)
The B subunit of the DNA polymerase a-primase complex in
Saccharomyces cerevisiae executes an essential function at the
initial stage of DNA replication. Mol. Cell. Biol. 14, 923–933.
12. Foiani, M., Liberi, G., Lucchini, G. & Plevani, P. (1995) Cell
cycle-dependent phosphorylation and dephosphorylation of the
yeast DNA polymerase a-primase B subunit. Mol. Cell. Biol. 15,
883–891.
13. Miyazawa, H., Izumi, M., Tada, S., Takada, R., Masutani, M.,
Ui, M. & Hanaoka, F. (1993) Molecular cloning of the cDNAs for
the four subunits of mouse DNA polymerase a-primase complex
and their gene expression during cell proliferation and the cell
cycle. J. Biol. Chem. 268, 8111–8122.
14. Wahl, A.F., Geis, A.M., Spain, B.H., Wong, S.W., Korn, D. &
Wang, T.S. (1988) Gene expression of human DNA polymerase
a during cell proliferation and the cell cycle. Mol. Cell. Biol. 8,
5016–5025.

15. Sakaguchi, K., Hotta, Y. & Stern, H. (1980) Chromatin-asso-
ciated DNA polymerase activity in meitic cells of lily and mouse;
its stimulation by meiotic helix-destabilizing protein. Cell. Struct.
Funct. 5, 323–334.
Ó FEBS 2003 Meiotic expression of Coprinus DNA polymerase a (Eur. J. Biochem. 270) 2145
16. Sakaguchi, K. & Lu, B.C. (1982) Meiosis in Coprinus:character-
ization and activities of two forms of DNA polymerase during
meiotic stages. Mol. Cell. Biol. 2, 752–757.
17. Matsuda, S., Takami, K., Sono, A. & Sakaguchi, K. (1993) A
meiotic DNA polymerase from Coprinus cinereus: further purifi-
cation and characterization. Chromosoma 102, 631–636.
18. Gomi, K. & Sakaguchi, K. (1994) A new meiotic protein factor
which enhances activity of meiotic DNA polymerase from
Coprinus cinereus. Biochem. Biophys. Res. Commun. 198, 1232–1239.
19. Takami, K., Matsuda, S., Sono, A. & Sakaguchi, K. (1994) A
meiotic DNA polymerase from a mushroom, Agaricus bisporus.
Biochem. J. 299 (2), 335–340.
20. Lu, B.C. & Jeng, D.Y. (1975) Meiosis in Coprinus VII. The pre-
karyogamy S-phase and the postkaryogamy DNA replication in
C. lagopus. J. Cell. Sci. 17, 461–470.
21. Sawado, T. & Sakaguchi, K. (1997) A DNA polymerase a cata-
lytic subunit is purified independently from the tissues at meiotic
prometaphase I of a basidiomycete, Coprinus cinereus. Biochem.
Biophys. Res. Commun. 232, 454–460.
22. Hotta, Y. & Stern, H. (1971) Analysis of DNA synthesis during
meiotic prophase in Lilium. J. Mol. Biol. 55, 337–355.
23. Hotta, Y., Tabata, S. & Stern, H. (1984) Replication and nicking
of zygotene DNA sequences. Control by a meiosis-specific protein.
Chromosoma 90, 243–253.
24. Hotta, Y., Tabata, S., Bouchard, R.A., Pinon, R. & Stern, H.

(1985) General recombination mechanisms in extracts of meiotic
cells. Chromosoma 93, 140–151.
25. Paques, F. & Haber, J.E. (1999) Multiple pathways of
recombination induced by double-strand breaks in Saccharomyces
cerevisiae. Microbiol. Mol. Biol. Rev. 63, 349–404.
26. Villeneuve, A.M. & Hillers, K.J. (2001) Whence meiosis? Cell 106,
647–650.
27. Nara,T.,Saka,T.,Sawado,T.,Takase,H.,Ito,Y.,Hotta,Y.&
Sakaguchi, K. (1999) Isolation of a LIM15/DMC1 homolog from
the basidiomycete Coprinus cinereus and its expression in relation
to meiotic chromosomepairing. Mol. General Genet. 262, 781–789.
28. Hamada, F., Namekawa, S., Kasai, N., Nara, T., Kimura, S.,
Sugawara, F. & Sakaguchi, K. (2002) Proliferating cell nuclear
antigen from a basidiomycete, Coprinus cinereus.Alternative
truncation and expression in meiosis. Eur. J. Biochem. 269,
164–174.
29. Celerin, M., Merino, S.T., Stone, J.E., Menzie, A.M. & Zolan,
M.E. (2000) Multiple roles of Spo11 in meiotic chromosome
behavior. EMBO J. 19, 2739–2750.
30. Tanabe,S.&Kamada,T.(1994)Theroleofastralmicrotubulesin
conjugate division in the dikaryon of Coprinus cinereus. Exp.
Mycol. 18, 338–348.
31. Terasawa, M., Shinohara, A., Hotta, Y., Ogawa, H. & Ogawa, T.
(1995) Localization of RecA-like recombination proteins on
chromosomes of the lily at various meiotic stages. Genes. Dev. 9,
925–934.
32. Seto, H., Hatanaka, M., Kimura, S., Oshige, M., Tsuya, Y.,
Mizushina, Y., Sawado, T., Aoyagi, N., Matsumoto, T., Hashi-
moto, J. & Sakaguchi, K. (1998) Purification and characterization
of a 100 kDa DNA polymerase from cauliflower inflorescence.

Biochem. J. 332 (2), 557–563.
33. Johnston, L.H., White, J.H., Johnson, A.L., Lucchini, G. &
Plevani, P. (1987) The yeast DNA polymerase I transcript is
regulatedinboththemitoticcellcycleandinmeiosisandis
also induced after DNA damage. Nucleic Acids. Res. 15,
5017–5030.
34. Johnston, L.H., White, J.H., Johnson, A.L., Lucchini, G. &
Plevani, P. (1990) Expression of the yeast DNA primase gene,
PRI1, is regulated within the mitotic cell cycle and in meiosis. Mol.
General Genet. 221, 44–48.
35. Kuroda, K. & Ueda, R. (1995) A 130 kDa polypeptide
immunologically related to the 180 kDa catalytic subunit of DNA
polymerase a-primase complex is detected in early embryos of
Drosophila. J. Biochem. (Tokyo) 117, 809–818.
36. Kaguni, L.S., Rossignol, J.M., Conaway, R.C. & Lehman, I.R.
(1983) Isolation of an intact DNA polymerase-primase from
embryos of Drosophila melanogaster. Proc. Nat. Acad. Sci. USA,
80, 2221–2225.
37. Yagura, T., Kozu, T. & Seno, T. (1982) Mouse DNA
polymerase accompanied by a novel RNA polymerase activity:
purification and partial characterization. J. Biochem. (Tokyo) 91,
607–618.
38. Tseng, B.Y. & Ahlem, C.N. (1983) A DNA primase from mouse
cells. Purification and partial characterization. J. Biol. Chem. 258,
9845–9849.
2146 S. Namekawa et al. (Eur. J. Biochem. 270) Ó FEBS 2003