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

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: [email protected]
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.

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

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
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

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

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%

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

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

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).

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

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

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

(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

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

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