Dissociation of DNA polymerase a-primase complex during meiosisSatoshi Namekawa, Fumika Hamada, Tomoyuki Sawado†, Satomi Ishii, Takayuki Nara‡, Takashi Ishizaki, Takashi Ohuchi, Takao Ar
Trang 1Dissociation of DNA polymerase a-primase complex during meiosis
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 cinereusDNA polymerase a catalytic subunit (p140) and
C cinereusprimase 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 I n situ immuno-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, DNA repair 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: kengo@rs.noda.tus.ac.jp
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.
Present address: Department of Food Science and Human Nutrition,
University of Illinois, 62801.
(Received 16 January 2003, revised 5 March 2003,
accepted 12 March 2003)
Trang 2allele, 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 noncrosscross-over pathways accompany DNA
synthe-sis [25,26] Despite the importance of DNA synthesynthe-sis 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 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 ofC 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 37C in total
darkness and from day 7 onward at 25C 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¢) ( N ¼ A, C, G or T, S ¼ C or G, W ¼ A or T,
R¼ A or G, Y ¼ C or T, H ¼ A, T or C) These primers were used for PCR of cDNA generated from total RNA isolated from meiotic tissues of C cinereus The PCR 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 NdeI and HindIII sites The coding region of p48 was cloned into the expression vector pET21a (Novagen) in the NdeI and XhoI sites 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 His6 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 His6protein After centrifugation 39 000 g
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 meioticC 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 mMNaH2PO4-HCl pH 6.5, 5 mMMgCl2, 5% (w/v) polyethylene glycol 8000 and 5 mMEGTA at room temperature for 20 min The gills were applied to glass slides, cover slips were affixed, and slides were placed in liquid N2for 10 s The cover slips were then removed and slides were dried for 2 h The slides were washed three times
in NaCl/Pi pH 7.4 for 10 min The cell walls were then digested with 0.4% (w/v) Novozyme 234 (Novo Nordisk) in
50 mM NaH2PO4-HCl pH 6.5, 5 mM MgCl2 for 3 min each, washed three times in NaCl/PipH 7.4 for 10 min, and then were soaked in a detergent solution (1% Triton X-100,
5 mM EGTA, 1 mM phenylmethanesulfonyl-fluoride, in NaCl/P (pH 7.4) at room temperature for 20 min The
Trang 3slides were then washed three times in NaCl/Pi(pH 7.4) for
10 min each and incubated overnight at 4C with a 1 : 100
dilution of either the anti-p140 Ig or the anti-p48 Ig The
next day, slides were washed three times with NaCl/Pi
(pH 7.4) containing 1% (w/v) BSA for 10 min and treated
at 37C 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/Pi(pH 8.4) for 10 min Slides were stained with a
solution of 20 gÆL)1DAPI Specimens were examined under
a fluorescence microscope (Olympus BH2)
Purification of DNA polymerase a from the fruiting
bodies at the meiotic prophase I ofC cinereus
TEMG buffer contains the following reagents: 50 mMTris/
HCl (pH 7.5), 1 mM EDTA, 5 mM 2-mercaptoethanol,
15% glycerol, plus protease inhibitors (1 lgÆmL)1leupeptin
and pepstatin, and 1 mMphenylmethanesulfonyl fluoride)
DNA polymerase a was purified using the protocol
described below All procedures were carried out at 4C
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)
containing 600 mMNaCl, 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 mMNaCl and dialyzed against TEMG buffer
contain-ing 300 mM NaCl This fraction was passed through a
DEAE–Sepharose column equilibrated with the same buffer
containing 300 mM 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 mM NaCl, and eluted with a 200-mL NaCl gradient
from 100 mMto 700 mMin TEMG buffer An active DNA
polymerase peak was eluted at 450 mM NaCl The active
fractions were dialyzed against TEMG buffer and were
loaded onto a DEAE–Sepharose column (2.5· 5 cm)
equilibrated with TEMG buffer containing 50 mM NaCl
Then proteins were eluted with a 90-mL NaCl gradient from
50 mMto 600 mMin TEMG buffer The DNA polymerase
activity was detected at 150 mMNaCl 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 mMNaCl The proteins were eluted with a
30-mL NaCl gradient from zero to 1Min TEMG buffer
The active fractions were eluted at 600 mM and dialyzed
against TEMG buffer Then the samples were loaded onto a
single strand DNA cellulose column (1.5· 4 cm) that had
been equilibrated with TEMG buffer The proteins were
eluted with a 30-mL NaCl gradient from zero to 600 mMin
TEMG buffer The active fraction was eluted at 150 mMof
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 mM NaCl The fractions were eluted with a 40-mL NaCl gradient from zero to 400 mMin TEMG buffer In the MonoQ column, DNA pol a-primase complex was eluted at 250 mM NaCl as a single peak The purified proteins were desalted, concentrated, and stored at)20 C
in a solution containing 50 mMTris/HCl (pH 7.5), 1 mM
EDTA, 5 mM b-mercaptoethanol, 50% glycerol, 0.01% Nonidet P-40, and 20% sucrose
DNA primase assay The DNA primase assay using a DE81 filter was the same as 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 mMTris/ HCl (pH 7.5) containing 5 mMMgCl2, 5 mMdithiothreitol,
2 mMATP, 0.02MdATP, 0.04 U Klenow fragment, 20 lM
of [3H]dATP (4800 c.p.m.Æpmol)1), 40 gÆmL)1of poly(dT), and 15% glycerol Incubation was carried out at 37C for
30 min
The primase activity was also tested as follows (Fig 5B) The reaction mixture (20 mL) contains 50 mM Tris/HCl (pH 7.5), 10 mM MgCl2, 5 mMdithiothreitol, 2 mMATP,
80 gÆmL)1of poly(dT), 20 lMdATP, 4 lCi of [a-32P]dATP (6000 CiÆmmol)1), and 4 lL of purified fraction Incubation was perfomed at 37C 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
95C for 5 min After separation on a 10% polyacryl-amide/7Murea 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 mMTris/HCl (pH 7.5), 300 mMNaCl, 10% glycerol, 1 mMEDTA, 5 mM2-mercaptoethanol, and proteinase inhibitors [1 mMphenylmethylsulfonyl fluoride,
1 lgÆmL)1leupeptin, 1 lgÆmL)1pepstatin A, and Protease Inhibitor Cocktail (Roche)] Aliquots of 100 lL containing
1 mg of crude extract protein were layered onto 1900 lL of
a linear 15–35% glycerol gradient in a buffer containing
50 mMTris/HCl (pH 7.5), 300 mMKCl, 1 mMEDTA 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
(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
Trang 4Immunostaining 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 inC 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¢- and 3¢-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 molecular mass of which is 161 kDa The cDNA for the 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 its eukaryotic 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 lg of total RNA 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).
Trang 5investigate 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.
Trang 6using 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 ofC 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.
Trang 7of 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.
Trang 8Mode 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
Fig 6 Characterization of C cinereus DNA polymerase a ( A and B)
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.
Trang 9The 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.
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