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: 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. 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. 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Dissociation of DNA polymerase a-primase complex during meiosis in Coprinus cinereus Satoshi Namekawa, Fumika Hamada,. 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