Báo cáo khoa học: Interaction between Lim15/Dmc1 and the homologue of the large subunit of CAF-1 – a molecular link between recombination and chromatin assembly during meiosis pot

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Interaction between Lim15/Dmc1 and the homologue ofthe large subunit of CAF-1 a molecular link betweenrecombination and chromatin assembly during meiosisSatomi Ishii*,†, Akiyo Koshiyama*, Fumika N. Hamada, Takayuki Y. Nara, Kazuki Iwabata,Kengo Sakaguchi and Satoshi H. NamekawaDepartment of Applied Biological Science, Faculty of Science and Technology, Tokyo University of Science, JapanKeywordschromatin assembly; chromatin assemblyfactor 1 (CAF-1); Lim15/Dmc1; meioticrecombination; proliferating cell nuclearantigen (PCNA)CorrespondenceK. Sakaguchi, Department of AppliedBiological Science, Faculty of Science andTechnology, Tokyo University of Science,2641 Yamazaki, Noda-shi, Chiba-ken278-8510, JapanFax: +81 4 7123 9767Tel: +81 4 7124 1501 (ext. 3409)E-mail: kengo@rs.noda.tus.ac.jpWebsite: http://www.tus.ac.jp/en/grad/riko_app_bio.htmlS. H. Namekawa, Department of MolecularBiology, Massachusetts General Hospital,and Department of Genetics, HarvardMedical School, Boston, MA 02114, USAFax: +1 617 726 6893Tel: +1 617 726 5966E-mail: namekawa@molbio.mgh.harvard.eduPresent address†Quantum Beam Science Directorate, JapanAtomic Energy Agency, Gunma, Japan*These authors contributed equally to this work(Received 7 January 2008, revised 24February 2008, accepted 25 February 2008)doi:10.1111/j.1742-4658.2008.06357.xIn eukaryotes, meiosis leads to genetically variable gametes through recom-bination between homologous chromosomes of maternal and paternal ori-gin. Chromatin organization following meiotic recombination is critical toensure the correct segregation of homologous chromosomes into gametes.However, the mechanism of chromatin organization after meiotic recombi-nation is unknown. In this study we report that the meiosis-specificrecombinase Lim15/Dmc1 interacts with the homologue of the largestsubunit of chromatin assembly factor 1 (CAF-1) in the basidiomyceteCoprinopsis cinerea (Coprinus cinereus). Using C. cinerea LIM15/DMC1(CcLIM15) as the bait in a yeast two-hybrid screen, we have isolated theC. cinerea homologue of Cac1, the largest subunit of CAF-1 in Saccharo-myces cerevisiae, and named it C. cinerea Cac1-like (CcCac1L). Two-hybridassays confirmed that CcCac1L binds CcLim15 in vivo. b-Galactosidaseassays revealed that the N-terminus of CcCac1L preferentially interactswith CcLim15. Co-immunoprecipitation experiments showed that theseproteins also interact in the crude extract of meiotic cells. Furthermore, wedemonstrate that, during meiosis, CcCac1L interacts with proliferating cellnuclear antigen (PCNA), a component of the DNA synthesis machineryrecently reported as an interacting partner of Lim15/Dmc1. Takentogether, these results suggest a novel role of the CAF-1–PCNA complexin meiotic events. We propose that the CAF-1–PCNA complex modulateschromatin assembly following meiotic recombination.AbbreviationsATCC, American Type Culture Collection; Cac1, chromatin assembly complex 1; CAF-1, chromatin assembly factor 1; CcCac1L,Coprinopsis cinerea Cac1-like; CPRG, chlorophenol red-b-D-galactopyranoside; DSB, double-strand break; IPTG, isopropyl thio-b-D-galactoside;PCNA, proliferating cell nuclear antigen; RLM-RACE, RNA ligase-mediated-RACE; RU, resonance unit; SPR, surface plasmon resonance.2032 FEBS Journal 275 (2008) 2032–2041 ª 2008 The Authors Journal compilation ª 2008 FEBSIn eukaryotes, sexual reproduction is achieved by theconjugation of genetically variable gametes, which aregenerated during meiosis in the parental germline. Mei-osis consists of two rounds of chromosome segrega-tion, resulting in gametes with half the number ofchromosomes in order to prepare for conjugation.During prophase of the first meiotic division, recombi-nation takes place between homologous chromosomesof maternal and paternal origin. This is followed bythe segregation of maternal and paternal copies ofeach chromosome. A physical connection at the site ofhomologous recombination, called the chiasma, orientshomologous chromosome pairs towards opposite spin-dle poles at meiosis I [1]. Therefore, chromatin organi-zation following meiotic recombination is required toestablish the chiasma and to segregate homologouschromosomes.Meiotic recombination comprises several stepsbeginning with meiosis-specific double-strand breaks(DSBs). A single-strand overhang is formed by exonu-clease activity and invades the homologous double-stranded region of the other allele. These stepsof homology search and recombination are catalysedby two bacterial RecA homologues, Rad51 andLim15/Dmc1. Rad51 catalyses both somatic andmeiotic recombination, whereas Lim15/Dmc1 is meio-sis-specific [2–5]. Rad51 and Lim15/Dmc1 are compo-nents of a multiprotein complex at the site ofrecombination [6,7]. In order to understand the mecha-nisms of meiotic recombination, much effort has beenmade to identify additional components of the Rad51and Lim15/Dmc1 complex, in particular Lim15/Dmc1interacting partners.Recent analysis has identified various interactingpartners of Lim15/Dmc1, which seem to be involved inhomology search and strand exchange. Tid1/Rdh54,an SWI2/SNF2 family of chromatin-remodellingfactors, promotes the co-localization of Rad51 andLim15/Dmc1 [8]. The heterodimeric complex of Hop2and Mnd1 stimulates strand exchange of Lim15/Dmc1[9–11]. The meiosis-specific proteins Mei5 and Sae3form a complex with Lim15/Dmc1 and are necessaryfor the assembly of Lim15/Dmc1 [12,13]. Furthermore,the DNA mismatch repair protein MSH4 (MutShomologue 4) [14], the tumor suppressor protein p53[15], DNA topoisomerase II [16], the sumoylation pro-tein Ubc9 [17] and the DNA synthesis-related factorproliferating cell nuclear antigen (PCNA) [18] havebeen reported to interact with Lim15/Dmc1. Theseproteins seem to participate in the modulation ofLim15/Dmc1. However, how chromatin is organizedfollowing meiotic recombination has not beendescribed.In order to explore chromatin organization aftermeiotic recombination, we designed experiments toinvestigate the possible interactions between recombi-nation proteins and chromatin assembly factors. Inthis article, we report that the largest subunit homo-logue of chromatin assembly factor 1 (CAF-1) is anovel interacting partner of Lim15/Dmc1. CAF-1 con-sists of three subunits that are highly conservedamongst yeast, plant, fly and human [19–23]. CAF-1deposits histones H3 and H4 onto newly synthesizedDNA after replication and repair [24–26]. In addition,the largest subunit of CAF-1 interacts with PCNAduring replication [27], in nucleotide excision repair[28] and in DSB repair [29,30]. Despite much accumu-lating evidence regarding the role of CAF-1 in chroma-tin assembly following various DNA synthesis events,its involvement in chromatin assembly following mei-otic recombination is unknown. In this study, we testthe involvement of CAF-1 in meiotic events. We pro-pose a novel role of the CAF-1–PCNA complex inchromatin assembly following meiotic recombination.ResultsIsolation of Coprinopsis cinerea Cac1-like(CcCac1L) by two-hybrid screening usingCcLim15 as baitTo isolate proteins that interact with CcLim15, we per-formed a yeast two-hybrid screen using CcLim15 asbait. A clone was isolated which had moderate aminoacid similarity with the largest subunit of humanCAF-1 (p150) [19] and the largest subunit of Saccharo-myces cerevisiae CAF-1 (Cac1, chromatin assemblycomplex 1) [20]. The sequence similarities of this clonewith human and S. cerevisiae homologues were foundto be 26% and 23%, respectively. Hence, this clonewas identified as C. cinerea Cac1-like (CcCac1L).CcCac1L encodes a predicted product of 812 aminoacid residues with a molecular mass of 120 kDa. Thehighly charged KER (lysine/glutamate/arginine-rich;242–360 amino acids) and ED (glutamate/aspartate-rich; 522–578 amino acids) domains in CcCac1L areconserved amongst human and S. cerevisiae homo-logues (Fig. 1A). The KER and ED domains areknown to interact directly with newly synthesized H3/H4 histones [19,21].CcCac1L interacts with CcLim15To confirm the specificity of interaction betweenCcCac1L and CcLim15, we performed yeast two-hybrid and b-galactosidase assays (Fig. 1C,D). Next,S. Ishii et al. Link between Lim15/Dmc1 and the CAF-1–PCNA complexFEBS Journal 275 (2008) 2032–2041 ª 2008 The Authors Journal compilation ª 2008 FEBS 2033we sought to determine which region of CcCac1Lwas responsible for binding to CcLim15. The N-ter-minus (CcCac1L-N; amino acids 1–381) containedthe KER domain, whereas the C-terminus (CcCac1L-C; amino acids 382–812) contained the ED domain(Fig. 1B). Two-hybrid assays demonstrated thatCcLim15 interacts with either of the truncatedmutants of CcCac1L in the mild selection medium[SD3: lacking histidine, leucine and tryptophan(–His/–Leu/–Trp)], and that CcLim15 preferentiallyinteracts with CcCac1L-N in the stringent selectionmedium [SD4: lacking adenine, histidine, leucineand tryptophan (–Ade/–His/–Leu/–Trp)] (Fig. 1C).The interaction between the truncated mutants ofCcCac1L and CcLim15 was confirmed by b-galactosi-dase assays, which demonstrated a higher bindingaffinity of CcCac1L-N than CcCac1L-C to CcLim15(Fig. 1D).Characterization of CcCac1L during meiosisThe data above strongly suggest a novel function ofCAF-1 as a binding partner of Lim15/Dmc1. How-ever, currently there are no observations availabledescribing the meiotic role of CAF-1. Therefore, wesought to examine the distribution of CcCac1L dur-ing meiosis. First, in order to determine the geneexpression profile of CcCac1L during meiotic develop-ment, we performed northern analyses at each stageduring meiotic development. Total RNA wasextracted from basidia in synchronous culture at 1 hintervals after the induction of meiosis. CcCac1L wasexpressed at the premeiotic S phase, at the time ofgenomic DNA replication (Fig. 2A). Homologouschromosomes start to align at the leptotene/zygotenestage. Then, fully synapsed homologues are observedat the pachytene stage. CcCac1L began to accumulateat the leptotene and zygotene stage, and decreasedafter the pachytene stage (Fig. 2A). This expressionprofile suggests the specific induction of CcCac1Ltranscription during the meiotic prophase. Interest-ingly, CcLIM15 showed specific expression during themeiotic prophase [16,31], suggesting that CcCac1Land CcLIM15 are expressed robustly at the samestage.Next, we examined the distribution of CcCac1L andCcLim15 in the meiotic nuclei by immunostaining. Weraised a specific antibody against CcCac1L using apurified fragment of CcCac1L, and confirmed its speci-ficity in crude extracts of meiotic cells by western anal-ysis (Fig. 2B). CcCac1L protein localized to nucleifrom the premeiotic S phase until the pachytene stage,and then disappeared at metaphase I (Fig. 2C).Consistent with our previous observations [16,17],CcLim15 localized within nuclei from the leptotene/zygotene stage to the pachytene stage, and disappearedat metaphase I (Fig. 2C). Importantly, significantamounts of CcCac1L and CcLim15 were localizedwithin the nuclei from the leptotene/zygotene stage tothe pachytene stage.To examine the interaction between CcCac1L andCcLim15 during meiosis, we performed co-immuno-precipitation analysis using cell extracts from themeiotic prophase in C. cinerea. CcLim15 wasco-immunoprecipitated by anti-CcCac1L IgG, but notby control rabbit IgG (Fig. 2D). The reciprocalA BC D Fig. 1. Molecular cloning of CcCac1L and its interaction with CcLim15. (A) Schematic diagram of the CAF-1 large subunits in human, C. cine-rea and S. cerevisiae. The KER and ED domains are represented by black and grey boxes, respectively. (B) Schematic diagram of the trunca-tion mutants of CcCac1L. (C) Interaction between CcCac1L and CcLim15 in a yeast two-hybrid assay. The inserts in the activation domain(AD) and DNA-binding domain (BK) are shown. +, binding; ), no binding. The mild selection medium (SD3: –His/–Leu/–Trp) and the stringentselection medium (SD4: –Ade/–His/–Leu/–Trp) were tested. (D) Interaction between CcCac1L and CcLim15 in yeast using quantitativeb-galactosidase assays. b-Galactosidase assays with the other vector pairs in (C) showed little activity below the detection limit ofabsorbance, and were not quantified.Link between Lim15/Dmc1 and the CAF-1–PCNA complex S. Ishii et al.2034 FEBS Journal 275 (2008) 2032–2041 ª 2008 The Authors Journal compilation ª 2008 FEBSexperiment confirmed the specific interaction ofCcCac1L and CcLim15 in the crude extracts of mei-otic tissues (Fig. 2E). Taken together, these resultssuggest that the interaction between CcLim15 andCcCac1L is related to specific events during the mei-otic prophase.Interaction between CcCac1L and CcPCNAduring meiosisCAF-1 forms a complex with PCNA to deposithistones at the site of newly synthesized DNA duringreplication and repair. The results above raised thenovel possibility that CAF-1 is involved in chromatinassembly following recombination-associated DNAsynthesis during meiosis. If so, CAF-1 must form acomplex with PCNA in the meiotic prophase. PCNAis expressed abundantly in meiotic prophase I [32].Interestingly, recent analysis has revealed that PCNAinteracts with Lim15/Dmc1 at the time of meioticrecombination [18]. To determine whether CcCac1Linteracts with CcPCNA during meiosis, we performedco-immunoprecipitation analysis using cell extractsfrom the meiotic prophase in C. cinerea. CcPCNA wasspecifically co-immunoprecipitated by anti-CcCac1LIgG, but not by control rabbit IgG (Fig. 3A). TheA C B D E Fig. 2. Interaction between CcCac1L and CcLim15 during meiosis. (A) Northern analysis of CcCac1L expression at various stages duringmeiosis. Each lane contained 20 lg of total RNA isolated from meiotic cells of C. cinerea at the premeiotic S phase and at every hour afterkaryogamy (the initiation of meiosis) to 9 h after karyogamy. The blot was hybridized with either CcCac1L (top panel) or C. cinerea glyceral-dehyde 3-phosphate dehydrogenase (CcG3PDH; bottom panel). (B) Western analysis of the rat anti-CcCac1L IgG. The cell extract at the mei-otic prophase was examined. (C) Nuclear localization of CcLim15 and CcCac1L in the nuclei of C. cinerea meiotic cells. Meiotic nuclei werestained with anti-CcCac1L IgG (red) and anti-CcLim15 IgG (green). The nuclei were then counterstained with 4¢,6-diamidino-2-phenylindole di-hydrochloride n-hydrate (DAPI). The meiotic stages are indicated on the left. (D, E) Immunoprecipitation of CcCac1L and CcLim15 from thecell extract at the meiotic prophase; 20 mg of cell extract was incubated with anti-CcLim15 IgG, anti-CcCac1L IgG or control rabbit serum-conjugated beads. After washing the beads, the bound proteins were eluted and analysed by western analysis using anti-CcLim15 IgG (D) oranti-CcCac1L IgG (E). Lane 1, 100 lg of crude extract was loaded.S. Ishii et al. Link between Lim15/Dmc1 and the CAF-1–PCNA complexFEBS Journal 275 (2008) 2032–2041 ª 2008 The Authors Journal compilation ª 2008 FEBS 2035reciprocal experiment confirmed the specific interactionof CcCac1L and CcPCNA in the crude extracts ofmeiotic tissues (Fig. 3B).Next, we sought to examine the binding affinity ofCcCac1L to CcPCNA by performing BIAcore analysiswith the truncated mutants of CcCac1L, as shown inFig. 1B. The BIAcore system enabled us to detect thesurface plasmon resonance (SPR), which measures theinteraction between a ligand on a detection surface(sensor chip) and a ligand that is injected. First, weconjugated CcPCNA to a sensor chip onto whicheither CcCac1L-N or CcCac1L-C was injected. Consis-tent with results from other organisms [27,33],CcCac1L-N specifically bound to CcPCNA (Fig. 3C),confirming the evolutionarily conserved CAF-1–PCNAcomplex. From these results, we suggest a novel roleof the CAF-1–PCNA complex during the meiotic pro-phase together with the meiosis-specific recombinase,Lim15/Dmc1.DiscussionIn this study, we identified CcCac1L as a novelinteracting partner of CcLim15. Furthermore, it wasshown that CcCac1L interacts with CcPCNA duringthe meiotic prophase. Several DNA synthesis eventstake place during the meiotic prophase, even aftergenome-wide replication at the premeiotic S phase[32,34]. In the current model, DNA synthesis isrequired in the molecular events of meiotic recombi-nation [35,36]. Meiotic DSBs are processed to single-strand overhangs, followed by single-strand invasionto the other allele. Recombination results in eithercrossover products (exchanging the flanking DNAarms between homologues) or non-crossover products(non-exchange of DNA arms). Both pathwaysaccompany DNA synthesis following recombination[35,36]. Given the coordination of CAF-1 and PCNAin various DNA synthesis events, a CAF-1–PCNAcomplex may be involved in chromatin assembly fol-lowing DNA synthesis events during the meiotic pro-phase. Based on the current model, we propose therole of the CAF-1–PCNA complex during meiosis(Fig. 4). PCNA recruits DNA polymerase at theend of single-strand regions that are coated byLim15/Dmc1 (Fig. 4A,B). Consistent with this model,DNA polymerases and DNA ligases are active dur-ing this stage [37–40]. After DNA synthesis, CAF-1is recruited to the site of the Lim15/Dmc1–PCNAcomplex and deposits histone H3 (or a histone vari-ant) and H4 on the naked DNA to restore thenucleosome structure (Fig. 4C). Because of the vari-ous interactions of Lim15/Dmc1–CAF-1–PCNA, wesuggest that they act in multiple ways at the site ofmeiotic recombination and contribute to the subse-quent assembly of chromatin. Therefore, there maybe coordination between meiotic recombination andCAF-1-dependent nucleosome assembly before theresolution of Holliday junctions (Fig. 4C).The CAF-1–PCNA complex senses DNA damageand subsequently contributes to chromatin assemblyat the site of DNA repair [33], including nucleotideexcision repair [28] and DSB repair [29,30]. Duringthe process of chromatin assembly, CAF-1 depositsnew H3.1 histones on the site of repair-associatedDNA synthesis without the recycling of parentalhistones; therefore, CAF-1-dependent chromatinassembly results in a chromatin memory of damageat a repair site [41]. Similarly, CAF-1 may establisha chromatin memory at the site of DNA synthesisABCFig. 3. Interaction between CcCac1L and CcPCNA during meiosis.(A, B) Co-immunoprecipitation of CcCac1L and PCNA in the cellextract at the meiotic prophase; 20 mg of cell extract was incu-bated with anti-CcPCNA IgG, anti-CcCac1L IgG or control rabbitserum-conjugated beads. After washing the beads, the boundproteins were eluted and analysed by western analysis with anti-PCNA IgG (A) or anti-CcCac1L IgG (B). Lane 1, 100 lg of crudeextract was loaded. (C) Detection of SPR using a Biacore assay.Truncation mutants of CcCac1L were injected onto a CcPCNAconjugated chip. The binding affinity is inversely related to thedissociation constant (KD), which is a ratio of the dissociation (Kd)and association (Ka) rates (KD= Kd/Ka). ND, not detected.Link between Lim15/Dmc1 and the CAF-1–PCNA complex S. Ishii et al.2036 FEBS Journal 275 (2008) 2032–2041 ª 2008 The Authors Journal compilation ª 2008 FEBSfollowing meiotic recombination. The site of cross-over recombination becomes the chiasma, requiredfor the appropriate segregation of homologous chro-mosomes. Chiasma formation involves the coordi-nated local change of DNA and the surroundingchromatin environment [42]. One tantalizing possibil-ity is that CAF-1-dependent chromatin memorydirects chiasma formation to newly synthesized DNAat the site of recombination. CAF-1-dependent his-tone deposition is an established key early step forchromatin organization in mitosis [19,24–26]. Multi-ple steps are involved in organizing the chromatinstructure after histone deposition by CAF-1. There-fore, the CAF-1–PCNA complex may be the centralplayer establishing the memory of recombination,leading to unique nuclear organization duringmeiosis.Materials and methodsCulture of C. cinerea and collection of fruitingbodiesThe basidiomycete Coprinopsis cinerea (Coprinus cinereus)(strain #56838) was purchased from the American TypeCulture Collection (ATCC), Manassas, VA, USA. Theculture methods and procedures for the photoinduction ofmeiosis were performed as described previously [38,43].Yeast two-hybrid screeningThe C. cinerea cDNA library in meiotic tissues was con-structed using a Time Saver cDNA Synthesis Kit (GEHealthcare UK Ltd, Little Chalfont, UK). Yeasttwo-hybrid screening was carried out using the MATCH-MAKER GAL4 Two-Hybrid System 3 (Clontech, Moun-tain View, CA, USA). The cDNA encoding full-lengthCcLim15 was fused in-frame with the GAL4 DNA-bindingdomain in the pBKDT7 vector as bait. The cDNA librarywas subsequently cloned into the pGADT7 vector encodingthe GAL4 activation domain, and used as prey in the two-hybrid experiments. Both the GAL4 fusion bait and theprey plasmids were transformed into the yeast strain,AH109 (Clontech), by standard lithium acetate transforma-tion. Putative interacting clones were subsequently isolatedbased on their ability to activate the expression of theGAL4 selectable marker genes, thus producing growth onSD minimal medium lacking adenine, histidine, leucineand tryptophan (SD4: –Ade/–His/–Leu/–Trp). To confirmgalactosidase activity, colonies that grew under this selectivecondition were plated onto SD4 medium with X-a-galacto-sidase. Purified plasmids from yeast clones were electropo-rated into Escherichia coli DH10B. After the plasmid DNAhad been prepared, the cDNA inserts were sequenced andthe corresponding gene was identified by blast analysis.cDNA cloning of CcCac1LOne of the interacting factors identified in our screen wasfound to encode the CcCac1L C-terminus, consisting ofthe amino acid region 382–812 (CcCac1L-C) (Fig. 1B). Toobtain the full-length CcCac1L cDNA, 5¢RNA ligase-medi-ated-RACE (5¢RLM-RACE) (Ambion, Austin, TX, USA)and 3¢RLM-RACE (Invitrogen, Carlsbad, CA, USA)experiments were performed, each according to the manu-facturer’s protocol. The DDBJ/EMBL/GenBank accessionnumber of the nucleotide sequence for CcCac1L reportedin this study is AB074897.ABCFig. 4. Model of chromatin assembly following meiotic recombina-tion. (A) After DSB formation, Lim15/Dmc1 coats the single-strandend during strand invasion. (B) PCNA recruits the DNA polymeraseto the site of Lim15/Dmc1. The broken line represents newly syn-thesized DNA. (C) CAF-1 forms a complex with Lim15/Dmc1 andPCNA. CAF-1 deposits histones H3 and H4 or other factors, suchas histone variants (indicated as ‘?’), on the newly synthesizedDNA.S. Ishii et al. Link between Lim15/Dmc1 and the CAF-1–PCNA complexFEBS Journal 275 (2008) 2032–2041 ª 2008 The Authors Journal compilation ª 2008 FEBS 2037Two-hybrid assayTo confirm the direct interaction between proteins or pro-tein fragments, the appropriate bait and prey constructswere co-transformed into yeast cells, and two-hybrid assayswere performed using the MATCH-MAKER Kit (Clon-tech), according to the manufacturer’s instructions. Thefull-length CcLim15, CcCac1L, CcCac1L-N and CcCac1L-C fragments were cloned into pGADT7 and pGBKT7. Thevector pairs indicated in Fig. 1C were co-transformed intothe yeast strain AH109. Controls for self-activating fusionproteins were carried out in each of these assays by trans-formation of specific expression constructs with a pGBKT7or pGADT7 empty vector. Transformants were then platedonto three types of selection medium: SD2, –Leu/–Trp;SD3, –His/–Leu/–Trp; SD4, –Ade/–His/–Leu/–Trp.b-Galactosidase assays were performed in chlorophenolred-b-d-galactopyranoside (CPRG)-based liquid cultureusing the individual colonies that grew in SD3 medium,according to the Yeast Protocols Handbook (Clontech).Northern blottingNorthern blotting was performed as described previously[44]. The region of the CcCac1L cDNA corresponding to1146–2346 bp was used as a probe.AntibodiesA polyclonal antibody against the CcCac1L protein wasraised in rabbit and rat using the purified 382–812 aminoacid fragment expressed as a His-CcCac1L-C protein inE. coli. The specificity of the antibodies was confirmed bywestern analysis as described previously [44,45]. A poly-clonal antibody against CcLim15 was also raised asdescribed previously [45]. Anti-CcPCNA IgGs and purifiedrecombinant His-tagged CcPCNA (His-CcPCNA) havebeen described previously [44].In vivo co-immunoprecipitationRabbit anti-CcCac1L polyclonal IgGs rabbit anti-CcLim15 polyclonal IgG or control rabbit serum wascoupled with CNBr-activated sepharose beads, accordingto the manufacturer’s instructions {20 mg aliquots ofcrude extracts from meiotic tissues were prepared in buf-fer D [buffer C, as described below, with 0.6 m NaCl andprotease inhibitors (1 mm phenylmethanesulfonyl fluoride,1 lm leupeptin and 1 lm pepstatin A)]}. The extracts inbuffer D were then incubated with either 70 lL of pri-mary antibody or with control rabbit serum-conjugatedbeads for 1 h at 4 °C. The beads were then collected bycentrifugation at 800 g for 30 s. After resuspension of thebeads in buffer E (0.15 m NaCl in buffer D), the superna-tant was removed by centrifugation at 9100 g for 30 s. Thebound material was eluted from the beads with 20 lLof buffer F (50 mm glycine/HCl, pH 2.5, and 0.01%Triton X-100). After neutralization of the pH by the additionof 1 m Tris/HCl, pH 7.5, the bound material was analysed byimmunoblotting with either anti-CcCac1L or anti-CcLim15IgG, both at a dilution of 1 : 1000. To test the interactionbetween CcCac1L and CcPCNA in vivo, anti-CcCac1L andanti-CcPCNA IgGs were used and in vivo immunoprecipita-tion experiments were performed as described previously[44]. The CcCac1L cDNA corresponding to 1146–2346 bpwas used as a probe.Immunostaining of nuclei of C. cinerea meioticcellsImmunostaining of nuclei of C. cinerea meiotic cells wasperformed as described previously [38]. A 1 : 100 dilutionwas used of both rabbit anti-CcLim15 and rat anti-CcCac1L primary IgGs. We also employed a 1 : 1000 dilu-tion of both anti-rabbit IgG conjugated with Alexa Fluoro488 (Invitrogen) for anti-CcLim15 and anti-rat IgG conju-gated with Alexa Fluoro 568 (Invitrogen) for anti-CcCac1Las secondary antibodies.ProteinsA truncated cDNA corresponding to the N-terminus (resi-dues 1–381, as shown in Fig. 1B) of CcCac1L (CcCac1L-N)was cloned into the Bam HI and NotI sites of the expressionvector pET21a(+) (Novagen, Gibbstown, NJ, USA). TheC-terminal insert of CcCac1L (CcCac1L-C, residues 382–812) was cloned into the NcoI and XhoI sites of thepET21d(+) expression vector (Novagen). The followingprimer pairs were used for subsequent PCR amplificationof these cDNAs. CcCac1L-N: 1F, 5¢-CGGGATCCATGTCGGGAGCAGATTCA; 381R, 5¢-TGCTACTTCTCTCAGCGGCCGCATTCTTAT. CcCac1L-C: 382F, 5¢-CATGCCATGGTGTCAGGGGATGTAGAAATG; 812R,5¢-GAGATTTCAGTTTCGTCACTCGAGCGG. To over-express N-terminal hexahistidine-tagged CcCac1L-N(His-CcCac1L-N) and CcCac1L-C (His-CcCac1L-C), E. coliBL21 cells (DE3) (Novagen) carrying the expression plasmidfor each gene were grown in 2 · YT medium (16 gÆL)1poly-peptone, 10 gÆL)1yeast extract, 5 gÆL)1NaCl) containing1 lgÆmL)1ampicillin at 37 °C. After reaching an absorbanceat 600 nm of 0.6, isopropyl thio-b-d-galactoside (IPTG) wasadded to these cultures at a final concentration of 1 mm,and the cells were incubated for an additional 5 h at 25 °C.The bacterial cells were then harvested by centrifugation at4500 g for 15 min, and the resulting cell pellet was resus-pended in 15 mL of ice-cold buffer A [20 mm Tris/HCl,pH 7.9, 10% glycerol, 0.5 m NaCl, 5 mm imidazole con-taining protease inhibitors (1 mm phenylmethanesulfonylLink between Lim15/Dmc1 and the CAF-1–PCNA complex S. Ishii et al.2038 FEBS Journal 275 (2008) 2032–2041 ª 2008 The Authors Journal compilation ª 2008 FEBSfluoride, 1 lm leupeptin and 1 lm pepstatin A)]. The cellswere then lysed by the addition of 1 mgÆmL)1lysozyme, stir-red on ice for 30 min and sonicated. Insoluble material wasremoved by centrifugation at 26 000 g for 15 min. Proteinswere loaded onto a 5 mL Hi-trap chelating column (GEHealthcare UK Ltd.), and bound proteins were eluted witha 20 mL linear gradient of 0.05–1 m imidazole in buffer B(buffer A with 0.1% Nonidet P40). The eluted protein frac-tion was then dialysed against buffer C (50 mm Tris/HCl,pH 7.5, 0.05 m NaCl, 1 mm EDTA, 5 mm 2-mercaptoetha-nol, 10% glycerol, 0.1% Nonidet P40), and the dialysatewas loaded onto a heparin column (GE Healthcare UKLtd.) equilibrated with 0.05 m NaCl in buffer B. Afterwashing, fractions were collected with a 20 mL linear gradi-ent of 0–0.5 m NaCl in buffer B. The eluted protein wasthen dialysed against 0.05 m NaCl in buffer B, and loadedonto a MonoQ HR5/5 column (GE Healthcare UK Ltd).After washing, the fractions were again collected with20 mL of a linear gradient of 0–0.5 m NaCl in buffer B.Fractions containing the recombinant proteins were verifiedby SDS-PAGE, pooled and then dialysed against storagebuffer (NaCl/Pi, pH 7.4, 50% glycerol). Recombinant His-tagged CcLim15 (His-CcLim15) was expressed in E. coliand purified as described previously [31].Surface plasmon resonanceAnalysis of both His-CcCac1L-N and His-CcCac1L-Cbinding to His-CcPCNA was performed using a BIAcoreBiosensor instrument (GE Healthcare Bio-Sciences,Uppsala, Sweden), according to the manufacturer’s proto-col. A sensor chip (CM 5 research grade) was activated bythe N-hydroxysuccinimide/N-ethyl-N¢-(dimethylaminopro-pyl)carbodiimide coupling reaction, and 55 lL of couplingbuffer (10 mm sodium acetate, pH 4.0) containing theHis-CcPCNA protein (625 nm) was injected over the chipat a rate of 20 lLÆmin)1. His-CcPCNA was covalentlybound to the sensor chip surface via carboxyl moieties onthe dextran. Unreacted N-hydroxysuccinimide ester groupswere inactivated using 1 m ethanolamine/HCl (pH 8.0).HBS-EP buffer (10 mm Hepes, pH 7.4, 150 mm NaCl,3mm EDTA, 0.005% Tween 20) was passed continuouslyover the sensor chip. The binding levels were measured inresonance units (RU); 1000 RU of protein corresponds toa surface concentration alteration of approximately1ngÆmm)2[46]. In this experiment, approximately 6600RU of His-CcPCNA was immobilized onto the chipsurface. The binding of His-CcPCNA to eitherHis-CcCac1L-N or His-CcCac1L-C was performed in areaction containing 20 lL of HBS-EP buffer with threedifferent concentrations of His-CcCac1L-N or His-CcCac1L-C (250 nm, 500 nm or 1 lm). The running buffer(HBS-EP buffer) flow rate was 5 lLÆmin)1at 37 °C. Alldata were monitored and analysed using the manufac-turer’s software (GE Healthcare Bio-Sciences).AcknowledgementsWe thank Montserrat Anguera, Jennifer Erwin andJanice Ahn for critical reading of the manuscript, andall members of Sakaguchi Laboratory for help and dis-cussions. S. H. N. is a research fellow of the JapanSociety for Promotion of Science.References1 Kleckner N (2006) Chiasma formation: chromatin/axisinterplay and the role(s) of the synaptonemal complex.Chromosoma 115, 175–194.2 Masson JY & West SC (2001) The Rad51 and Dmc1recombinases: a non-identical twin relationship. 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Xem thêm: Báo cáo khoa học: Interaction between Lim15/Dmc1 and the homologue of the large subunit of CAF-1 – a molecular link between recombination and chromatin assembly during meiosis pot, Báo cáo khoa học: Interaction between Lim15/Dmc1 and the homologue of the large subunit of CAF-1 – a molecular link between recombination and chromatin assembly during meiosis pot, Báo cáo khoa học: Interaction between Lim15/Dmc1 and the homologue of the large subunit of CAF-1 – a molecular link between recombination and chromatin assembly during meiosis pot