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

Namekawa Department of Applied Biological Science, Faculty of Science and Technology, Tokyo University of Science, Japan Keywords chromatin assembly; chromatin assembly factor 1 CAF-1; L

the large subunit of CAF-1 – a molecular link between

recombination and chromatin assembly during meiosis

Satomi Ishii*,†, Akiyo Koshiyama*, Fumika N Hamada, Takayuki Y Nara, Kazuki Iwabata,

Kengo Sakaguchi and Satoshi H Namekawa

Department of Applied Biological Science, Faculty of Science and Technology, Tokyo University of Science, Japan

Keywords

chromatin assembly; chromatin assembly

factor 1 (CAF-1); Lim15/Dmc1; meiotic

recombination; proliferating cell nuclear

antigen (PCNA)

Correspondence

K Sakaguchi, Department of Applied

Biological Science, Faculty of Science and

Technology, Tokyo University of Science,

2641 Yamazaki, Noda-shi, Chiba-ken

278-8510, Japan

Fax: +81 4 7123 9767

Tel: +81 4 7124 1501 (ext 3409)

E-mail: kengo@rs.noda.tus.ac.jp

Website: http://www.tus.ac.jp/en/grad/

riko_app_bio.html

S H Namekawa, Department of Molecular

Biology, Massachusetts General Hospital,

and Department of Genetics, Harvard

Medical School, Boston, MA 02114, USA

Fax: +1 617 726 6893

Tel: +1 617 726 5966

E-mail: namekawa@molbio.mgh.harvard.edu

Present address

†Quantum Beam Science Directorate, Japan

Atomic Energy Agency, Gunma, Japan

*These authors contributed equally to this work

(Received 7 January 2008, revised 24

February 2008, accepted 25 February 2008)

doi:10.1111/j.1742-4658.2008.06357.x

In 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 to ensure 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-specific recombinase Lim15/Dmc1 interacts with the homologue of the largest subunit of chromatin assembly factor 1 (CAF-1) in the basidiomycete Coprinopsis cinerea (Coprinus cinereus) Using C cinerea LIM15/DMC1 (CcLIM15) as the bait in a yeast two-hybrid screen, we have isolated the

C cinerea homologue of Cac1, the largest subunit of CAF-1 in Saccharo-myces cerevisiae, and named it C cinerea Cac1-like (CcCac1L) Two-hybrid assays confirmed that CcCac1L binds CcLim15 in vivo b-Galactosidase assays revealed that the N-terminus of CcCac1L preferentially interacts with CcLim15 Co-immunoprecipitation experiments showed that these proteins also interact in the crude extract of meiotic cells Furthermore, we demonstrate that, during meiosis, CcCac1L interacts with proliferating cell nuclear antigen (PCNA), a component of the DNA synthesis machinery recently reported as an interacting partner of Lim15/Dmc1 Taken together, these results suggest a novel role of the CAF-1–PCNA complex

in meiotic events We propose that the CAF-1–PCNA complex modulates chromatin assembly following meiotic recombination

Abbreviations

ATCC, 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.

In eukaryotes, sexual reproduction is achieved by the

conjugation of genetically variable gametes, which are

generated during meiosis in the parental germline

Mei-osis consists of two rounds of chromosome

segrega-tion, resulting in gametes with half the number of

chromosomes in order to prepare for conjugation

During prophase of the first meiotic division,

recombi-nation takes place between homologous chromosomes

of maternal and paternal origin This is followed by

the segregation of maternal and paternal copies of

each chromosome A physical connection at the site of

homologous recombination, called the chiasma, orients

homologous chromosome pairs towards opposite

spin-dle poles at meiosis I [1] Therefore, chromatin

organi-zation following meiotic recombination is required to

establish the chiasma and to segregate homologous

chromosomes

Meiotic recombination comprises several steps

beginning 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 steps

of homology search and recombination are catalysed

by two bacterial RecA homologues, Rad51 and

Lim15/Dmc1 Rad51 catalyses both somatic and

meiotic recombination, whereas Lim15/Dmc1 is

meio-sis-specific [2–5] Rad51 and Lim15/Dmc1 are

compo-nents of a multiprotein complex at the site of

recombination [6,7] In order to understand the

mecha-nisms of meiotic recombination, much effort has been

made to identify additional components of the Rad51

and Lim15/Dmc1 complex, in particular Lim15/Dmc1

interacting partners

Recent analysis has identified various interacting

partners of Lim15/Dmc1, which seem to be involved in

homology search and strand exchange Tid1/Rdh54,

an SWI2/SNF2 family of chromatin-remodelling

factors, promotes the co-localization of Rad51 and

Lim15/Dmc1 [8] The heterodimeric complex of Hop2

and Mnd1 stimulates strand exchange of Lim15/Dmc1

[9–11] The meiosis-specific proteins Mei5 and Sae3

form a complex with Lim15/Dmc1 and are necessary

for the assembly of Lim15/Dmc1 [12,13] Furthermore,

the DNA mismatch repair protein MSH4 (MutS

homologue 4) [14], the tumor suppressor protein p53

[15], DNA topoisomerase II [16], the sumoylation

pro-tein Ubc9 [17] and the DNA synthesis-related factor

proliferating cell nuclear antigen (PCNA) [18] have

been reported to interact with Lim15/Dmc1 These

proteins seem to participate in the modulation of

Lim15/Dmc1 However, how chromatin is organized

following meiotic recombination has not been

described

In order to explore chromatin organization after meiotic recombination, we designed experiments to investigate the possible interactions between recombi-nation proteins and chromatin assembly factors In this article, we report that the largest subunit homo-logue of chromatin assembly factor 1 (CAF-1) is a novel interacting partner of Lim15/Dmc1 CAF-1 con-sists of three subunits that are highly conserved amongst yeast, plant, fly and human [19–23] CAF-1 deposits histones H3 and H4 onto newly synthesized DNA after replication and repair [24–26] In addition, the largest subunit of CAF-1 interacts with PCNA during 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 test the involvement of CAF-1 in meiotic events We pro-pose a novel role of the CAF-1–PCNA complex in chromatin assembly following meiotic recombination

Results Isolation of Coprinopsis cinerea Cac1-like (CcCac1L) by two-hybrid screening using CcLim15 as bait

To isolate proteins that interact with CcLim15, we per-formed a yeast two-hybrid screen using CcLim15 as bait A clone was isolated which had moderate amino acid similarity with the largest subunit of human CAF-1 (p150) [19] and the largest subunit of Saccharo-myces cerevisiae CAF-1 (Cac1, chromatin assembly complex 1) [20] The sequence similarities of this clone with human and S cerevisiae homologues were found

to be 26% and 23%, respectively Hence, this clone was identified as C cinerea Cac1-like (CcCac1L) CcCac1L encodes a predicted product of 812 amino acid residues with a molecular mass of 120 kDa The highly charged KER (lysine/glutamate/arginine-rich; 242–360 amino acids) and ED (glutamate/aspartate-rich; 522–578 amino acids) domains in CcCac1L are conserved amongst human and S cerevisiae homo-logues (Fig 1A) The KER and ED domains are known to interact directly with newly synthesized H3/ H4 histones [19,21]

CcCac1L interacts with CcLim15

To confirm the specificity of interaction between CcCac1L and CcLim15, we performed yeast two-hybrid and b-galactosidase assays (Fig 1C,D) Next,

we sought to determine which region of CcCac1L

was responsible for binding to CcLim15 The

N-ter-minus (CcCac1L-N; amino acids 1–381) contained

the KER domain, whereas the C-terminus

(CcCac1L-C; amino acids 382–812) contained the ED domain

(Fig 1B) Two-hybrid assays demonstrated that

CcLim15 interacts with either of the truncated

mutants of CcCac1L in the mild selection medium

[SD3: lacking histidine, leucine and tryptophan

(–His/–Leu/–Trp)], and that CcLim15 preferentially

interacts with CcCac1L-N in the stringent selection

medium [SD4: lacking adenine, histidine, leucine

and tryptophan (–Ade/–His/–Leu/–Trp)] (Fig 1C)

The interaction between the truncated mutants of

CcCac1L and CcLim15 was confirmed by

b-galactosi-dase assays, which demonstrated a higher binding

affinity of CcCac1L-N than CcCac1L-C to CcLim15

(Fig 1D)

Characterization of CcCac1L during meiosis

The data above strongly suggest a novel function of

CAF-1 as a binding partner of Lim15/Dmc1

How-ever, currently there are no observations available

describing the meiotic role of CAF-1 Therefore, we

sought to examine the distribution of CcCac1L

dur-ing meiosis First, in order to determine the gene

expression profile of CcCac1L during meiotic

develop-ment, we performed northern analyses at each stage

during meiotic development Total RNA was

extracted from basidia in synchronous culture at 1 h

intervals after the induction of meiosis CcCac1L was

expressed at the premeiotic S phase, at the time of

genomic DNA replication (Fig 2A) Homologous chromosomes start to align at the leptotene/zygotene stage Then, fully synapsed homologues are observed

at the pachytene stage CcCac1L began to accumulate

at the leptotene and zygotene stage, and decreased after the pachytene stage (Fig 2A) This expression profile suggests the specific induction of CcCac1L transcription during the meiotic prophase Interest-ingly, CcLIM15 showed specific expression during the meiotic prophase [16,31], suggesting that CcCac1L and CcLIM15 are expressed robustly at the same stage

Next, we examined the distribution of CcCac1L and CcLim15 in the meiotic nuclei by immunostaining We raised a specific antibody against CcCac1L using a purified fragment of CcCac1L, and confirmed its speci-ficity in crude extracts of meiotic cells by western anal-ysis (Fig 2B) CcCac1L protein localized to nuclei from 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 disappeared

at metaphase I (Fig 2C) Importantly, significant amounts of CcCac1L and CcLim15 were localized within the nuclei from the leptotene/zygotene stage to the pachytene stage

To examine the interaction between CcCac1L and CcLim15 during meiosis, we performed co-immuno-precipitation analysis using cell extracts from the meiotic prophase in C cinerea CcLim15 was co-immunoprecipitated by anti-CcCac1L IgG, but not

by control rabbit IgG (Fig 2D) The reciprocal

A

B

C 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) Interactrunca-tion between CcCac1L and CcLim15 in a yeast two-hybrid assay The inserts in the activatrunca-tion domain (AD) and DNA-binding domain (BK) are shown +, binding; ), no binding The mild selection medium (SD3: –His/–Leu/–Trp) and the stringent selection medium (SD4: –Ade/–His/–Leu/–Trp) were tested (D) Interaction between CcCac1L and CcLim15 in yeast using quantitative b-galactosidase assays b-Galactosidase assays with the other vector pairs in (C) showed little activity below the detection limit of absorbance, and were not quantified.

experiment confirmed the specific interaction of

CcCac1L and CcLim15 in the crude extracts of

mei-otic tissues (Fig 2E) Taken together, these results

suggest that the interaction between CcLim15 and

CcCac1L is related to specific events during the

mei-otic prophase

Interaction between CcCac1L and CcPCNA

during meiosis

CAF-1 forms a complex with PCNA to deposit

histones at the site of newly synthesized DNA during

replication and repair The results above raised the

novel possibility that CAF-1 is involved in chromatin assembly following recombination-associated DNA synthesis during meiosis If so, CAF-1 must form a complex with PCNA in the meiotic prophase PCNA

is expressed abundantly in meiotic prophase I [32] Interestingly, recent analysis has revealed that PCNA interacts with Lim15/Dmc1 at the time of meiotic recombination [18] To determine whether CcCac1L interacts with CcPCNA during meiosis, we performed co-immunoprecipitation analysis using cell extracts from the meiotic prophase in C cinerea CcPCNA was specifically co-immunoprecipitated by anti-CcCac1L IgG, but not by control rabbit IgG (Fig 3A) The

A

C

B

D

E

Fig 2 Interaction between CcCac1L and CcLim15 during meiosis (A) Northern analysis of CcCac1L expression at various stages during meiosis Each lane contained 20 lg of total RNA isolated from meiotic cells of C cinerea at the premeiotic S phase and at every hour after karyogamy (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 meimei-otic cells Meimei-otic nuclei were stained 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 the cell 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) or anti-CcCac1L IgG (E) Lane 1, 100 lg of crude extract was loaded.

reciprocal experiment confirmed the specific interaction

of CcCac1L and CcPCNA in the crude extracts of

meiotic tissues (Fig 3B)

Next, we sought to examine the binding affinity of

CcCac1L to CcPCNA by performing BIAcore analysis

with the truncated mutants of CcCac1L, as shown in

Fig 1B The BIAcore system enabled us to detect the

surface plasmon resonance (SPR), which measures the

interaction between a ligand on a detection surface

(sensor chip) and a ligand that is injected First, we

conjugated CcPCNA to a sensor chip onto which

either 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–PCNA

complex From these results, we suggest a novel role

of the CAF-1–PCNA complex during the meiotic

pro-phase together with the meiosis-specific recombinase, Lim15/Dmc1

Discussion

In this study, we identified CcCac1L as a novel interacting partner of CcLim15 Furthermore, it was shown that CcCac1L interacts with CcPCNA during the meiotic prophase Several DNA synthesis events take place during the meiotic prophase, even after genome-wide replication at the premeiotic S phase [32,34] In the current model, DNA synthesis is required in the molecular events of meiotic recombi-nation [35,36] Meiotic DSBs are processed to single-strand overhangs, followed by single-single-strand invasion

to the other allele Recombination results in either crossover products (exchanging the flanking DNA arms between homologues) or non-crossover products (non-exchange of DNA arms) Both pathways accompany DNA synthesis following recombination [35,36] Given the coordination of CAF-1 and PCNA

in various DNA synthesis events, a CAF-1–PCNA complex may be involved in chromatin assembly fol-lowing DNA synthesis events during the meiotic pro-phase Based on the current model, we propose the role of the CAF-1–PCNA complex during meiosis (Fig 4) PCNA recruits DNA polymerase at the end of single-strand regions that are coated by Lim15/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-1

is recruited to the site of the Lim15/Dmc1–PCNA complex and deposits histone H3 (or a histone vari-ant) and H4 on the naked DNA to restore the nucleosome structure (Fig 4C) Because of the vari-ous interactions of Lim15/Dmc1–CAF-1–PCNA, we suggest that they act in multiple ways at the site of meiotic recombination and contribute to the subse-quent assembly of chromatin Therefore, there may

be coordination between meiotic recombination and CAF-1-dependent nucleosome assembly before the resolution of Holliday junctions (Fig 4C)

The CAF-1–PCNA complex senses DNA damage and subsequently contributes to chromatin assembly

at the site of DNA repair [33], including nucleotide excision repair [28] and DSB repair [29,30] During the process of chromatin assembly, CAF-1 deposits new H3.1 histones on the site of repair-associated DNA synthesis without the recycling of parental histones; therefore, CAF-1-dependent chromatin assembly results in a chromatin memory of damage

at a repair site [41] Similarly, CAF-1 may establish

a chromatin memory at the site of DNA synthesis

A

B

C

Fig 3 Interaction between CcCac1L and CcPCNA during meiosis.

(A, B) Co-immunoprecipitation of CcCac1L and PCNA in the cell

extract at the meiotic prophase; 20 mg of cell extract was

incu-bated with anti-CcPCNA IgG, anti-CcCac1L IgG or control rabbit

serum-conjugated beads After washing the beads, the bound

proteins were eluted and analysed by western analysis with

anti-PCNA IgG (A) or anti-CcCac1L IgG (B) Lane 1, 100 lg of crude

extract was loaded (C) Detection of SPR using a Biacore assay.

Truncation mutants of CcCac1L were injected onto a CcPCNA

conjugated chip The binding affinity is inversely related to the

dissociation constant (K D ), which is a ratio of the dissociation (K d )

and association (K a ) rates (K D = K d /K a ) ND, not detected.

following meiotic recombination The site of

cross-over recombination becomes the chiasma, required

for the appropriate segregation of homologous

chro-mosomes Chiasma formation involves the

coordi-nated local change of DNA and the surrounding

chromatin environment [42] One tantalizing

possibil-ity is that CAF-1-dependent chromatin memory

directs chiasma formation to newly synthesized DNA

at the site of recombination CAF-1-dependent

his-tone deposition is an established key early step for

chromatin organization in mitosis [19,24–26]

Multi-ple steps are involved in organizing the chromatin

structure after histone deposition by CAF-1

There-fore, the CAF-1–PCNA complex may be the central

player establishing the memory of recombination, leading to unique nuclear organization during meiosis

Materials and methods Culture of C cinerea and collection of fruiting bodies

The basidiomycete Coprinopsis cinerea (Coprinus cinereus) (strain #56838) was purchased from the American Type Culture Collection (ATCC), Manassas, VA, USA The culture methods and procedures for the photoinduction of meiosis were performed as described previously [38,43]

Yeast two-hybrid screening

The C cinerea cDNA library in meiotic tissues was con-structed using a Time Saver cDNA Synthesis Kit (GE Healthcare UK Ltd, Little Chalfont, UK) Yeast two-hybrid screening was carried out using the MATCH-MAKER GAL4 Two-Hybrid System 3 (Clontech, Moun-tain View, CA, USA) The cDNA encoding full-length CcLim15 was fused in-frame with the GAL4 DNA-binding domain in the pBKDT7 vector as bait The cDNA library was subsequently cloned into the pGADT7 vector encoding the GAL4 activation domain, and used as prey in the two-hybrid experiments Both the GAL4 fusion bait and the prey plasmids were transformed into the yeast strain, AH109 (Clontech), by standard lithium acetate transforma-tion Putative interacting clones were subsequently isolated based on their ability to activate the expression of the GAL4 selectable marker genes, thus producing growth on

SD minimal medium lacking adenine, histidine, leucine and tryptophan (SD4: –Ade/–His/–Leu/–Trp) To confirm galactosidase activity, colonies that grew under this selective condition 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 DNA had been prepared, the cDNA inserts were sequenced and the corresponding gene was identified by blast analysis

cDNA cloning of CcCac1L

One of the interacting factors identified in our screen was found to encode the CcCac1L C-terminus, consisting of the amino acid region 382–812 (CcCac1L-C) (Fig 1B) To obtain 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 accession number of the nucleotide sequence for CcCac1L reported

in this study is AB074897

A

B

C

Fig 4 Model of chromatin assembly following meiotic

recombina-tion (A) After DSB formation, Lim15/Dmc1 coats the single-strand

end during strand invasion (B) PCNA recruits the DNA polymerase

to the site of Lim15/Dmc1 The broken line represents newly

syn-thesized DNA (C) CAF-1 forms a complex with Lim15/Dmc1 and

PCNA CAF-1 deposits histones H3 and H4 or other factors, such

as histone variants (indicated as ‘?’), on the newly synthesized

DNA.

Two-hybrid assay

To confirm the direct interaction between proteins or

pro-tein fragments, the appropriate bait and prey constructs

were co-transformed into yeast cells, and two-hybrid assays

were performed using the MATCH-MAKER Kit

(Clon-tech), according to the manufacturer’s instructions The

full-length CcLim15, CcCac1L, N and

CcCac1L-C fragments were cloned into pGADT7 and pGBKT7 The

vector pairs indicated in Fig 1C were co-transformed into

the yeast strain AH109 Controls for self-activating fusion

proteins were carried out in each of these assays by

trans-formation of specific expression constructs with a pGBKT7

or pGADT7 empty vector Transformants were then plated

onto three types of selection medium: SD2, –Leu/–Trp;

SD3, –His/–Leu/–Trp; SD4, –Ade/–His/–Leu/–Trp

b-Galactosidase assays were performed in chlorophenol

red-b-d-galactopyranoside (CPRG)-based liquid culture

using the individual colonies that grew in SD3 medium,

according to the Yeast Protocols Handbook (Clontech)

Northern blotting

Northern blotting was performed as described previously

[44] The region of the CcCac1L cDNA corresponding to

1146–2346 bp was used as a probe

Antibodies

A polyclonal antibody against the CcCac1L protein was

raised in rabbit and rat using the purified 382–812 amino

acid fragment expressed as a His-CcCac1L-C protein in

E coli The specificity of the antibodies was confirmed by

western analysis as described previously [44,45] A

poly-clonal antibody against CcLim15 was also raised as

described previously [45] Anti-CcPCNA IgGs and purified

recombinant His-tagged CcPCNA (His-CcPCNA) have

been described previously [44]

In vivo co-immunoprecipitation

Rabbit anti-CcCac1L polyclonal IgGs rabbit

anti-CcLim15 polyclonal IgG or control rabbit serum was

coupled with CNBr-activated sepharose beads, according

to the manufacturer’s instructions {20 mg aliquots of

crude extracts from meiotic tissues were prepared in

buf-fer D [bufbuf-fer C, as described below, with 0.6 m NaCl and

protease inhibitors (1 mm phenylmethanesulfonyl fluoride,

1 lm leupeptin and 1 lm pepstatin A)]} The extracts in

buffer D were then incubated with either 70 lL of

pri-mary antibody or with control rabbit serum-conjugated

beads for 1 h at 4C The beads were then collected by

centrifugation at 800 g for 30 s After resuspension of the

beads in buffer E (0.15 m NaCl in buffer D), the

superna-tant was removed by centrifugation at 9100 g for 30 s The bound material was eluted from the beads with 20 lL

of buffer F (50 mm glycine/HCl, pH 2.5, and 0.01% Triton X-100) After neutralization of the pH by the addition

of 1 m Tris/HCl, pH 7.5, the bound material was analysed by immunoblotting with either anti-CcCac1L or anti-CcLim15 IgG, both at a dilution of 1 : 1000 To test the interaction between CcCac1L and CcPCNA in vivo, anti-CcCac1L and anti-CcPCNA IgGs were used and in vivo immunoprecipita-tion experiments were performed as described previously [44] The CcCac1L cDNA corresponding to 1146–2346 bp was used as a probe

Immunostaining of nuclei of C cinerea meiotic cells

Immunostaining of nuclei of C cinerea meiotic cells was performed as described previously [38] A 1 : 100 dilution was used of both rabbit CcLim15 and rat anti-CcCac1L primary IgGs We also employed a 1 : 1000 dilu-tion of both anti-rabbit IgG conjugated with Alexa Fluoro

488 (Invitrogen) for anti-CcLim15 and anti-rat IgG conju-gated with Alexa Fluoro 568 (Invitrogen) for anti-CcCac1L

as secondary antibodies

Proteins

A truncated cDNA corresponding to the N-terminus (resi-dues 1–381, as shown in Fig 1B) of CcCac1L (CcCac1L-N) was cloned into the BamHI and NotI sites of the expression vector pET21a(+) (Novagen, Gibbstown, NJ, USA) The C-terminal insert of CcCac1L (CcCac1L-C, residues 382– 812) was cloned into the NcoI and XhoI sites of the pET21d(+) expression vector (Novagen) The following primer pairs were used for subsequent PCR amplification

TGTCGGGAGCAGATTCA; 381R, 5¢-TGCTACTTCTC TCAGCGGCCGCATTCTTAT CcCac1L-C: 382F, 5¢-CA

5¢-GAGATTTCAGTTTCGTCACTCGAGCGG To over-express N-terminal hexahistidine-tagged CcCac1L-N (His-CcCac1L-N) and CcCac1L-C (His-CcCac1L-C), E coli BL21 cells (DE3) (Novagen) carrying the expression plasmid for each gene were grown in 2· YT medium (16 gÆL)1 poly-peptone, 10 gÆL)1 yeast extract, 5 gÆL)1 NaCl) containing

1 lgÆmL)1ampicillin at 37C After reaching an absorbance

at 600 nm of 0.6, isopropyl thio-b-d-galactoside (IPTG) was added to these cultures at a final concentration of 1 mm, and the cells were incubated for an additional 5 h at 25C The bacterial cells were then harvested by centrifugation at

4500 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 phenylmethanesulfonyl

fluoride, 1 lm leupeptin and 1 lm pepstatin A)] The cells

were then lysed by the addition of 1 mgÆmL)1lysozyme,

stir-red on ice for 30 min and sonicated Insoluble material was

removed by centrifugation at 26 000 g for 15 min Proteins

were loaded onto a 5 mL Hi-trap chelating column (GE

Healthcare UK Ltd.), and bound proteins were eluted with

a 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 dialysate

was loaded onto a heparin column (GE Healthcare UK

Ltd.) equilibrated with 0.05 m NaCl in buffer B After

washing, fractions were collected with a 20 mL linear

gradi-ent of 0–0.5 m NaCl in buffer B The eluted protein was

then dialysed against 0.05 m NaCl in buffer B, and loaded

onto a MonoQ HR5/5 column (GE Healthcare UK Ltd)

After washing, the fractions were again collected with

20 mL of a linear gradient of 0–0.5 m NaCl in buffer B

Fractions containing the recombinant proteins were verified

by SDS-PAGE, pooled and then dialysed against storage

buffer (NaCl/Pi, pH 7.4, 50% glycerol) Recombinant

His-tagged CcLim15 (His-CcLim15) was expressed in E coli

and purified as described previously [31]

Surface plasmon resonance

Analysis of both His-CcCac1L-N and His-CcCac1L-C

binding to His-CcPCNA was performed using a BIAcore

Biosensor instrument (GE Healthcare Bio-Sciences,

Uppsala, Sweden), according to the manufacturer’s

proto-col A sensor chip (CM 5 research grade) was activated by

the

N-hydroxysuccinimide/N-ethyl-N¢-(dimethylaminopro-pyl)carbodiimide coupling reaction, and 55 lL of coupling

buffer (10 mm sodium acetate, pH 4.0) containing the

His-CcPCNA protein (625 nm) was injected over the chip

at a rate of 20 lLÆmin)1 His-CcPCNA was covalently

bound to the sensor chip surface via carboxyl moieties on

the dextran Unreacted N-hydroxysuccinimide ester groups

were inactivated using 1 m ethanolamine/HCl (pH 8.0)

HBS-EP buffer (10 mm Hepes, pH 7.4, 150 mm NaCl,

3 mm EDTA, 0.005% Tween 20) was passed continuously

over the sensor chip The binding levels were measured in

resonance units (RU); 1000 RU of protein corresponds to

a surface concentration alteration of approximately

1 ngÆmm)2 [46] In this experiment, approximately 6600

RU of His-CcPCNA was immobilized onto the chip

His-CcCac1L-N or His-CcCac1L-C was performed in a

reaction containing 20 lL of HBS-EP buffer with three

different 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)1 at 37C All

data were monitored and analysed using the

manufac-turer’s software (GE Healthcare Bio-Sciences)

Acknowledgements

We thank Montserrat Anguera, Jennifer Erwin and Janice Ahn for critical reading of the manuscript, and all members of Sakaguchi Laboratory for help and dis-cussions S H N is a research fellow of the Japan Society for Promotion of Science

References

1 Kleckner N (2006) Chiasma formation: chromatin/axis interplay and the role(s) of the synaptonemal complex Chromosoma 115, 175–194

2 Masson JY & West SC (2001) The Rad51 and Dmc1 recombinases: a non-identical twin relationship Trends Biochem Sci 26, 131–136

3 Namekawa SH, Iwabata K, Sugawara H, Hamada FN, Koshiyama A, Chiku H, Kamada T & Sakaguchi K (2005) Knockdown of LIM15/DMC1 in the mushroom Coprinus cinereusby double-stranded RNA-mediated gene silencing Microbiology 151, 3669–3678

4 Neale MJ & Keeney S (2006) Clarifying the mechanics

of DNA strand exchange in meiotic recombination Nature 442, 153–158

5 Villeneuve AM & Hillers KJ (2001) Whence meiosis? Cell 106, 647–650

6 Bishop DK (1994) RecA homologs Dmc1 and Rad51 interact to form multiple nuclear complexes prior to meiotic chromosome synapsis Cell 79, 1081–1092

7 Tarsounas M, Morita T, Pearlman RE & Moens PB (1999) RAD51 and DMC1 form mixed complexes asso-ciated with mouse meiotic chromosome cores and syn-aptonemal complexes J Cell Biol 147, 207–220

8 Shinohara M, Gasior SL, Bishop DK & Shinohara A (2000) Tid1/Rdh54 promotes colocalization of rad51 and dmc1 during meiotic recombination Proc Natl Acad Sci USA 97, 10814–10819

9 Chen YK, Leng CH, Olivares H, Lee MH, Chang YC, Kung WM, Ti SC, Lo YH, Wang AH, Chang CS et al (2004) Heterodimeric complexes of Hop2 and Mnd1 function with Dmc1 to promote meiotic homolog juxta-position and strand assimilation Proc Natl Acad Sci USA 101, 10572–10577

10 Enomoto R, Kinebuchi T, Sato M, Yagi H, Kurumi-zaka H & Yokoyama S (2006) Stimulation of DNA strand exchange by the human TBPIP/Hop2-Mnd1 complex J Biol Chem 281, 5575–5581

11 Petukhova GV, Pezza RJ, Vanevski F, Ploquin M, Masson JY & Camerini-Otero RD (2005) The Hop2 and Mnd1 proteins act in concert with Rad51 and Dmc1 in meiotic recombination Nat Struct Mol Biol

12, 449–453

12 Hayase A, Takagi M, Miyazaki T, Oshiumi H, Shinohara M & Shinohara A (2004) A protein complex containing Mei5 and Sae3 promotes the assembly of the

meiosis-specific RecA homolog Dmc1 Cell 119, 927–

940

13 Tsubouchi H & Roeder GS (2004) The budding yeast

mei5 and sae3 proteins act together with dmc1 during

meiotic recombination Genetics 168, 1219–1230

14 Neyton S, Lespinasse F, Moens PB, Paul R, Gaudray

P, Paquis-Flucklinger V & Santucci-Darmanin S (2004)

Association between MSH4 (MutS homologue 4) and

the DNA strand-exchange RAD51 and DMC1 proteins

during mammalian meiosis Mol Hum Reprod 10, 917–

924

15 Habu T, Wakabayashi N, Yoshida K, Yomogida K,

Nishimune Y & Morita T (2004) p53 Protein interacts

specifically with the meiosis-specific mammalian

RecA-like protein DMC1 in meiosis Carcinogenesis 25, 889–

893

16 Iwabata K, Koshiyama A, Yamaguchi T, Sugawara H,

Hamada FN, Namekawa SH, Ishii S, Ishizaki T, Chiku

H, Nara T et al (2005) DNA topoisomerase II interacts

with Lim15/Dmc1 in meiosis Nucleic Acids Res 33,

5809–5818

17 Koshiyama A, Hamada FN, Namekawa SH, Iwabata

K, Sugawara H, Sakamoto A, Ishizaki T & Sakaguchi

K (2006) Sumoylation of a meiosis-specific RecA

homo-log, Lim15/Dmc1, via interaction with the small

ubiqu-itin-related modifier (SUMO)-conjugating enzyme

Ubc9 Febs J 273, 4003–4012

18 Hamada FN, Koshiyama A, Namekawa SH, Ishii S,

Iwabata K, Sugawara H, Nara TY, Sakaguchi K &

Sawado T (2007) Proliferating cell nuclear antigen

(PCNA) interacts with a meiosis-specific RecA

homo-logue, Lim15/Dmc1, but does not stimulate its strand

transfer activity Biochem Biophys Res Commun 352,

836–842

19 Kaufman PD, Kobayashi R, Kessler N & Stillman B

(1995) The p150 and p60 subunits of chromatin

assembly factor I: a molecular link between newly

synthesized histones and DNA replication Cell 81,

1105–1114

20 Kaufman PD, Kobayashi R & Stillman B (1997)

Ultra-violet radiation sensitivity and reduction of telomeric

silencing in Saccharomyces cerevisiae cells lacking

chro-matin assembly factor-I Genes Dev 11, 345–357

21 Tyler JK, Collins KA, Prasad-Sinha J, Amiott E, Bulger

M, Harte PJ, Kobayashi R & Kadonaga JT (2001)

Inter-action between the Drosophila CAF-1 and ASF1

chro-matin assembly factors Mol Cell Biol 21, 6574–6584

22 Verreault A, Kaufman PD, Kobayashi R & Stillman B

(1996) Nucleosome assembly by a complex of CAF-1

and acetylated histones H3/H4 Cell 87, 95–104

23 Kaya H, Shibahara KI, Taoka KI, Iwabuchi M,

Still-man B & Araki T (2001) FASCIATA genes for

chro-matin assembly factor-1 in Arabidopsis maintain the

cellular organization of apical meristems Cell 104, 131–

142

24 Gaillard PH, Martini EM, Kaufman PD, Stillman B, Moustacchi E & Almouzni G (1996) Chromatin assem-bly coupled to DNA repair: a new role for chromatin assembly factor I Cell 86, 887–896

25 Smith S & Stillman B (1989) Purification and character-ization of CAF-I, a human cell factor required for chro-matin assembly during DNA replication in vitro Cell

58, 15–25

26 Stillman B (1986) Chromatin assembly during SV40 DNA replication in vitro Cell 45, 555–565

27 Shibahara K & Stillman B (1999) Replication-depen-dent marking of DNA by PCNA facilitates CAF-1-cou-pled inheritance of chromatin Cell 96, 575–585

28 Green CM & Almouzni G (2003) Local action of the chromatin assembly factor CAF-1 at sites of nucleotide excision repair in vivo EMBO J 22, 5163–5174

29 Linger J & Tyler JK (2005) The yeast histone chaperone chromatin assembly factor 1 protects against double-strand DNA-damaging agents Genetics 171, 1513–1522

30 Nabatiyan A, Szuts D & Krude T (2006) Induction of CAF-1 expression in response to DNA strand breaks in quiescent human cells Mol Cell Biol 26, 1839–1849

31 Nara T, Yamamoto T & Sakaguchi K (2000) Charac-terization of interaction of C- and N-terminal domains

in LIM15/DMC1 and RAD51 from a basidiomycete, Coprinus cinereus Biochem Biophys Res Commun 275, 97–102

32 Hotta Y, Ito M & Stern H (1966) Synthesis of DNA during meiosis Proc Natl Acad Sci USA 56, 1184–1191

33 Moggs JG, Grandi P, Quivy JP, Jonsson ZO, Hubscher

U, Becker PB & Almouzni G (2000) A CAF-1–PCNA-mediated chromatin assembly pathway triggered by sensing DNA damage Mol Cell Biol 20, 1206–1218

34 Lu BC & Jeng DY (1975) Meiosis in Coprinus VII The prekaryogamy S-phase and the postkaryogamy DNA replication in C lagopus J Cell Sci 17, 461–470

35 Allers T & Lichten M (2001) Differential timing and control of noncrossover and crossover recombination during meiosis Cell 106, 47–57

36 Hunter N & Kleckner N (2001) The single-end invasion:

an asymmetric intermediate at the double-strand break

to double-Holliday junction transition of meiotic recom-bination Cell 106, 59–70

37 Namekawa S, Hamada F, Ishii S, Ichijima Y, Yamagu-chi T, Nara T, Kimura S, Ishizaki T, Iwabata K, Ko-shiyama A et al (2003) Coprinus cinereus DNA ligase I during meiotic development Biochim Biophys Acta

1627, 47–55

38 Namekawa S, Hamada F, Sawado T, Ishii S, Nara T, Ishizaki T, Ohuchi T, Arai T & Sakaguchi K (2003) Dissociation of DNA polymerase alpha-primase com-plex during meiosis in Coprinus cinereus Eur J Biochem

270, 2137–2146

39 Sakaguchi K & Lu BC (1982) Meiosis in Coprinus: characterization and activities of two forms of DNA

polymerase during meiotic stages Mol Cell Biol 2, 752–

757

40 Sawado T & Sakaguchi K (1997) A DNA polymerase

alpha catalytic subunit is purified independently from

the tissues at meiotic prometaphase I of a

basidiomy-cete, Coprinus cinereus Biochem Biophys Res Commun

232, 454–460

41 Polo SE, Roche D & Almouzni G (2006) New histone

incorporation marks sites of UV repair in human cells

Cell 127, 481–493

42 Blat Y, Protacio RU, Hunter N & Kleckner N (2002)

Physical and functional interactions among basic

chro-mosome organizational features govern early steps of

meiotic chiasma formation Cell 111, 791–802

43 Nara T, Saka T, Sawado T, Takase H, Ito Y, Hotta Y

& Sakaguchi K (1999) Isolation of a LIM15/DMC1

homolog from the basidiomycete Coprinus cinereus and

its expression in relation to meiotic chromosome pair-ing Mol Gen Genet 262, 781–789

44 Hamada F, Namekawa S, Kasai N, Nara T, Kimura S, Sugawara F & Sakaguchi K (2002) Proliferating cell nuclear antigen from a basidiomycete, Coprinus

cinere-us Alternative truncation and expression in meiosis Eur J Biochem 269, 164–174

45 Nara T, Hamada F, Namekawa S & Sakaguchi K (2001) Strand exchange reaction in vitro and DNA-dependent ATPase activity of recombinant LIM15/ DMC1 and RAD51 proteins from Coprinus cinereus Biochem Biophys Res Commun 285, 92–97

46 Wong CW, Komm B & Cheskis BJ (2001) Structure– function evaluation of ER alpha and beta interplay with SRC family coactivators ER selective ligands Biochemistry 40, 6756–6765