Interaction between Lim15/Dmc1 and the homologue of
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: [email protected]
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: [email protected]

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.
2032 FEBS Journal 275 (2008) 2032–2041 ª 2008 The Authors Journal compilation ª 2008 FEBS
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

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

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

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.
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 FEBS
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

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-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-

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.
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 FEBS
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

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-

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 [buffer 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 4 °C. 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

(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
)1
ampicillin at 37 °C. 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 25 °C.
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
Link 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 FEBS
fluoride, 1 lm leupeptin and 1 lm pepstatin A)]. The cells
were then lysed by the addition of 1 mgÆmL
)1
lysozyme, 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

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,
3mm 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
1ngÆmm
)2
[46]. In this experiment, approximately 6600
RU of His-CcPCNA was immobilized onto the chip
surface. The binding of His-CcPCNA to either
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 37 °C. 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-

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