Carcinogenesis vol.25 no.6 pp.889--893, 2004
DOI: 10.1093/carcin/bgh099
p53 protein interacts specifically with the meiosis-specific mammalian RecA-like
protein DMC1 in meiosis
Toshiyuki Habu1,4, Nobunao Wakabayashi1,5, Kayo
Yoshida3, Kenntaro Yomogida2, Yoshitake Nishimune2
and Takashi Morita3,6
1
Department of Molecular Embryology, Research Institute for Microbial
Diseases, Osaka University, 3-1 Yamadaoka, Suita, Osaka 565, Japan,
Department of Science for Laboratory Animal Experimentation, Research
Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka,
Suita, Osaka 565, Japan and 3Department of Molecular Genetics
Graduate School of Medicine, Osaka City University, 1-4-3 Asahimachi,
Abeno, Osaka 545-8585, Japan
2
Present addresses: 4Radiation Biology Center, Kyoto University,
Yoshidakonoe-cho, Sakyou-ku, Kyoto 606-8501, Japan and 5Johns Hopkins
University, Bloomberg School of Public Health, Division of
Toxicological Sciences, 615 North Wolfe Street, Room 7032, Baltimore,
MD 21205, USA
6
To whom correspondence should be addressed
Email: [email protected]
The tumor suppressor protein p53 is specifically expressed
during meiosis in spermatocytes. Subsets of p53 knockout
mice exhibit testicular giant cell degenerative syndrome,
which suggests p53 may be associated with meiotic cell
cycle and/or DNA metabolism. Here, we show that p53
binds to the mouse meiosis-specific RecA-like protein Mus
musculus DMC1 (MmDMC1). The C-terminal domain
(amino acid 234--340) of MmDMC1 binds to DNA-binding
domain of p53 protein. p53 might be involved in homologous recombination and/or checkpoint function by directly
binding to DMC1 protein to repress genomic instability in
meiotic germ cells.
Introduction
The tumor suppressor protein p53 is a multifunctional molecule that regulates cell-cycle progression, DNA repair and
apoptosis by interacting with other molecules responding to
DNA damage (1--7). A lack of p53 function results in elevated
levels of gene amplification, and homologous recombination
and repair (8--10). Furthermore, p53 protein interacts directly
with human Rad51 protein (11,12), which is a homolog of
Escherichia coli RecA and yeast RAD51 gene, and catalyzes
DNA strand transfer and recombination (13--15).
p53 gene is expressed in tetraploid primary spermatocytes at
the meiotic pachytene stage of spermatogenesis, and its
expression is enhanced by g-irradiation (16,17). Impairment
of p53 expression results in giant cell degenerative syndrome
(18). This suggests, that p53 protein may be involved in meiotic cell-cycle progression and/or recombinational repair. We
speculated that during meiosis, p53 interacts with recombinational machinery as in the case of mitosis.
In meiotic cells of eukaryotes, two RecA homologs are
expressed: Rad51 and Dmc1 (19,20). Both proteins have
Abbreviations: GST, glutathione S-transferase; MmDMC1, Mus musculus
DMC1.
Carcinogenesis vol.25 no.6 # Oxford University Press 2004; all rights reserved.
similar structures to the central core region of the RecA protein
(domain II), including two nucleotide-binding motifs. An
in vitro study revealed that purified human DMC1 protein
has DNA-dependent ATPase activity, single-stranded DNAbinding activity, and strand exchange activity (21). Thus,
DMC1 protein is expected to catalyze DNA mediated recombination in meiosis, as observed for RecA and Rad51 proteins.
Another in vivo study showed that mouse DMC1 protein is
expressed in leptotene-to-zygotene spermatocytes, and that a
defect in the Dmc1 gene results in meiotic arrest caused by the
impairment of homologous chromosome synapsis (22,23).
Here, we examined the interaction of p53 protein with the
recombination proteins RAD51 and DMC1 during meiosis.
We demonstrate that the RecA-homolog, which interacts
with p53 in spermatocytes is DMC1. This suggests, that p53
and DMC1 proteins are involved in meiotic cell-cycle progression, homologous synapsis or recombinational repair to maintain genomic integrity during meiosis.
Materials and methods
Protein extraction and immunoprecipitation
Cell extracts from mouse testes or 293T cells transfected with appropriate
expression plasmids were prepared as follows. Seminiferous tubules from
adult Balb/c mouse testes were lysed in 1 ml of Harlow buffer (50 mM
HEPES, pH 7.5, 0.2 mM EDTA, 10 mM NaF, 0.5% Nonidet-40, 250 mM
NaCl), and kept on ice for 10 min. Cell debris was spun down at 10 000 r.p.m.
for 5 min, and the supernatant was used in an immunoprecipitation assay.
Transfected 293T cells were washed twice with ice-cold PBS, and lysed with
1 ml of Harlow buffer. After storing on ice for 10 min, cell debris was spun
down at 10 000 r.p.m. for 5 min. The protein concentration of each extract was
measured with a Protein Assay Kit (Bio-Rad, CA). Equal amounts (15 mg) of
each extract were analyzed by SDS--PAGE and western blotting followed by
detection with an ECL detection system according to the manufacturer's
instructions (Amersham Pharmacia Biotech, Buckinghamshire, UK). For
immunoprecipitation, non-specific binding in the cell extracts was removed
by adding 50 ml of protein G--Sepharose beads (Amersham Pharmacia Biotech)
equilibrated with Harlow or RIPA buffer to the extracts and rocking for 2 h at
4 C. After removing the beads, cell extract containing 15 mg of protein was
incubated with anti-p53 monoclonal antibody (pAb1; CALBIOCHEM, CA),
anti-Mus musculus DMC1 (MmDMC1) rabbit polyclonal antibody or anti-Mus
musculus RAD51 (MmRAD51) rabbit polyclonal antibody for 6 h at 4 C.
Then, 10 ml of protein G--Sepharose beads equilibrated with the same buffer
was added to each reaction mixture and rocked for 6 h at 4 C. The beads were
washed five times with Harlow buffer, and analyzed by western blotting using
anti-p53 monoclonal antibody (pAb1), anti-MmDMC1 polyclonal antibody, or
anti-MmRAD51 polyclonal antibody.
Expression plasmids
Expression plasmids for wild-type (WT393) N-terminal and C-terminal deletion mutants (CT213, NT115) of p53 were constructed by PCR, and subsequently cloned into pcDNAI vector (Invitrogen, CA) under the CMV
promoter. The primers we used for PCR of WT393 were: 50 -GGAATTCAATGGAGGAGCCGCAGTCAGA-30 and 50 -GCTCTAGAGTCTGAGTCAGGCCCTTCTG-30 ; primers for CT213, 50 -GGAATTCAATGGAGGAGCCGCAGTCAGA-30 and 50 -GCTCTAGAAAAAGTGTTTCTGTCATCCA30 ; and for NT115, 50 -GGAATTCTTGCATTCTGGGACAGCCAA-30 and
50 -GGAATTCAATGGAGGAGCCGCAGTCAGA-30 . Each primer was
linked with an EcoRI or XbaI site for cloning into pcDNAI vector.
For in vivo expression of MmDMC1 and MmRad51 in mammalian cells, we
amplified MmDMC1 and MmRAD51 coding sequences using mouse cDNAs
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T.Habu et al.
Fig. 1. p53 protein interacts with MmDMC1 protein, but not with MmRAD51 protein in the testis. (A) Co-immunoprecipitation analysis of p53 and MmDMC1
proteins using mouse testis extracts. Mouse testis extracts were immunoprecipitated with the indicated antibodies. Immunoprecipitated proteins were separated on
SDS--polyacrylamide gels and analyzed by western blotting using anti-p53 monoclonal antibody (lanes 2 and 4), anti-DMC1 (lane 6) or anti-MmRAD51
polyclonal antibody (lane 7). p53 (53 kDa), MmDMC1 (37 kDa) and MmRAD51 (37 kDa) proteins are indicated by arrow heads. (B) Transient expression of p53,
MmDMC1 and MmRAD51 proteins in 293T cells. The proteins were separated on SDS--polyacrylamide gel and detected by western blotting with anti-p53
monoclonal antibody, anti-MmDMC1 or anti-MmRAD51 polyclonal antibody. (C) Co-immunoprecipitation analysis of p53, MmDMC1, and MmRAD51 proteins
in 293T cells. Immunoprecipitated proteins were detected with anti-p53 monoclonal antibody, anti-MmDMC1 polyclonal antibody, and anti-MmRAD51
polyclonal antibody.
as templates. An EcoRI or XbaI site was artificially inserted into the primer
sequences for cloning into pcDNAI.
The primer sequences for MmRAD51 were 50 -GGAATTCATGGCTATGCAAATGCAGCT-30 and 50 -GCTCTAGATCAGTCTTTGGCATCGCCCA-30 ,
and for DMC1 were 50 -GGAATTCATGAAGGAGGATCAAGTTGT-30 and
50 -GCTCTAGACTACTCCTTGGCATCCCCGA-30 , respectively.
To make glutathione S-transferase (GST)-fusion proteins with full-length
p53 and N- and C-terminal deletion proteins, PCR-generated DNA fragments
were digested by BamHI and EcoRI restriction enzymes, and subcloned into
the BamHI and EcoRI sites of pGEX-2T (Amersham Pharmacia Biotech).
Purification of GST-fusion protein
To purify GST-fusion proteins, the GST-capture procedure was carried out
following the manufacturer's instructions (Amersham Pharmacia Biotech).
Escherichia coli HB101 (recAÿ) induced to produce GST, GST115-213
890
(A3), GST115-190 (A2), GST115-165 (A1), GST141-213 (B3), GST165-213
(B2) or GST165-190 (B1) proteins was lysed by the freeze-thawing procedure
in Start buffer (PBS, pH 7.4, plus 5 mM DTT). After sonication, lysates
containing A2, B3, B1 or GST protein were directly applied to 1 ml of
glutathione--Sepharose 4B (Amersham Pharmacia Biotech) equilibrated by
Start buffer. After washing with the buffer, GST-fusion proteins were eluted
by elution buffer (10 mM Tris--HCl, pH 8.0, 10 mM glutathione, 5 mM DTT).
The other proteins (GST--p53, A3, A1, B2) were insoluble in Start buffer, so
they were separated by SDS--PAGE and extracted from the gels with denaturing buffer (PBS, pH 7.4, plus 6 M guanidine--HCl and 5 mM DTT) for 20 h at
4 C followed by sequential dialyses against denaturing buffer containing 3, 2,
1 and 0 M guanidine--HCl. Solubilized GST-fusion proteins were dialyzed
against store buffer (PBS, pH 7.4, 50% glycerol and 5 mM DTT) and applied to
a glutathione--Sepharose column. After washing, GST-fusion proteins were
eluted with elution buffer. Each eluted fraction was dialyzed twice against
p53 interacts with DMC1 in meiosis
Fig. 2. MmDMC1 protein interacts with the DNA-binding domain of p53 protein. (A) Schematic presentation of mutant proteins of p53 transiently co-expressed
with MmDMC1 in 293T cells. (B) Deletion mutant proteins of p53 were expressed in 293T cells. Cell extracts were immunoprecipitated with anti-MmDMC1
polyclonal antibody, and precipitated proteins were analyzed by western blotting using anti-p53 antibody. (C) In vitro binding assay of p53 fragments to
MmDMC1 protein. Schematic diagram of GST--p53-fusion proteins. (D) Production of GST--p53-fusion proteins in E.coli. Affinity purified GST-tagged p53
proteins were analyzed by SDS--PAGE and stained with Coomassie brilliant blue. (E) GST-pull-down assay. MmDMC1 proteins that physically interacted with
GST--p53 peptides linked to the glutathione column were eluted and detected by western blotting with anti-MmDMC1 antibody.
store buffer for 12 h at 4 C. To remove non-specific binding materials, cell
extracts containing DMC1 protein were incubated with glutathione--Sepharose
beads (Amersham Pharmacia Biotech) for 2 h at 4 C. After removing the
beads, GST-fusion proteins were added to 100 ml of cell extract and rocked
for 6 h at 4 C. Then, 10 ml of glutathione--Sepharose beads was added to the
reaction mixture and rocked for 6 h at 4 C. The beads were washed five times
by Harlow buffer, and directly analyzed by SDS--PAGE and western blotting
using anti-MmDMC1 polyclonal antibody.
Results and discussion
We examined the interaction between MmDMC1 and p53
protein in mouse testicular cells. Using anti-p53 monoclonal
antibody, MmDMC1 protein was co-immunoprecipitated from
the whole protein lysate of the testis (Figure 1A, lane 2).
In contrast, MmRAD51 protein was not detected (Figure 1A,
lane 4) by anti-p53 immunoprecipitation. Anti-MmDMC1
polyclonal antibody co-precipitated p53 protein (Figure 1A,
lane 6), whereas anti-MmRAD51 antibody did not (Figure 1A,
lane 7). These results indicated that MmDMC1 and p53 proteins formed a complex that did not involve the MmRAD51
protein in testicular cells.
To confirm the interaction in cultured cells, we transiently
co-expressed p53, MmDMC1 and MmRAD51 proteins
(Figure 1B) in the human embryonic kidney 293T cells (Figure
1B). We examined the expression of p53, RAD51 and DMC1
proteins in the cells (lanes 1--4) in various combinations. We
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T.Habu et al.
extracted whole cell lysates and performed co-immunoprecipitation followed by western blot analysis. When p53 was
expressed with MmRAD51 or MmDMC1 protein, p53 coimmunoprecipitated with MmRAD51 or MmDMC1, respectively (Figure 1C, lanes 2 and 4). When MmDMC1 and
MmRad51 were expressed simultaneously, p53 protein coprecipitated with MmDMC1, but not with MmRAD51 protein
(Figure 1C, lane 1). This may reflect the small amount of
RAD51 protein produced in transfected cells. Alternatively,
it might indicate that p53 forms a protein complex exclusively
with MmDMC1, but not with MmRAD51 protein, when the
three proteins (p53, MmDMC1 and MmRAD51) are expressed
together as in the testis in vivo.
To define the region of p53 responsible for MmDMC1
binding, we first used two deletion mutants of p53 (CT213
and NT115) (Figure 2A). They were expressed transiently in
293T cells along with wild-type MmDMC1 protein. Whole
cell proteins were immunoprecipitated with anti-DMC1 antibody and detected by western blotting using anti-p53 antibody.
When MmDMC1 protein was expressed with wild-type p53
(WT393), we detected normal-sized p53 protein in the precipitate. By transfection with the mutant p53 genes, two p53
deletion mutant proteins (CT213 and NT115) were detected
along with endogenous wild-type p53 protein (Figure 2B) by
anti-DMC1 co-immunoprecipitation. This suggested that the
MmDMC1 bound between amino acids (aa) 115 and 213 in
p53. To more precisely define the interaction domain, we
employed a pull-down assay for six GST--p53 mutant proteins
(A1--A3 and B1--B3) (Figure 2C and D). We analyzed their
interaction with purified MmDMC1 in vitro using the GSTpull-down assay. We tested six GST--p53 deletion mutants,
and found that A1, A2, A3 and B3 mutant proteins of p53
bound to purified MmDMC1 protein (Figure 2E). B1 and B2
proteins were negative in this experiment (Figure 2E). These
results indicated that the region between aa 141 and 165 in p53
protein where the sequence-specific DNA-binding domain
was located was responsible for MmDMC1 binding. However,
weak binding of the B3 fragment suggests the region between
115 and 141 aa was necessary for full binding activity.
The p53-binding site on MmDMC1 protein was examined
using a series of C-terminal deleted MmDMC1 proteins
(CT234, CT89 and D-CT228) (Figure 3A) together with alternatively spliced forms of DMC1 (DMC1-D; D-284) (20),
which lacks a highly conserved region, the homologous core.
This region is shared by members of the RecA family and is
critical for their biological and biochemical activities in homologous recombination (24--26). DMC1 mutant proteins were
transiently expressed in 293T cells with wild-type p53 protein
and co-immunoprecipitated by anti-p53 antibody (Figure 3B).
Wild-type DMC1 (WT340) and DMC1-D (D-284) proteins coimmunoprecipitated with p53. However, the C-terminal
deleted proteins (CT234, CT89 and D-CT228) did not
co-immunoprecipitate (Figure 3B). From these results, we concluded that the region in DMC1 protein involved in p53 interaction was the C-terminal part (234--340 aa region). This result
differs from those obtained for the RAD51 protein. Sturzbecher
et al. reported that the region of RAD51 responsible for
p53-binding resides within the homologous core (125 and 220
aa for Homo sapiens RAD51 and HsRAD51 proteins) (11,12).
Thus, p53 targets a different portion of DMC1 compared with
RAD51 for its binding, indicating a different mode of
involvement of p53 in recombinational and/or biochemical
activities (27,28).
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Fig. 3. Mapping of the p53-binding site on DMC1 protein. (A) Schematic
presentation of deletion mutants of MmDMC1 protein. Wild-type MmDMC1
(WT340), DMC1-D (D-284), deletion mutant proteins of MmDMC1
(CT234, CT89, and D-CT228) were expressed together with p53 protein in
293T cells. (B) Co-immunoprecipitation of MmDMC1 protein derivatives
with anti-p53 antibody. Cell extracts were immunoprecipitated with the
anti-p53 monoclonal antibody and precipitated proteins were analyzed by
western blotting with anti-MmDMC1 polyclonal antibody.
Since DMC1 protein is involved in homologous synapsis
during meiosis (22), we suggest that the direct interaction
between p53 and DMC1 proteins plays a role in a cell--cycle
checkpoint to detect chromosome aberrations in meiotic
synapsis or recombination (29--31). Alternatively, p53 protein
may directly influence meiotic recombination or synaptonemal
complex formation by binding to DMC1 protein (32--35).
p53 inhibits tumorigenesis through a variety of functions
such as the mediation of cell-cycle arrest, premature senescence and apoptosis. p53 also associates with proteins
involved in homologous recombination such as Rad51 and
Rad54 (36). These proteins are involved in DNA damage
repair induced by environmental factors and replication during
the cell cycle. If the system is not properly regulated, genetic
instability and carcinogenesis may occur. In fact, dominantnegative Rad51 stimulates tumorigenesis in mammalian cells
defective in p53 (37). Our result that meiosis-specific homologous recombination protein DMC1 binds to p53 protein
suggests that DMC1 plays a role similar to RAD51 in DNA
repair during meiosis to repress carcinogenesis in testicular
and ovarian cells.
Acknowledgements
This work was supported by the Grant-in-Aid from the Ministry of Education,
Culture, Sports, Science and Technology (No. 15770113).
p53 interacts with DMC1 in meiosis
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Received August 1, 2003; revised January 15, 2004;
accepted January 17, 2004
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