Biochemical analysis of the human DMC1-I37N
polymorphism
Juri Hikiba
1
, Yoshimasa Takizawa
1
, Shukuko Ikawa
2
, Takehiko Shibata
2
and Hitoshi Kurumizaka
1
1 Laboratory of Structural Biology, Waseda University, Tokyo, Japan
2 Cellular & Molecular Biology Laboratory, RIKEN Advanced Science Institute, Saitama, Japan
Two successive rounds of nuclear division, meiosis I
and meiosis II, occur with a single round of DNA rep-
lication during meiosis. These processes produce
haploid gametes from diploid cells in eukaryotes [1].
Homologous recombination occurs between homolo-
gous chromosomes during meiosis I. This process
forms chiasma which ensure the correct segregation
of homologous chromosomes into daughter cells at
meiosis I [1–4].
The DMC1 protein was first discovered as a mei-
osis-specific gene, whose mutants are defective in
meiotic homologous recombination in the yeast
Saccharomyces cerevisiae [5]. The DMC1 protein is a
homolog of the bacterial RecA protein, and is highly
conserved from yeast to human [6]. In eukaryotes, a
second RecA homolog, the RAD51 protein, has also
been found [7–12]. The RAD51 protein is produced in

E-mail:
(Received 5 September 2008, revised 6
November 2008, accepted 10 November
2008)
doi:10.1111/j.1742-4658.2008.06786.x
The DMC1 protein, a meiosis-specific DNA recombinase, promotes homol-
ogous pairing and strand exchange. The I37N single nucleotide polymor-
phism of the human DMC1 protein was reported as a result of human
genome sequencing projects. In this study, we purified the human DMC1-
I37N variant, as a recombinant protein. The DMC1 protein is known to
require DNA for efficient ATP hydrolysis. By contrast, the DMC1-I37N
variant efficiently hydrolyzed ATP in the absence of DNA. Like the con-
ventional DMC1 protein, the DMC1-I37N variant promoted strand
exchange, but it required a high Ca
2+
concentration (4–8 mm), a condition
that inactivates the strand-exchange activity of the conventional DMC1
protein. These biochemical differences between the DMC1 and DMC1-
I37N proteins suggest that the DMC1-I37N polymorphism may be a
source of improper meiotic recombination, causing meiotic defects in
humans.
Abbreviations
dsDNA, double-stranded DNA; ssDNA, single-stranded DNA.
FEBS Journal 276 (2009) 457–465 ª 2008 The Authors Journal compilation ª 2008 FEBS 457
reactions occur between the 3¢ single-stranded DNA
(ssDNA) tails generated at DSB sites and the double-
stranded DNA (dsDNA) of the other, intact homo-
logous chromosome. An autosomal-dominant dmc1
mutation, containing a single amino acid substitution,
was isolated as a recombination-defective allele causing

ome-sequencing projects (NCBI rfSNP ID: rs1129426).
To elucidate the biochemical consequences of this
single amino acid substitution, we purified the human
DMC1-I37N variant, as a bacterially expressed recom-
binant protein, using a method including Ni-NTA
agarose column chromatography, removal of the
hexahistidine tag from the DMC1 portion with throm-
bin protease and heparin Sepharose column chroma-
tography (Fig. 1A). CD analysis revealed that the
DMC1-I37N variant was thermally unstable, com-
pared with the conventional DMC1 protein (Fig. 1B).
We then tested the ATPase activity of the DMC1-
I37N variant. The human DMC1 protein efficiently
hydrolyzes ATP in the presence of ssDNA. In the
125 mm KCl concentration used in this assay, the
ATPase activity of the DMC1 protein was not detected
in the absence of ssDNA (Fig. 2A). By contrast, we
found that the DMC1-I37N variant exhibited detect-
able ATPase activity in the absence of ssDNA
(Fig. 2B). To eliminate the possibility that the ATPase
activity of the DMC1-I37N variant may be stimulated
by contaminating DNA, we subjected the DMC1-I37N
Markers
I37N
WT
132
A
–12
–10
–8

280
⁄ A
260
ratio (1.29) of the purified DMC1-I37N pro-
tein was exactly the same as that of the conventional
DMC1 protein, indicating that the DMC1-I37N prepa-
ration did not contain contaminating nucleotides. In
addition, DNaseI treatment did not affect the ATPase
activity of the DMC1-I37N variant (Fig. 2C). There-
fore, the DNA-independent ATPase activity of the
DMC1-I37N variant may not be due to DNA contam-
ination during protein purification.
The ATPase activity of the DMC1 protein is also
stimulated under high salt conditions without DNA.
As shown in Fig. 2D, the DMC1 protein hydrolyzed
ATP in the presence of salt concentrations > 0.5 m
KCl, but did not under low salt conditions (0–0.3 m
KCl). By contrast, the DMC1-I37N variant hydrolyzed
ATP in the presence of low salt concentrations
(< 0.5 m KCl), but did not under high salt conditions
(> 1.0 m KCl) (Fig. 2D). These results indicated that
the DMC1-I37N variant possesses ATPase activity,
but it is very different from that of the conventional
human DMC1 protein.
Strand-exchange and homologous-pairing
activities of the human DMC1-I37N variant
We next tested the strand-exchange activity of the
DMC1-I37N variant. In this assay, /X174 phage
circular ssDNA (5386 bp) and linearized /X174
dsDNA (5386 bp) were used as DNA substrates. Both

did not under low Ca
2+
conditions (Fig. 3C, lanes 3
and 5). Consistent with this, the DMC1-I37N variant
promoted homologous pairing only under high Ca
2+
conditions, in contrast to the conventional DMC1
protein (Fig. 4B). In this assay, a single-stranded oli-
gonucleotide 50-mer and supercoiled dsDNA were
used as the substrates for homologous pairing
(Fig. 4A). These results indicated that the optimal
conditions for homologous pairing and strand
0
0.05
0.1
0.15
0.2
0.25
0.3
No DNA
ssDNA
Time (min)
Phosphate (mM)
Time (min)
Phosphate (mM)
0
0.04
0.08
0.12
0.16

I37N variant. (A) Time course experiments
of ATP hydrolysis by the conventional
DMC1 protein. Open and closed circles indi-
cate the experiments in the presence and
absence of ssDNA, respectively. (B) Time
course experiments of ATP hydrolysis by
the DMC1-I37N variant. Open and closed
circles indicate the experiments in the pres-
ence and absence of ssDNA, respectively.
(C) DNaseI treatment. The ATPase assay
with the DMC1-I37N variant was performed
in the presence of DNaseI. Open and closed
circles indicate the experiments in the pres-
ence and absence of DNaseI, respectively.
(D) KCl titration. The ATPase assays were
performed with the indicated amounts of
KCl. Open and closed circles indicate the
experiments with the DMC1-I37N variant
and the DMC1 protein, respectively.
J. Hikiba et al. Activity of the human DMC1-I37N variant
FEBS Journal 276 (2009) 457–465 ª 2008 The Authors Journal compilation ª 2008 FEBS 459
exchange are very different between the DMC1 and
DMC1-I37N proteins.
No synergistic or additive action between the
DMC protein and the DMC1-I37N variant for
strand exchange
We then tested strand exchange under conditions
where the conventional DMC1 and DMC1-I37N
proteins co-existed in various stoichiometries. These
conditions may mimic the heterozygous DMC1 ⁄

ssDNA dsDNA
JM
(joint molecule)
NC
(nicked circular
dsDNA)
A
+
ssDNA
No protein
WT
I37N
dsDNA
NC
JM
ssDNA
B
WT
I37N
891011
1234567
1234567
CaCl
2
(mM)
WT
WT
WT
WT
I37N

Discussion
Human genome sequencing projects have identified
many SNPs in the DMC1 gene locus. The DMC1-
I37N polymorphism contains an amino acid substi-
tution in the coding region of the DMC1 gene. The
distribution of the DMC1-I37N polymorphism in the
human population has not yet been elucidated. In this
study, we purified the DMC1-I37N variant, and com-
pared its biochemical properties with those of the con-
ventional DMC1 protein. We have previously studied
the human DMC1-M200V variant [29], which is
suspected to be a source of infertility [27,28]. The
DMC1-M200V variant was moderately defective in the
recombinase activity in vitro and in vivo; however, its
biochemical and structural characteristics are not sig-
nificantly different from those of the DMC1 protein
[29]. In contrast to the DMC1-M200V variant, in this
study, we found that the DMC1-I37N variant pos-
sesses quite different biochemical characteristics from
those of the DMC1 protein. The DMC1 protein
requires ssDNA or a high salt concentration for effi-
cient ATP hydrolysis. We found that the DMC1-I37N
variant hydrolyzed ATP in the absence of DNA or the
presence of a low salt concentration. The DMC1-I37N
variant promoted homologous pairing and strand
exchange under high Ca
2+
conditions, but was com-
pletely defective under the conditions suitable for the
conventional DMC1 protein. Conversely, under such

I37N
I37N
I37N
I37N
No protein
8 9 10 11
1234567
01248
ssDNA dsDNA
D-loop
A
ssDNA
D-loops
Fig. 4. The homologous-pairing activity of the DMC1-I37N variant.
(A) A schematic representation of the homologous-pairing assay.
(B) The DMC1 (4 l
M) or DMC1-I37N (4 lM) protein was incubated
with the
32
P-labeled single-stranded oligonucleotide 50-mer in the
presence of the indicated amounts of CaCl
2
(final concentration
1–8 m
M), and the reaction was initiated by adding the supercoiled
dsDNA. The reactions were conducted for 8 min, and the deprotei-
nized products were resolved by 0.8% agarose gel electrophoresis
in 1· TAE buffer at 3.3 VÆcm
)1
for 2.5 h. Bands were visualized by

M KCl
8 m
M CaCl
2
dsDNA
NC
JM
ssDNA
Fig. 5. The strand-exchange assay in the presence of various
amounts of the DMC1 protein and the DMC1-I37N variant. The
indicated amounts of the DMC1 and DMC1-I37N proteins (total
6 l
M) were incubated with /X174 circular ssDNA at 37 °C for
10 min. After the addition of RPA, /X174 linear dsDNA was added
to initiate the reaction. The reactions were continued for 60 min.
J. Hikiba et al. Activity of the human DMC1-I37N variant
FEBS Journal 276 (2009) 457–465 ª 2008 The Authors Journal compilation ª 2008 FEBS 461
strand-exchange reaction promoted by the DMC1
protein when it co-existed, the heterozygous
DMC1 ⁄ DMC1-I37N situation may not cause serious
meiotic defects, if a sufficient amount of the DMC1 pro-
tein is produced. An infertile woman, who is homozy-
gous for the human DMC1-M200V polymorphism, has
been identified in a set of infertile patients [27,28], and a
mouse DMC1 mutant containing a single amino acid
substitution (Ala272 to pro, A272P) causes male-specific
infertility [26]. These DMC1-M200V and DMC1-A272P
proteins are moderately and significantly defective in the
recombinase activity in vitro, respectively [26,29]. We
report here that the DMC1-I37N protein is significantly

fied by the method described previously [40]. Briefly, cells
producing the proteins were harvested and lyzed by sonica-
tion in buffer A (50 mm Tris ⁄ HCl buffer pH 8.0, containing
0.5 m NaCl, 2 mm 2-mercaptoethanol, 10% glycerol and
5mm imidazole) on ice. The cell lysate was centrifuged at
27 700 g for 20 min, and the supernatant was gently mixed
by the batch method with 4 mL of Ni-NTA agarose beads
(QIAGEN, Hilden, Germany) at 4 °C for 1 h. The protein-
bound beads were packed into an Econo-column (Bio-Rad
Laboratories, Hercules, CA, USA) and washed with 30 col-
umn volumes of buffer A. The human DMC1 protein was
eluted in a 20 column volume linear gradient of 5–500 mm
imidazole in buffer A. The peak fractions were collected, and
thrombin protease (2 unitsÆmg
)1
of the human DMC1 pro-
tein; GE Healthcare Biosciences, Uppsala, Sweden) was
added to remove the His-tag. The samples were then immedi-
ately dialyzed overnight at 4 °C in buffer B (20 mm Tris ⁄ HCl
buffer pH 8.0, containing 0.2 m KCl, 0.25 mm EDTA, 2 mm
2-mercaptoethanol and 10% glycerol). The human DMC1
protein, which now lacked the His-tag, was subjected to
chromatography on a 4 mL Heparin-Sepharose (GE Health-
care Biosciences) column. The column was washed with 20
column volumes of buffer B, and the protein was eluted with
a 20 column volume linear gradient of 0.2–1.0 m KCl in
buffer B. The purified proteins were concentrated with a
centrifugal cartridge, and the buffer was exchanged with
BA
1234567812345678

The strand-exchange assay
The human DMC1 protein or the DMC1-I37N variant was
incubated at 37 °C for 10 min with 20 lm /X174 circular
ssDNA, in 10 lLof20mm Hepes–KOH buffer (pH 7.5),
containing 1 mm ATP, 1 mm MgCl
2
, 0.1 mgÆmL
)1
BSA,
20 mm creatine phosphate and 75 lgÆmL
)1
creatine kinase.
After this incubation, 2 lm RPA and the indicated amounts
of KCl and CaCl
2
were added to the reaction mixture,
which was incubated at 37 °C for 10 min. The reactions
were then initiated by the addition of 20 lm /X174 linear
dsDNA, and were continued for 1 h. The reactions were
stopped by the addition of 0.1% SDS and 1.7 mgÆmL
)1
proteinase K (Roche Applied Science, Basel, Switzerland),
and the samples were further incubated at 37 °C for
20 min. The deproteinized reaction products were separated
by 1% agarose gel electrophoresis in 1· TAE buffer (ice-
cold, 40 mm Tris-acetate and 1 mm EDTA) at 3.0 VÆcm
)1
for 4 h. The products were visualized by SYBR Gold (Invi-
trogen, Carlsbad, CA, USA) staining.
The D-loop assay

proteinase K (Roche Applied
Science), and the samples were further incubated at 37 °C for
15 min. The products were resolved by 0.8% agarose gel elec-
trophoresis in 1· TAE buffer at 3.3 VÆcm
)1
for 2.5 h, and
were visualized and quantitated by an FLA-7000 imaging
analyzer (Fujifilm, Tokyo, Japan).
Assays for DNA binding
The /X174 circular ssDNA (20 lm) or the supercoiled
/X174 dsDNA (10 lm) was mixed with the human DMC1
protein or the DMC1-I37N variant in 10 lL of a standard
reaction solution, containing 20 mm Hepes–KOH (pH 7.5),
1mm dithiothreitol, 0.1 mgÆmL
)1
BSA, 1 mm MgCl
2
,
250 mm KCl, 5% glycerol and 1 mm ATP. The reaction
mixtures were incubated at 37 °C for 10 min, and then
analyzed by 0.8% agarose gel electrophoresis in 1· TAE
buffer at 3.3 VÆcm
)1
for 2.5 h. The bands were visualized
by ethidium bromide staining.
ATPase activity
The human DMC1 protein or the DMC1-I37N variant was
incubated in 20 mm Hepes–KOH (pH 7.5), 125 mm KCl,
1mm MgCl
2

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