Báo cáo khoa học: A DNA-binding surface of SPO11-1, an Arabidopsis SPO11 orthologue required for normal meiosis docx

A DNA-binding surface of SPO11-1, an Arabidopsis SPO11
orthologue required for normal meiosis
Yoshinori Shingu
1
, Tsutomu Mikawa
2
, Mariko Onuma
1
, Takashi Hirayama
3
and Takehiko Shibata
1
1 Cellular & Molecular Biology Laboratory, RIKEN Advanced Science Institute, 2-1 Hirosawa, Wako-shi, Saitama, Japan
2 Biometal Science Laboratory, RIKEN Spring-8 Center, Mikazuki cho, Hyogo, Japan
3 Molecular Membrane Biology Laboratory, RIKEN Advanced Science Institute, 2-1 Hirosawa, Wako-shi, Saitama, Japan
Introduction
In eukaryotes, homologous recombination contributes
to genetic diversity, assists with the meiotic segregation
of homologous chromosomes and promotes the repair
of double-strand breaks to maintain genome stability.
Numerous studies have used Saccharomyces cerevisiae
to demonstrate that meiotic recombination is initiated
by DNA double-stranded breaks [1], which are intro-
duced by the SPO11 protein [2,3]. The SPO11 gene
was first isolated from S. cerevisiae [4]. SPO11 is found
in virtually all eukaryotes and shares homology with
the A-subunit (i.e. Top6A) of topoisomerase VI in the
archaeon, Sulfolobus shibatae [3]. SPO11 orthologues
Keywords
AtSPO11-1; homology modelling; meiotic
recombination; topoisomerase VI; transgenic

Arabidopsis
Correspondence
T. Shibata, Cellular & Molecular Biology
Laboratory, RIKEN Advanced Science
Institute, 2-1 Hirosawa, Wako-shi, Saitama
351-0198, Japan
Fax: +81 48 462 1227
Tel: +81 48 467 9528
E-mail:
(Received 28 November 2009, revised 9
March 2010, accepted 15 March 2010)
doi:10.1111/j.1742-4658.2010.07651.x
Meiotic recombination is initiated by DNA double-stranded breaks intro-
duced by the SPO11 protein. Despite a decade of research, the biochemical
functions of SPO11 remain largely unknown, perhaps because of difficulties
in studying the functionally active SPO11. Arabidopsis thaliana encodes
three SPO11-related proteins, two of which (SPO11-1 and SPO11-2) are
required for, and cooperate in, meiosis. We isolated soluble SPO11-1, fused
with or free of a trigger factor-tag at its N terminus. The tag-free SPO11-1
needed to interact physically with soluble SPO11-1 to maintain its solubil-
ity, suggesting a multimeric active form including a solubilizing protein
cofactor. An N-terminal fragment of PRD1, a SPO11-1-interacting protein
required for normal meiosis, but not SPO11-2, forms a soluble complex
with trigger factor-tagged SPO11-1, but the trigger factor-tag was required
for the solubility. Formation of the complex is not sufficient to express
endonuclease activity. Trigger factor-tagged SPO11-1 exhibited DNA-bind-
ing activities: Glu substitutions of the invariant Gly215 and Arg222 and of
the nonconserved Arg223 and Arg226 in a conserved motif (G215E,
R222E, R223E, R226E) reduced the DNA-binding ability in vitro, but sub-
stitutions of the conserved Arg130 and invariant Tyr103 (a residue in the

putative endonuclease-active center) and of Arg residues outside conserved
motifs by Glu or Phe (R130E, Y103F, R207E and R254E), did not. Tests
for the ability of mutant spo11-1 proteins to complement the silique-defec-
tive phenotype of a spo11-1-homozygous mutant in vivo revealed that
R222E and G215E induced serious deficiencies, while R130E caused a
partial defect in silique formation. Thus, the Gly215, Arg222 and Arg223
residues of SPO11-1 form a DNA-binding surface that is functional in
meiosis.
Abbreviations
CV1, co-expression verctor 1; CV2, co-expression vector 2; EV-S1, expression vector of Spo11-1; IPTG, isopropyl thio-b-
D-galactoside;
PRD1N, N-terminal domain of PRD1; TF, trigger factor; TF-SPO11, SPO11 fusion protein with a TF tag attached to its N terminus.
2360 FEBS Journal 277 (2010) 2360–2374 ª 2010 The Authors Journal compilation ª 2010 FEBS
responsible for the initiation of meiotic recombination
or recombination-related events have been identified in
Schizosaccharomyces pombe (i.e. the Rec12 protein)
[2,5], Drosophila melanogaster (i.e. the mei-W68
protein) [6], Coprinus cinereus [7], mouse [8–13] and
Arabidopsis thaliana [14,15]. Yeast, flies, nematodes
and mammals encode a single SPO11; however, plants
(e.g. Arabidopsis and rice Oryza sativa) encode at least
three SPO11 paralogues [16,17]. In Arabidopsis,
SPO11-1 and SPO11-2 are considered to be the ‘true’
SPO11 proteins that function in the initiation of mei-
otic recombination, as demonstrated by the sterile and
reduced meiotic recombination phenotypes of spo11-1
and spo11-2 homozygous mutants, respectively [14,15].
SPO11-3, the third Arabidopsis SPO11 paralogue, is
highly expressed in somatic cells and contributes
to somatic development (i.e. endoreduplication) by

forming a complex with Top6B, a homologue of the
B-subunit of topoisomerase VI [18–20]. SPO11-2 and
SPO11-3 interacted with Top6B in a yeast two-hybrid
system, while SPO11-1 did not [16]. These findings
imply that SPO11-1 and SPO11-2 perform different
functions.
In S. cerevisiae, meiotic double-strand break forma-
tion to initiate meiotic recombination requires the
products of at least nine genes (i.e. REC102, SKI8,
REC104, REC114, MEI4, MER2, MRE11, RAD50
and XRS2) in addition to that of the SPO11 gene (see
refs. 21,22 for review). Among these, only three
homologous genes (SKI8, MRE11 and RAD50) have
been identified; however, their corresponding proteins
are not required for meiotic double-strand break for-
mation in Arabidopsis (23,24, see ref. 25 for review).
By contrast, in Arabidopsis, the PRD1 protein, which
is unique to plants, is required for meiotic double-
strand break formation [26]. These observations and
the presence of two or more SPO11 paralogues suggest
that plants and yeast differ in their regulation of mei-
otic double-strand break formation. In Arabidopsis,
a genetic study revealed a genetic interaction between
SPO11-1 and SPO11-2 [15], while another study
showed that spo11-1 and spo11-2 were epistatic with
respect to meiotic functions and that a spo11-1 or a
spo11-2 mutation abolished meiotic double-strand
cleavage [27]. These studies suggested that SPO11-1
and SPO11-2 function as members of the same com-
plex for the double-strand cleavage.

Although various studies have concluded that
SPO11 plays important roles in meiotic double-strand
break formation and the initiation of meiotic recombi-
nation, there is little biochemical evidence of the role
of this protein in meiotic double-strand break forma-
tion. The lack of biochemical data probably stems
from the difficulty in isolating the active form of
SPO11 by a method avoiding a renaturation process
or a refolding process. Only one study has described
the purification of SPO11 from S. pombe (i.e. Rec12)
by refolding a denatured protein that had been
expressed in Escherichia coli [28].
A number of mutations have been reported in
SPO11, and certain amino acid residues have been
suggested to play a role in the biochemical functions
of SPO11 [3,29,30]. However, for the same reason
mentioned above, the biochemical defects caused by
these mutations have not yet been determined. Func-
tional characterization of a purified and active form of
SPO11 and the complexes of SPO11 with regulatory
proteins would greatly enhance our understanding of
the molecular functions and regulation of SPO11 pro-
teins. This would be especially helpful in plants,
because genetic studies in plants are more time-con-
suming than those in yeast. Here, we developed a
method for purifying the active form of SPO11-1, and
a complex of SPO11-1 and the N-terminal domain of
PRD1, from E. coli cells without the need for a rena-
turation process, and studied a DNA-binding surface
that contributes to the meiotic function of SPO11-1

in vivo. We found that the meiotic defects in vivo
caused by single amino acid-substitution mutations in
SPO11-1 were correlated with defects in the in vitro
DNA-binding activities of the mutant spo11-1 proteins
(hereafter denoted as spo11-1 to discriminate them
from wild-type SPO11-1). Using this information, we
identified a DNA-binding surface on SPO11-1. To our
knowledge, this is the first report of the characteriza-
tion of a soluble and active form of SPO11 in vitro,in
a decade since the discovery of the function of SPO11
in the meiotic double-stranded breakage [2,3], and of
the correlation between the in vivo defects caused by
mutations in SPO11 and the in vitro biochemical activ-
ities of SPO11 mutant proteins purified in an active
form.
Results
Maintenance of soluble forms of SPO11-1 and a
SPO11-1-associated protein, PRD1
After making various attempts to express and isolate
the Arabidopsis SPO11-1 protein in a soluble form, we
tried to use the trigger factor (TF), which reportedly
helps with the folding of co-expressed proteins [31,32].
Thus, we constructed the co-expression vector 1
(CV1)–SPO11-1 (Fig. 1A). Protein expression in trans-
formed E. coli BL21 cells was induced by exposure to
cold shock and isopropyl thio-b-d-galactoside (IPTG),
Y. Shingu et al. DNA-binding surface of Arabidopsis SPO11 protein
FEBS Journal 277 (2010) 2360–2374 ª 2010 The Authors Journal compilation ª 2010 FEBS 2361
A
B

C
Fig. 1. Expression of soluble TF-tagged
SPO11-1 and co-expression of tag-free
SPO11-1 and related proteins. (A) Expres-
sion vectors. TF-SPO11-1 and free TF were
expressed under the control of the cold
shock promoter (CsP), while TF-tag-free
SPO11-1, SPO11-2 and PRD1N were under
the control of the T7 promoter (T7P). Note
that the His-tag of the TF-SPO11-1 on
CV2-PR (PRDIN) was removed in order to
avoid detection of TF-SPO11, because the
band of PRD1N (92 kDa) and that of
TF-SPO11-1 (94 kDa) would overlap. (B)
Immunoblotting analyses of TF-tag-free
proteins co-expressed with free TF or
TF-SPO11-1. Proteins were detected with
an anti-His-tag IgG. All proteins have a
His-tag at the N terminus unless otherwise
noted (see Fig. 1A). Cell-free lysates
obtained from cells expressing each expres-
sion vector were separated by centrifugation
into supernatant fractions (sup) and precipi-
tates (ppt). The samples were then
subjected to PAGE under denaturing condi-
tions. Arrows indicate TF-SPO11-1 (94 kDa),
TF (53 kDa), SPO11-1 (44 kDa), SPO11-2
(45 kDa), TF-SPO11-2 (95 kDa) and PRD1N
(92 kDa). (C) Quantification of the results
shown in panel B. The percentages of solu-

ble tag-free SPO11-1, SPO11-2 and PRD1N
were calculated by densitometric measure-
ments of the photographs shown in Fig. 1B.
Each bar represents the mean value ± stan-
dard deviation from three independent
experiments.
DNA-binding surface of Arabidopsis SPO11 protein Y. Shingu et al.
2362 FEBS Journal 277 (2010) 2360–2374 ª 2010 The Authors Journal compilation ª 2010 FEBS
and the cell lysates were fractionated by centrifugation
into supernatants (soluble form) and precipitates
(insoluble form). Co-expression with TF greatly
improved the recovery of the expressed SPO11-1 in the
soluble form (i.e. under these conditions, 43% of the
total expressed SPO11-1 was obtained in a soluble
form, but the majority of SPO11-1 remained in the
precipitates) (Fig. 1B, lane 1 versus lane 2, and
Fig. 1C).
Next, we tried to express SPO11-1 as a fusion pro-
tein with a TF tag attached at the N terminus (i.e.
TF-SPO11-1) by the use of the expression vector of
SPO11-1, EV-S1 (Fig. 1A), and found that fusion to
TF greatly improved the recovery of SPO11-1 in a sol-
uble fraction (Fig. 1B,C). Then, we successfully estab-
lished a method to isolate an active and soluble form
of TF-SPO11-1 in a sufficient amount for biochemical
studies, without the need for a refolding step during
the purification process, as described in the Materials
and methods. The heparin column facilitated the
separation of TF-SPO11-1 (94 kDa) from the partially
degraded TF-SPO11-1 and free TF, which were pro-

duced, along with the intact fusion protein, in E. coli
cells (Fig. 2A). Most of the purified TF-SPO11-1 was
retained by the Superdex 200 column and eluted as
multimers with heterogeneous sizes larger than dimers
(Fig. 2C). We obtained  1 mg of purified TF-SPO11-1
from a 2 L culture.
To remove the TF tag after purification, a throm-
bin-digestion site was inserted between TF and SPO11-
1 in the fusion protein (EV-S1; Fig. 1A). After purifi-
cation, the 94 kDa protein was treated with thrombin,
and denatured samples were separated by SDS ⁄ PAGE.
We detected a fragment of  55 kDa and a weak sig-
nal from a protein of  40 kDa (Fig. 2B), while the
expected sizes of free TF and free SP11-1 were 53 and
42 kDa, respectively. The 40 kDa fragment was identi-
fied, by N-terminal sequence analyses, as SPO11-1,
and this result further confirmed that the 94 kDa
protein was TF-SPO11-1. While the TF-tag-free
A
B
C
Fig. 2. Purification of TF-tagged SPO11-1
and co-expression of tag-free SPO11-1.
(A) Purification of TF-SPO11-1. The samples
from each of the purification steps were
analyzed by PAGE under denaturing
conditions. Lanes from left to right:
molecular mass markers (M), supernatant
(sup) and insoluble fractions (ppt) of the
cell-free lysates from Escherichia coli cells

expressing TF-SPO11-1, the fraction from a
TALON column (TALON) and the fraction
from a heparin column (Heparin).
(B) Polyacrylamide gel electrophoretic
analysis of thrombin-treated TF-SPO11-1
under denaturing conditions. TF-SPO11-1
treated with thrombin was centrifuged to
obtain a supernatant (sup) and a precipitate
(ppt). Arrows indicate TF-SPO11-1, free TF
and TF-tag-free SPO11-1, from top to
bottom. (C) The gel-filtration profiles of
purified TF-SPO11-1 alone and the complex
of TF-SPO11-1 and SPO11-1. The gray and
black lines indicate absorbance at 280 nm in
the gel filtration of TF-SPO11-1 and that of
the complex of TF-SPO11-1 and SPO11-1,
respectively.
Y. Shingu et al. DNA-binding surface of Arabidopsis SPO11 protein
FEBS Journal 277 (2010) 2360–2374 ª 2010 The Authors Journal compilation ª 2010 FEBS 2363
SPO11-1 was obtained in a soluble form through
partial digestion with thrombin (Fig. 2B; 3 h thrombin
cleavage), all of the tag-free SPO11-1 became insoluble
upon complete digestion (Fig. 2B; overnight thrombin
cleavage).
A possible explanation for the results described
above is that TF-tag-free SPO11 is maintained as a
soluble form by direct interactions with a soluble form
of SPO11-1 (i.e. TF-SPO11-1). To test this possibility,
we modified CV1 to co-express TF-SPO11-1 and the
TF-tag-free SPO11-1 (CV2-S1; Fig. 1A), and the

expression experiments revealed that the majority of
the TF-tag-free SPO11-1 was in a soluble form (i.e.
79% of the total protein; Fig. 1B, lane 3 versus lane 4,
and Fig. 1C). Moreover, Superdex 200 gel filtration of
the cell-free extracts prepared from cells co-expressing
TF-SPO11-1 and TF-tag-free SPO11-1 on CV2-S1
showed that almost all of the TF-SPO11-1 and
TF-tag-free SPO11-1 were in a complex in the void
volume fraction (Fig. 2C). These results, and those
following solubilization of the N-terminal domain of
PRD1 (PRD1N) co-expressed with TF-SPO11-1,
support the explanation about the solubilization by a
direct interaction with a soluble form.
Spo11-2 and PRD1 co-operate with SPO11-1 in mei-
osis in Arabidopsis (see Introduction). A yeast two-
hybrid analysis showed that PRD1 physically interacts
with SPO11-1 at its N-terminal domain (802 residues:
ref. 26), while it is not clear whether SPO11-2 interacts
directly with SPO11-1 [15,27]. To test for the possible
direct interactions of these two proteins with SPO11-1
in vitro, we co-expressed SPO11-2 or PRD1N with
TF-SPO11-1 or TF on CV2 and CV1, respectively,
considering that these proteins were mostly expressed
in an insoluble form in E. coli cells (Fig. 1A). We have
not solved the problem with the expression of the full-
length PRD1 in E. coli cells and thus were not able to
study the full-length PRD1. As expected by the known
interactions that would maintain its own solubility,
PRD1N showed a significant improvement in the
recovery (91%) as a soluble form when co-expressed

with TF-SPO11-1 on CV2 (Fig. 1B,C). By contrast,
the solubility of Spo11-2 was not improved by the
co-expression of TF-SPO11-1 on CV2 (Fig. 1B,C),
while TF-tagged SPO11-2 was soluble (Fig. 1B). These
results suggest that SPO11-2 does not directly interact
with SPO11-1.
DNA-binding activities of TF-SPO11-1
As described above, TF-tag-free SPO11 is not soluble
without the associated TF-SPO11-1 and therefore
we mainly used TF-SPO11-1 in the subsequent
biochemical analyses. We then assessed the DNA-bind-
ing activities of SPO11-1 using a native gel-mobility
assay. Negatively supercoiled pUC18 closed-circular
dsDNA was incubated with TF-SPO11-1 for 5 min at
37 °C and then analyzed by agarose-gel electrophore-
sis. A slower mobility shift was observed in the pUC18
dsDNA as the amount of protein was increased, while
TF alone caused only a slight change in the DNA
mobility (Fig. 3A). DNA-binding analyses using
M13mp18 ssDNA indicated that TF-SPO11-1 bound
to the ssDNA with a slightly lower affinity (approxi-
mately twofold) than to dsDNA, as judged by the
amounts of proteins giving the same extents of band-
shift (Fig. 3B). These results indicate that the soluble
form of SPO11-1 is active in DNA binding.
DNA binding was indicated by a broadened band
that gradually decreased in mobility; thus, we had to
assess the DNA binding by monitoring the decreases in
the mobility and amount of DNA in the bands. This
made it difficult to quantify the DNA-binding ability

of mutant SPO11-1. When we tested the 180 bp
dsDNA as a substrate for DNA binding, we found that
the free 180 bp dsDNA and the Spo11-1-bound DNA
formed discrete bands (Fig. 3C), and this allowed us to
quantify the DNA-binding ability clearly (see Fig. 5B
below). Again, TF alone did not bind to the 180 bp
dsDNA molecule (Fig. 3C). We conclude, from these
results, and from those described above, that the
SPO11 domain of TF-SPO11-1 binds to dsDNA.
The binding of TF-SPO11-1 to DNA remained sta-
ble in the presence of up to 150 mm NaCl, as little free
DNA was detected (Fig. 3D). When the NaCl concen-
tration was increased above 150 mm, another signal
for DNA binding, appearing between the fully bound
form and the free DNA, became more significant. In
the presence of 600 mm NaCl, about half of the DNA
still remained in the bound forms, and the amount of
the second signal for the DNA binding was compara-
ble to that of the fully bound form (Fig. 3D). The sec-
ond signal for the DNA binding was weak at a low
concentration of SPO11-1, even without a high concen-
tration of NaCl (Fig. 3C; see Fig. 5A). This result
shows that there are two distinct forms of the com-
plexes of DNA and SPO11-1.
We did not detect DNase or topoisomerase activities
on the dsDNAs of pUC18, pBR322 or E. coli phage
lambda, or on the dsDNA containing a topoisomerase
target sequence (Pryv, TopoGen), a yeast hypersensi-
tive sequence to yeast SPO11 [33] or an Arabidopsis
hot-spot for meiotic recombination [34], when these

DNA species were incubated with TF-SPO11-1 under
these and modified conditions (Fig. S1). We did not
detect any activity for covalent attachment to the
DNA-binding surface of Arabidopsis SPO11 protein Y. Shingu et al.
2364 FEBS Journal 277 (2010) 2360–2374 ª 2010 The Authors Journal compilation ª 2010 FEBS
DNA terminus (Figs 3C and S1; with SDS treatment).
These results suggest that either the endonucleolytic
activity of SPO11-1 is not expressed by itself or the
attachment of the TF-tag at the N terminus prevents
the expression of the activity. Genetic complementa-
tion tests using the cDNA encoding TF-SPO11-1 were
unsuccessful, but this does not mean that all biochemi-
cal functions of SPO11-1 are inactivated by the attach-
ment of a TF-tag.
Amino acid residues required for DNA binding by
SPO11-1 in vitro
We tried to identify a surface on SPO11-1, involved in
DNA binding, by analyzing the effects of a series of
single amino acid replacements. SPO11-1 shares
 30% amino acid sequence homology with Metha-
nococcus jannaschii Top6A, suggesting structural simi-
larity between these proteins [35]. Therefore, we
performed homology modelling of SPO11-1, based on
the crystal structure of Top6A [36], to consider
candidates of basic amino acid residues composing a
DNA-binding surface. The modelled SPO11-1 struc-
ture without the 70 N-terminal residues is shown in
Fig. 4A, in which the basic amino acid residues are
shown in blue. Gly202 is reportedly essential for DNA
binding by S. pombe Spo11 (Rec12); however, no

biochemical data have been described to support this
observation (see ref. 30). Gly215 (i.e. equivalent to the
Gly202 residue of S. pombe Rec12) was found in the
AB
CD
Fig. 3. DNA-binding activities of TF-SPO11-1. (A) Binding to pUC18 negatively supercoiled closed-circular dsDNA. pUC18 closed-circular
dsDNA (7.5 n
M) was incubated with free TF or with TF-SPO11-1 at 37 °C for 5 min. The products were separated by electrophoresis at
8.3 V ⁄ cm for 1 h, at room temperature through a 0.8% agarose gel, then stained with ethidium bromide. Lane M contains a smart ladder
DNA marker (Nippon Gene). The amounts of proteins are 0, 0.24, 0.47 and 0.71 l
M (from left to right). (B) Binding to ssDNA. M13mp18
ssDNA (2.2 n
M) was incubated with free TF or with TF-SPO11-1 at 37 °C for 5 min. The products were analyzed as described above, for
panel A. The amounts of proteins are 0, 0.24, 0.47 and 0.71 l
M (from left to right). (C) Binding to the 180 bp dsDNA. The dsDNA (9 nM) was
incubated with free TF or TF-SPO11-1 at 37 °C for 5 min. Lane S indicates the addition of SDS (final concentration 1%) after the binding
reaction with 0.71 l
M TF-SPO11-1. The products were separated by electrophoresis through a 0.8% agarose gel at 8.3 V ⁄ cm for 1 h, at
room temperature, and were detected by Southern hybridization using the 180 bp [
33
P]DNA probe. The amounts of proteins are 0, 0.24,
0.47 and 0.71 l
M (from left to right). (D) The NaCl sensitivity of the DNA binding of TF-SPO11-1. The 180 bp dsDNA (9 nM) was incubated in
the presence of NaCl with 0.47 l
M TF-SPO11-1 at 37 °C for 5 min. The NaCl concentrations were 0, 150, 300, 450 and 600 mM (from left
to right). After the agarose gel electrophoresis, the products were detected by Southern hybridization, as described above for panel C. 2nd
protein-bound DNA is a minor species of protein-bound DNA.
Y. Shingu et al. DNA-binding surface of Arabidopsis SPO11 protein
FEBS Journal 277 (2010) 2360–2374 ª 2010 The Authors Journal compilation ª 2010 FEBS 2365
vicinity of a basic surface (Fig. 4A). Proteins in the

SPO11 family contain five conserved amino acid
sequence motifs (i.e. motifs I to V; Fig. 4B). Consider-
ing these two series of structural features, we con-
structed a series of DNA fragments that each encoded
a mutant spo11-1 with a single amino acid substitu-
tion, wherein a conserved amino acid residue was
replaced with an acidic residue. These mutations
included R130E (where the first letter followed by the
number designates the original amino acid residue
replaced with the amino acid residue indicated by the
last letter) in motif II, and G215E and R222E in motif
IV (Fig. 4B). As controls for the in vitro DNA-binding
activity of these mutant spo11-1 proteins, to exclude
possible non-specific negative effects caused by the
replacement of a basic amino acid residue with an
acidic amino acid residue, we constructed a series of
coding DNAs in which the non-conserved basic amino
acid residues surrounding the putative DNA-binding
surface (i.e. the Arg223 and Arg226 residues in motif
IV, and the Arg207 and Arg254 residues outside the
conserved motifs) were substituted with acidic Glu res-
idues (Fig. 4B). In addition, the substitution, by Phe,
of the invariant Tyr103 residue (Y103F) in motif I of
the putative endonuclease-active center of SPO11-1
was constructed as a negative control [3,27,29].
All of the constructed mutants (i.e. R130E, R207E,
G215E, R222E, R223E, R226E, R254E and Y103F) of
TF-SPO11-1 were expressed in E. coli in soluble forms,
and were purified from the cell-free lysates (Fig. S2A).
Quantitative analyses of the DNA-binding activities of

the mutant spo11-1 proteins to the 180 bp dsDNA
molecule revealed that the mutations of invariant
amino acid residues (i.e. G215E and R222E) and non-
conserved residues (i.e. R223E and R226E) in motif
IV reduced the DNA-binding ability of SPO11-1
(Fig. 5A,B). Substitution of the invariant Tyr103
residue in the putative endonuclease-active centre
of SPO11-1 (i.e. Y103F) did not alter the DNA-
binding ability of SPO11-1 (Fig. 5A,B). The remaining
A
B
Fig. 4. Amino acid residues of SPO11-1
substituted in the mutant spo11-1 proteins.
(A) A 3D structural model of SPO11-1
obtained by homology modelling. This
SPO11-1 model consists of amino acid
residues 71–363. The electrostatic potential
was calculated using MolFeat. Positive and
negative potentials are indicated in blue and
red, respectively. The Gly215
,
Arg222,
Arg223 and Arg226 residues that were
found to function in DNA binding are shown
in light blue (see Fig. 5). The Tyr103,
Arg130, Arg207 and Arg254 residues that
are not involved in DNA binding are shown
in white. (B) Amino acid alignments of
regions containing mutated amino acid
residues. Numbers indicate the positions of

amino acid residues. The SPO11-1
(accession no.: AJ251989) and SPO11-2
(accession no.: AJ251990) proteins from
Arabidopsis thaliana, DmSpo11 from
Drosophila melanogaster (mei-W68;
accession no.: AAC61735), HsSpo11 from
Homo sapiens (accession no.: AAD52562),
ScSpo11 from Saccharomyces cerevisiae,
SpSpo11 from Schizosaccharomyces pombe
(rec12; accession no.: CAB11511) and
MjTop6A from Methanococcus jannaschii
(accession no.: AAB98358) are shown.
Amino acids that were conserved among at
least six of the seven homologues are
shaded in gray.
DNA-binding surface of Arabidopsis SPO11 protein Y. Shingu et al.
2366 FEBS Journal 277 (2010) 2360–2374 ª 2010 The Authors Journal compilation ª 2010 FEBS
mutations (i.e. R130E, R207E and R254E) outside
motif IV, including the substitution of the conserved
Arg130 residue in motif II, exhibited normal DNA-
binding activity (Fig. 5A,B). As the concentration of
DNA was very low relative to the concentration of
protein, the apparent K
d
values, which are equal to the
protein concentration at which 50% DNA is bound,
were estimated to be  0.35 lm for wild-type, Y103F,
R130E, R207E and R254E,  0.8 lm for G215E and
R226E and much larger than 0.8 lm (generally esti-
mated to be  1.5 lm) for R222E and R223E. These

results revealed that the reduced DNA binding of
mutant spo11-1 proteins (i.e. G215E, R222E, R223E
and R226E) observed in vitro was not the result of a
global change in the net charge (i.e. basic or neutral to
acidic) of the mutant proteins. Thus, Gly215, Arg222,
Arg223 and Arg226, but not Arg130, Arg207, Arg254
and Tyr103, play an important role in the DNA-bind-
ing activity of SPO11-1 in vitro.
We confirmed that this deficiency was not caused
by misfolding of the mutant proteins. First, the
spo11-1 mutants and the wild-type SPO11-1 showed
the same changes in florescence emission from aro-
matic residues (generally reflecting a change in protein
tertiary structure) when they were subjected to dena-
turation (data not shown). We further analyzed the
far-UV CD spectra (in the region between 200 and
250 nm) for the most severely DNA-binding defective
mutant, R222E, in comparison with the wild-type,
and found that both showed negative peaks at
around 210 and 220 nm (Fig. 5C), which are charac-
teristic of the secondary structure of proteins. The
signal intensity of the R222E mutant spo11-1 was
almost identical to that of the wild-type (Fig. 5C),
indicating that the spo11-1-R222E exhibits normal
protein folding. In addition, we compared the gel-
filtration profile of the spo11-1-R222E with that of
the wild-type SPO11-1, and both profiles were identi-
cal (Fig. S2). Thus, the higher-order structures of the
spo11-1-R222E mutant are normal, and the DNA-
binding deficiency is quite likely to be a direct effect

of the R222E substitution.
A
B
C
Fig. 5. dsDNA-binding activities of mutant TF-spo11-1 proteins.
(A) The TF-spo11-1 mutants were incubated with 9 n
M 180 bp
dsDNA at 37 °C for 5 min. The amounts of proteins are 0, 0.24,
0.47 and 0.71 l
M (from left to right). After the reaction, DNA
binding was analyzed as described in Fig. 3C. The symbols * and
** indicate protein-bound DNA and total input DNA, respectively.
(B) Quantification of the DNA-binding experiments described above,
in the legend to panel A. The wild-type and mutant proteins are
indicated within the figure. (C) Far-UV CD spectra of TF-SPO11-1
and TF-spo11-1-R222E. The spectra of TF-SPO11-1 and TF-spo11-1-
R222E (both 10 l
M) are represented as black and gray lines,
respectively, and were measured using a 1 mm cell in 50 m
M
sodium phosphate buffer, at 25 °C.
Y. Shingu et al. DNA-binding surface of Arabidopsis SPO11 protein
FEBS Journal 277 (2010) 2360–2374 ª 2010 The Authors Journal compilation ª 2010 FEBS 2367
Effects of single amino acid substitutions on
in vivo functions of SPO11-1
We sought to address whether the Gly and Arg resi-
dues responsible for the DNA-binding activity of
SPO11-1 in vitro were also required for the in vivo
functions of the protein. First of all, we expressed wild-
type SPO11-1 cDNA under the control of the SPO11-1

promoter in a spo11-1-3 homozygous A. thaliana
mutant. This construct complemented spo11-1-3,as
evidenced by the formation of normal siliques in all
five transformants (Fig. 6A). By contrast, the expres-
sion of spo11-1–Y103F cDNA did not complement
spo11-1-3 in any of the four transformants tested
(Fig. 6A), as described previously [27]. Then, using a
common vector, we expressed spo11-1 cDNAs harbor-
ing the glutamic acid substitution of the invariant
Gly215 (G215E) as a partially defective DNA-binding
mutant, the glutamic acid-substitution of invariant
Arg222 (R222E) as a DNA-binding defective mutant,
and the glutamic acid-substitution of invariant Arg130
(R130E), or of the non-conserved Arg207 (R207E),
as DNA-binding proficient variants (Table 1). We
observed no complementation in the three G215E
transformants and the three R222E transformants, and
observed full complementation in two of the three
R207E transformants (Fig. 6A). As no SPO11-1-spe-
cific antiserum is currently available to test for the
expression of these proteins, we analyzed the expres-
sion and the sequence of each mutant spo11-1 cDNA
transgene by RT-PCR, and found that the amounts of
SPO11-1 mRNA were not affected by any of the single
substitution mutations (Fig. 6B). We then determined
the number of seeds produced by 20 randomly selected
siliques from the transformants. The number of seeds
produced by the G215E and R222E transformants was
similar to that produced by the spo11-1-3 homozygous
mutant (Table 1).

A
B
Fig. 6. Complementation of the meiotic
defects of an Arabidopsis spo11-1-3 homo-
zygous mutant by the expressed wild-type
and mutant SPO11-1 cDNAs. (A) Panels
show the flowering stems of the wild-type
strain, the spo11-1-3 homozygous mutant
and spo11-1-3 mutants transformed with a
vector carrying wild-type SPO11-1 or a
mutant spo11-1 (i.e. Y103F, R130E, R207E,
G215E, or R222E). Arrows indicate siliques.
The number of complementation-positive
lines and the total number of lines analyzed
are denoted under each picture. (B) RT-PCR
analyses of the spo11-1 transgenes. Lane 1,
wild type strain; lane 2, spo11-1-3 homozy-
gous mutant; lanes 3–8, the spo11-1-3
homozygous transformants containing the
transgene of SPO11-1, Y103F, R130E,
R207E, G215E and R222E, respectively.
DNA-binding surface of Arabidopsis SPO11 protein Y. Shingu et al.
2368 FEBS Journal 277 (2010) 2360–2374 ª 2010 The Authors Journal compilation ª 2010 FEBS
We did not initially observe complementation in
five R130E transformants, but closer examination
revealed that the siliques of the R130E mutants were
slightly thicker than those of the spo11-1-3 homozy-
gous mutant (Fig. 6A). The R130E transformants
produced a much smaller number of seeds than the
wild-type and R207E transformants, but they

produced a significantly larger number of seeds in
each silique, as compared with the Y103F, G215E or
R222E transformants and the spo11-1-3 homozygous
mutant (Table 1). From these results, we concluded
that the invariant Gly215 and Arg222 residues, the
conserved Arg130 residue, as well as the invariant
Tyr103 residue, are required for the in vivo function
of SPO11-1 during meiosis, and that a spo11-1
mutant containing the R130E substitution retained
partial in vivo function.
Discussion
In this study, we isolated an active form of SPO11
with functional DNA-binding activity. TF-SPO11-1 is
able to bind to dsDNA and to ssDNA (Fig. 3A,B);
however, it possesses approximately twofold higher
binding affinity towards dsDNA than towards ssDNA.
As free TF did not bind to either form of DNA, the
DNA-binding activities of TF-SPO11-1 reside on the
SPO11-1-domain. SPO11 is a homologue of the type II
topoisomerases [3], which change the topological states
of closed-circular dsDNA through transient double-
stranded cleavage. Thus, it is intriguing that SPO11-1
can bind to ssDNA. However, it was shown previously
that the type II topoisomerase of E. coli phage T4, the
prototype type II topoisomerase, bound and cleaved
ssDNA [37]. The biological significance of this charac-
teristic remains to be studied.
We detected two discrete signals for TF-SPO11-1-
bound 180-bp dsDNA: a major signal of a larger com-
plex that was detected in all experiments; and a minor

signal of a smaller complex that was detected only in
the presence of a certain amount of NaCl (Fig. 3D) or
in the presence of a smaller amount of TF-SPO11-1
(Figs 3C and 5A). The DNA-binding surface of the
homology modelled SPO11-1 is a channel of  20 A
˚
in
diameter and  30 A
˚
in width, and therefore each
SPO11-1 would accommodate eight to nine base pairs
of dsDNA (Fig. 4A). As the DNA-binding experi-
ments were performed in the presence of a molar
excess of the protein (Fig. 3C), and TF-SPO11 forms
multimers consisting of various numbers of protein
molecules (Fig. 2C), it is likely that a number of
SPO11-1 proteins can be simultaneously bound to a
180 bp dsDNA molecule as a multimer. The observed
two discrete bands of TF-SPO11-1-bound dsDNA
might reflect two distinct states of the multimer. One
may speculate that each TF-SPO11 multimer binds to
one side of a dsDNA molecule, but we have no further
results with which to discuss each state of the protein–
DNA complex.
The solubility of SPO11-1 was greatly enhanced by
using TF, especially when TF was attached to the N
terminus of SPO11-1 (TF-SPO11-1, Fig. 1). TF has
been shown to associate with the ribosomal 50S sub-
unit, and to catalyze the proline-limited refolding of an
RNase T1 variant and other proteins in vitro [31,32].

The refolding activity of TF and the solubility of TF
itself (especially in fused SPO11-1) might enhance the
solubility of the attached SPO11-1.
Almost all of the TF-tag-free SPO11-1 was expressed
in a soluble form via co-expression with TF-SPO11-1
(Fig. 1B,C), and the co-expressed TF-SPO11 and
TF-tag-free SPO11-1 form much larger complexes than
TF-SPO11-1 alone (Fig. 2C). Thus, the solubility of
SPO11-1 is maintained via the direct interaction with a
soluble form of SPO11-1 (i.e. the SPO11-1 domain of
TF-SPO11-1). This large complex of the co-expressed
TF-SPO11 and TF-tag-free SPO11-1 may exist as
soluble aggregates of Spo11-1 simply coated by soluble
TF-SPO11-1. However, it is more likely that the forma-
tion of the large soluble complexes is explained by
co-operativity in the proper folding of SPO11-1, and
this suggests that a solubilizing protein cofactor is
Table 1. Effect of amino acid substitutions within Spo11-1 upon
DNA-binding affinity and seed production. ND, not determined.
Strain Transgene
a
DNA binding
b
Seeds
c
Wild-type ++++ 36.9 ± 2.7
spo11-1-3 None 1.9 ± 0.5
Y103F ++++ 2.0 ± 0.6
R130E ++++ 5.3 ± 1.6
R207E ++++ 34.5 ± 3.5

G215E ++ 1.9 ± 0.8
R222E + 2.0 ± 0.8
R223E + ND
R226E ++ ND
R254E ++++ ND
a
spo11-1 containing the indicated amino acid substitution.
b
The
DNA-binding ability in vitro of the proteins containing the indicated
amino acid-substitution: ++++, as good as that of the wild-type pro-
tein (K
d
’ 0.35 lM); ++, partially defective (K
d
’ 0.8 lM); +, almost
defective (K
d
>> 0.8 lM;or 1.5 lM). See Fig. 5B for quantitative
results. The K
d
values were estimated by the protein concentra-
tions at which 50% of the DNA is or would be bound in the pres-
ence of excessive amounts of the protein (see the text).
c
The
average number of seeds per silique (i.e. among 20 randomly
selected siliques from the transformants).
Y. Shingu et al. DNA-binding surface of Arabidopsis SPO11 protein
FEBS Journal 277 (2010) 2360–2374 ª 2010 The Authors Journal compilation ª 2010 FEBS 2369

required for the proper folding and maintenance of a
soluble or active form of SPO11-1. In Arabidopsis, no
protein factors, except for PRD1, were shown to have
functions to activate SPO11s in meiosis [24,26], and
PRD1N alone was not soluble and did not help to sol-
ubilize SPO11-1 (data not shown).
Unlike yeast and other organisms, Arabidopsis
expresses a second SPO11 [i.e. SPO11-2, [38]], which is
required for meiotic recombination [15,27]. A genetic
study suggested that SPO11-1 and SPO11-2 exercise
their meiotic functions as components of the same
complex (see the Introduction). We isolated SPO11-2
in a soluble, DNA-binding active form by attaching a
TF tag at the N terminus of SPO11-2 (Fig. 1B and
unpublished observations). While the co-expression of
an SPO11-1-interacting protein, PRD1N (the N-termi-
nal domain of PRD1 [26]), with TF-SPO11-1 main-
tained almost all of the PRD1N in a soluble form, the
co-expression of SPO11-2 with TF-SPO11-1 did not
improve the solubility of SPO11-2, compared to the
co-expression with the free TF-tag (Fig. 1B,C). There-
fore, SPO11-1 and SPO11-2 may not interact directly.
These results also lend biochemical support to the pre-
vious finding that the PRD1 N-terminal region inter-
acts directly with SPO11-1 in a yeast two-hybrid
system [26].
The meiotic defects of the spo11-1-3 mutant were
fully complemented in homozygous mutant plants by a
DNA vector, in which wild-type SPO11-1 cDNA was
transcribed under the control of the SPO11-1 pro-

moter. However, a vector expressing spo11-1-Y103F
(i.e. a mutation intended to displace Tyr103 from the
putative endonuclease-active centre of the protein
[3,27,29], as a negative control) did not (Fig. 6A).
Thus, this system was an appropriate tool with which
to assess the in vivo effects of amino acid replacements
within SPO11-1. Using the in vivo and in vitro experi-
mental systems, we examined the in vivo and in vitro
effects of single replacement mutations in conserved or
invariant amino acid residues within a putative DNA-
binding surface of Arabidopsis SPO11-1. With controls
with the same type of amino acid-substitution mutant
spo11-1s, we showed that the Gly215, Arg222, Arg223
and Arg226 residues, but not Arg130, Arg207, Arg254
and Tyr103, are members of a DNA-binding surface
of SPO11-1 (Figs 4 and 5). Both Gly215 and Arg222
residues are required for in vivo meiotic function of
SPO11-1 (Fig. 6A). Thus, our results suggest that the
DNA-binding surface containing Gly215 and Arg222
residues is required for the meiotic function of SPO11-
1 in vivo. Previously, other amino acid residues in yeast
SPO11 were identified as being involved in meiotic
dsDNA cleavage in vivo [29]; in Spo11 of S. cerevisiae,
Glu233 and Asp288 reside in a conserved structural
motif called the Toprim domain, while an invariant
arginine (Arg131) exists within a second conserved
structural motif known as the 5Y-CAP domain. It
would be worthwhile to test the corresponding amino
acid residues in Arabidopsis SPO11 for their DNA-
binding activities and effects on meiosis in Arabidopsis.

We found that the Tyr103 residue in the putative
endonuclease-active center of SPO11-1, which is
known to be involved in meiotic double-stranded
cleavage and meiotic recombination, is not involved in
DNA binding in vitro. Furthermore, the DNA-binding
surface of SPO11-1 is located in a region that does not
include Tyr103, suggesting that the endonuclease-active
center and the DNA-binding surface involved in dou-
ble-stranded cleavage are spatially separated. This is
not surprising if one considers that the substrate DNA
is a long macromolecule. In addition, the spatial rela-
tionship between Tyr103 and the DNA-binding surface
determined on SPO11-1 is conserved, in comparison
with that of archaeal topoisomerase VI A-subunit, a
SPO11 homologue. In the case of the topoisomerase
VI A-subunit, a crystallographic study including small
angle X-ray scattering analyses suggested that Tyr in
the active site initially resides too far from the site for
DNA cleavage to occur, and that Topoisomerase VI
B-subunit dimerization places the Tyr residue adjacent
to the active center [39]. Thus, it is also likely that the
binding of an interacting protein(s) to SPO11-1 is
required to place Tyr103 at the appropriate location
for DNA cleavage, and this could provide an explana-
tion for the absence of a detectable endonuclease activ-
ity in our preparation of TF-SPO11-1.
This study has identified a group of spo11-1 mutants
with defects in meiosis that specifically correlate with
the defects in DNA binding at an identified DNA-
binding surface on SPO11-1.

Materials and methods
Nucleic acids
A 180 bp dsDNA fragment was obtained by PCR amplifi-
cation of pUC18 dsDNA, using the primers 180L and
180R (Table S1). Closed-circular pUC18 dsDNA and
M13mp18 ssDNA were purchased from TAKARA Bio Inc.
(Shiga, Japan).
Detailed procedure to construct vectors for
expression of soluble SPO11-1 in E. coli
SPO11-1 cDNA was amplified from the A. thaliana ecotype
Columbia cDNA library using primers At1F-N and
DNA-binding surface of Arabidopsis SPO11 protein Y. Shingu et al.
2370 FEBS Journal 277 (2010) 2360–2374 ª 2010 The Authors Journal compilation ª 2010 FEBS
At1R-B containing the NdeI and BamHI restriction sites at
the underlined sequences, respectively (Table S1), and then
inserted into the pColdTF vector (TAKARA Bio Inc.,
Shiga, Japan) and the pET14b vector (Merck KGaA,
Darmstadt, Germany) via the NdeI and BamHI sites. It
should be noted that, because of the presence of an NdeI
site in the SPO11-1 coding region, the SPO11-1-bearing
DNA was only partially digested with NdeI, in order to
ensure the cloning of its entire coding region. The SPO11-2
and PRD1N cDNAs were amplified using primers At2F-N
and At2R-B, and primers PRD1F-N and PRD1R802-B,
respectively (Table S1), and were then inserted into the
pET14b vector via the NdeI and BamHI sites. These inserts
containing the T7 promoter were re-amplified using primers
T7p Up and T7p Down, which contain the EcoRI and PstI
restriction sites at the underlined sequences, respectively
(Table S1), and then were inserted into the pColdTF-

Spo11-1 vector. In CV2-PR, the TF-SPO11-1 and the
TF-tag free PRD1N co-expression vector (Fig. 1A), the
DNA region in the TF-SPO11-1 N-terminal His-tag was
removed using a QuikChange Site-Directed Mutagenesis kit
(Stratagene, La Jolla, CA, USA) and the following primer
pairs CV2-PR-F and CV2-PR-R.
Expression of soluble SPO11-1 in E. coli
The SPO11-1, SPO11-2 and PRD1N (encoding the 5¢ ter-
minal amino acid residues 1–802 of PRD1) cDNAs were
obtained via RT-PCR using total RNA isolated from
A. thaliana, ecotype Columbia. The detailed procedures
used to construct vectors for the expression of soluble
SPO11-1 in E. coli are described in the Supporting informa-
tion. E. coli strain BL21 (DE3) was transformed with the
constructed vectors. The transformed cells were cultured at
37 °C to an attenuance of 0.4 at 600 nm, as determined
using an UltroSpec 4300 pro spectrophotometer (optical
length, 10 mm; slit width, 1 mm; GE Healthcare, Little
Chalfont, UK). The cells were then incubated at 15 °C for
24 h in the presence of 0.4 mm IPTG. Cell pellets obtained
from the cultures were lysed in 50 mm phosphate buffer,
pH 7.0, containing 300 mm NaCl, 0.6 mgÆmL
)1
of lysozyme
and 0.5% Brij58. The suspensions were sonicated on ice
and centrifuged (22 000 g, 30 min, 4 °C). Under reducing
and denaturing conditions, the crude cell-extracts were elec-
trophoresed on a 12.5% polyacrylamide gel containing
0.1% SDS, and visualized by staining with Coomassie
Brilliant Blue R-250.

Purification of TF-tagged SPO11-1
The supernatants of cell-free extracts obtained from cells
(cell pellets of 7 g) overexpressing TF-tagged SPO11-1
(denoted TF-SPO11-1) were applied to a TALON CellThru
Resin column (1.5 cm diameter · 20 cm: TAKARA Bio
Inc.) and eluted by applying a 100 mL linear gradient of
0–150 mm imidazole. The TALON-purified fraction was
diluted three times in 50 mm sodium phosphate buffer, pH
7.0, and further applied to a Heparin Sepharose 6 Fast Flow
column (1.5 cm/ · 20 cm: GE Healthcare), which was
developed using a 100 mL linear gradient of 0–1000 mm
NaCl. TF-Spo11-1 was recovered at around 700–1000 mm
NaCl from the column. The collected TF-SPO11-1 was
desalted and concentrated using a centriprep YM-30 column
(Millipore, Bedford, MA, USA), and centrifuged (20 000 g,
30 min, 4 °C). The supernatant fraction was applied to a
Superdex200 column (1 cm/ · 30 cm: GE Healthcare) that
was eluted at a flow rate of 0.5 mLÆmin
)1
with 23 mL of
50 mm sodium phosphate buffer, pH 7.0, 300 mm NaCl.
Fractions containing TF-AtSPO11-1 (0.2 mg of proteinsÆ
mL
)1
; 5 mL) were collected.
Thronbin treatment
TF-SPO11-1 (0.76 lm) was incubated with 0.013 U of
thrombin at 37 °C for 3 h or overnight. Samples were then
centrifuged at 22 000 g for 10 min to obtain a supernatant
fraction and a precipitate.

Immunoblotting assays
The proteins expressed in E. coli were subjected to electro-
phoresis on a 10% polyacrylamide gel containing SDS,
electroblotted onto poly(vinylidene difluoride) (PVDF)
membranes and detected using the SuperSignal West
HisProbe kit (Pierce Biotechnology, Rockford, IL, USA).
Molecular mass markers consisted of Precision Plus Protein
Standards (Bio-Rad Laboratories, Hercules, CA, USA).
The positive signal bands were quantified using the ATTO
CS analyzer version 2.0 software.
Gel-retardation assays
Aliquots of TF-SPO11-1 (0.24, 0.47, 0.71 lm) were incu-
bated with 7.5 nm (125 ng) pUC18 closed-circular dsDNA,
9nm 180 bp dsDNA (10 ng) or 2.2 nm M13mp18 ssDNA
(50 ng) in a 10 lL reaction mixture, containing 20 mm
Hepes (pH 7.4), 150 mm NaCl, 1 mm dithiothreitol and
0.2 mgÆmL
)1
of BSA, at 37 °C for 5 min. The protein–
DNA complexes were directly loaded onto 0.8% agarose
gels. The complexes were then separated by electrophoresis
and stained with ethidium bromide.
In the binding experiment with the 180 bp dsDNA, the
separating agarose gel was blotted onto a Hybond N+
membrane (GE Healthcare) for detection of the DNA by
hybridization. The 180 bp [
33
P]DNA probe was prepared
by using a DNA 5¢-end labelling kit, MEGALABEL
(TAKARA Bio Inc.), and was purified using CHROMA

SPIN+STE-10 Columns (TAKARA Bio Inc.). Hybridiza-
tion with the labelled probe was performed in the presence
Y. Shingu et al. DNA-binding surface of Arabidopsis SPO11 protein
FEBS Journal 277 (2010) 2360–2374 ª 2010 The Authors Journal compilation ª 2010 FEBS 2371
of 0.5 m Na
2
HPO
4
(pH 7.2), 1 mm EDTA and 7% SDS
[40] at 65 °C for 16 h. The membranes were washed twice
with 0.2 · NaCl ⁄ Cit (SSC) containing 0.1% SDS at 65 °C
and then exposed to an imaging plate (Fuji Film, Tokyo,
Japan). The bound DNA was detected and quantified using
a BAS 2500 imager and the Multi Gauge software, version
3.1 (Fuji Film).
Detection of possible endonuclease- and
topoisomerase activities and covalent
attachment of protein at DNA termini
TF-AtSPO11-1 (0.5 lm) was incubated with pUC18
dsDNA (7.5 nm)in10mm Tris ⁄ HCl (pH 7.5), 5 mm
MgCl
2
,1mm dithiothreitol and 0–150 mm NaCl or KCl, at
22 or 37 °C for 1 h. The reaction was terminated by adding
only SDS (1%), or SDS (1%) and Proteinase K to
50 lgÆmL
)1
, followed by incubation at 37 °C for 15min.
The DNA was then loaded onto a 0.8% agarose gel,
separated by electrophoresis and stained with ethidium

bromide.
Homology modelling
A structural model of SPO11-1 was generated using the com-
parative homology modelling software, modeller, version
7.7 [41]. The subunit structure of Top6A (1D3Y) was
obtained from the Protein Data Bank and was employed as
the basal structure. A molecular diagram was generated using
molfeat, version 2.2.1.8 (FiatLux Co., Tokyo, Japan).
Generation of mutant spo11-1 proteins
Mutant spo11-1 cDNAs harboring single amino acid substi-
tutions (R207E, G215E, R222E, R223E, R226E, R254E and
Y103F), were constructed by using the QuikChange Site-
Directed Mutagenesis kit, and the primer pairs are listed in
Table S1. Proteins containing spo11-1 point mutations were
overexpressed in E. coli as TF-tagged forms, and were puri-
fied as described for wild-type TF-tagged SPO11-1.
CD measurements
The far-UV CD measurements were performed in a 1 mm
cell at 25 °C using a Jasco spectropolarimeter, model J-720
(Jasco, Tokyo, Japan).
Complementation of a homozygous mutant of
A. thaliana spo11-1
A T-DNA insertional mutant (spo11-1-3)ofA. thaliana
SPO11-1 (SALK_146172: ecotype Columbia: ref. 42) was
purchased from the Arabidopsis Biological Resource Center
(Ohio State University, Columbus, OH, USA). A heterozy-
gous spo11-1-3 mutant [14] was used in assays to examine
the abilities of wild-type and mutant spo11-1 transgenes to
complement the sterility of homozygous mutant progeny.
The SPO11-1 gene was expressed in vivo downstream of an

868 bp fragment that contained an upstream sequence of
the SPO11-1 start codon (i.e. as a putative SPO11-1
promoter). The CaMV 35S promoter and the GUS region
of the pBI121 binary vector (Clontech) were replaced with
this putative SPO11-1 promoter, as well as with the wild-
type SPO11-1 cDNA or the mutant spo11-1 cDNA, respec-
tively. A heterozygous spo11-1-3 mutant was transformed
with these SPO11 expression vectors using the floral dip
method in Agrobacterium tumefaciens [43]. After screening
for kanamycin resistance of the T1 seeds, the genotypes of
the transformants were tested by PCR with primers for
the T-DNA insertion and the SPO11-1 intron regions.
Only plants that were homozygous for the spo11-1-3
mutation were analyzed further. Transgenes were analyzed
using RT-PCR with At1F-N and At1F-B, as previously
described, and the amplified DNA fragments were
sequenced.
Acknowledgements
The authors would like to thank the Arabidopsis Bio-
logical Resource Center for the T-DNA insertion lines.
We thank Prof. Yoshifumi Nishimura for his generous
support in measuring CD by the use of the Jasco J-720
spectropolarimeter. This research was supported by a
grant from the Program for the Promotion of Basic
Research Activities for Innovative Biosciences, Bio-ori-
ented Technology Research Advancement Institution
(BRAIN), and in part by a Grant in Aid from the
Ministry of Education, Culture, Sports, Science and
Technology of Japan.
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Supporting information
The following supplementary material is available:
Fig. S1. Tests for biochemical activities of SPO11-1.
Fig. S2. Gel filtration analysis of the mutant R222E.
Table S1. Primers for PCR experiments.
This supplementary material can be found in the
online version of this article.

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