Published online 10 April 2007Nucleic Acids Research, 2007, Vol. 35, No. 82719–2733
Stimulation of fission yeast and mouse Hop2-Mnd1
of the Dmc1 and Rad51 recombinases
Mickae ¨l Ploquin1, Galina V. Petukhova2, Dany Morneau1, Ugo De ´ry1, Ali Bransi1,
Andrzej Stasiak3, R. Daniel Camerini-Otero2and Jean-Yves Masson1,*
1Genome Stability Laboratory, Laval University Cancer Research Center, Ho ˆtel-Dieu de Que ´bec,
9 McMahon, Quebec city, QC, Canada G1R 2J6,2Genetics and Biochemistry Branch, National Institute
of Diabetes, Digestive and Kidney Diseases, National Institutes of Health, 5 Memorial Drive, Bethesda,
MD 20892, USA and3Laboratory of Ultrastructural Analysis, Faculty of Biology and Medicine, University of
Lausanne, 1015 Lausanne, Switzerland
Received December 18, 2006; Revised March 6, 2007; Accepted March 7, 2007
Genetic analysis of fission yeast suggests a role for
the spHop2–Mnd1 proteins in the Rad51 and Dmc1-
In order to gain biochemical insights into this
process, we purified Schizosaccharomyces pombe
Hop2-Mnd1 to homogeneity. spHop2 and spMnd1
interact by co-immunoprecipitation and two-hybrid
analysis. Electron microscopy reveals that S. pombe
30-tailed DNA. Interestingly, spHop2-Mnd1 pro-
single-strand DNA and catalyses strand exchange
reactions with short oligonucleotides. Importantly,
we show that spHop2-Mnd1 stimulates spDmc1-
dependent strand exchange and strand invasion.
Ca2þalleviate the requirement for the order of
addition of the proteins on DNA. We also demon-
strate that while spHop2-Mnd1 affects spDmc1
specifically, mHop2 or mHop2-Mnd1 stimulates
both the hRad51 and hDmc1 recombinases in
strand exchange assays. Thus, our results suggest
a crucial role for S. pombe and mouse Hop2-Mnd1
in homologous pairing and strand exchange and
reveal evolutionary divergence in their specificity for
the Dmc1 and Rad51 recombinases.
haploid gametes or spores through meiosis. This is
achieved by one round of DNA replication followed
by two successive rounds of nuclear division, meiosis I
and meiosis II. These divisions are preceded by a
distinctive meiotic prophase during which homologous
chromosomes synapse and undergo genetic recombination
between allelic and non-allelic positions (1,2). Reciprocal
recombination events, named crossovers, establish chias-
mata, which are physical connections between homologs
that ensure proper chromosome segregation at meiosis I.
Meiosis II involves the segregation of sister chromatids
and is therefore analogous to a mitotic division. Meiotic
interhomolog recombination leads to an exchange of
genetic material, contributes to genetic diversity and is
required for proper chromosome segregation (3).
Meiotic recombination in Saccharomyces cerevisiae is
initiated by the creation of double-strand breaks by Spo11
(Rec12 in Schizosaccharomyces pombe), a type II topo-
isomerase (4). Spo11 knock-out mice display chromosome
synapsis defects suggesting that the initiation of recombi-
nation precedes and is required for normal chromosome
synapsis during meiosis I (5,6). In S. pombe, the Rec6,
Rec7, Rec12, Rec14 and Rec15 proteins are essential for
the creation of DNA double-strand breaks and meiotic
recombination (7). The Mre11, Rad50 and Nbs1 proteins
are involved in the processing of DSBs to form
recombinogenic 30-single-stranded tailed DNA (8). The
resected DSBs are then used to invade homologous duplex
DNA, leading to the exchange of genetic information, a
process that requires the RAD51 and DMC1 genes.
Rad51 and the meiosis-specific protein Dmc1 are homo-
logs of the bacterial recombinase RecA. In vitro, these
proteins are, however, less efficient than their bacterial
counterpart. Hence, there exist accessory cofactors that
assist Dmc1 and Rad51. Dmc1 is helped specifically by
Mei5 and Sae3 (9) and Rad51 is aided by the members of
the Rad52 group including Rad52, Rad54, Rad55 and
Rad57 (10–12), whereas Tid1/Rdh54 assists both recom-
Several lines of evidence implicate Hop2 and Mnd1 in
meiotic recombination. Saccharomyces cerevisiae Hop2
plays a crucial role in the proper alignment of homologs
*To whom correspondence should be addressed. Tel: þ1-418-525-4444; Fax: þ1-418-691-5439; Email: Jean-Yves.Masson@crhdq.ulaval.ca
? 2007 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/
by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
during meiotic prophase (15). Hop2 knock-out mice and
S. cerevisiae mutants show resemblance in their pheno-
types such as spermatocyte arrest at the pachytene-like
chromosome condensation and the failure in meiotic
DSBs to be repaired (16). Although the recombination
defects of the Hop2 and Mnd1 mutants were thought to be
an indirect consequence of the lack of chromosome
pairing, studies have indicated that Hop2 and Mnd1 are
directly involved in recombination. In budding yeast, hop2
and mnd1 mutants, meiotic cells arrest at the pachytene
stage, double-strand breaks are not repaired properly and
the Dmc1 and Rad51 recombinases accumulate on meiotic
invasion, heteroduplex DNA or joint molecules are
detected (15,17–19). A decrease in intragenic recombina-
tion and homologous pairing is observed in S. pombe
meu13 mutants (a Hop2 homolog) (20). Mnd1 is suggested
to be involved in strand invasion since mnd1-1 mutant
cells initiate recombination but do not form heteroduplex
DNA or Holliday Junctions (19). Genetic analysis of the
fission yeast ortholog Mcp7, revealed functions necessary
for meiotic recombination (21). Because of the very similar
phenotypes of the hop2 and mnd1 mutants, the Hop2–
Mnd1 proteins are thought to be involved in a common
pathway leading to homology search during meiotic
recombination. In fact, several lines of evidence suggest
that Hop2 and Mnd1 both functions in a Dmc1-
dependent pathway. First, the recombination phenotypes
of the hop2 and mnd1 mutants are very similar to those of
suppresses the meiotic defects of the dmc1 and hop2
mutants (22). Third, in every genome analyzed to date, the
presence of the HOP2 and MND1 genes correlates with
that of DMC1. In particular, the genome of C. elegans or
Drosophila does not contain Dmc1, Hop2 or Mnd1 while
Rad51 is present.
Biochemical studies of Hop2-Mnd1 have led to several
paradoxes. Purified budding yeast Hop2-Mnd1 only
stimulates the strand invasion activity of Dmc1 by a
modest 3-fold (23). Purified mHop2-Mnd1 stimulated
strand invasion of hRad51 by about 10-fold and hDmc1
by 35-fold (24) while in another study mouse Hop2 had no
effect on human Rad51 in vitro (25). Finally, S. cerevisiae
and human Hop2-Mnd1 was found to bind dsDNA
preferentially (23,26), which is not consistent with its role
in strand invasion.
In this manuscript, we present biochemical evidence
that fission yeast Hop2-Mnd1 binds ssDNA of 30-tailed
molecules specifically by electron microscopy. spHop2-
Mnd1 show properties reminiscent of recombinases, it can
anneal long complementary single-strand DNAs and
promote strand exchange. Moreover, D-loop and strand
exchange assays revealed that spHop2-Mnd1 can activate
spDmc1 efficiently. In addition, we show that mouse Hop2
and Hop2-Mnd1 can stimulate both human Rad51 and
Dmc1 recombinases in strand exchange assays in accor-
dance with the Yokohama group (26). These results show
that Hop2-Mnd1 participates in both the homologous
pairing and strand exchange steps of homologous
MATERIALS AND METHODS
Nucleic acids andS. pombe techniques
Single-strand and double-strand øX174 DNA was pur-
chased from New England Biolabs (Beverly, MA, USA).
pPB4.3 (27) was prepared using Qiafilter plasmid maxi kit
(Qiagen, Valencia, CA, USA). Linear duplex DNA with
30-single-stranded tails at both ends were prepared as
described previously (28). For D-loop analysis, pUC18
plasmid DNA was purified by Triton X-100 lysis followed
by CsCl banding (24). DNA concentrations are expressed
in moles of nucleotides.
Schizosaccharomyces pombe methods were performed
according to Moreno et al. (29). Schizosaccharomyces
pombe mRNA was isolated using RNeasy (Qiagen). The
diploid strain JN628 (kindly provided by K. Tanaka) was
induced in meiosis and cells were collected at 2h intervals.
Protein extracts were subjected to western blot analysis
with anti-mouse Hop2 that cross-recognize the S. pombe
protein. The Meu13þ cDNA (S. pombe hop2þ) was
amplified by RT-PCR from S. pombe mRNA isolated
from meiotic cells using primers JYM110 (50-tcgcaattg
tagtttaagtcaattggtccctccgt) bearing NdeI and BamHI
sites, respectively. The PCR product was subjected to
digestion with NdeI and BamHI and cloned into pET16b
and pET28b (Novagen, Madison, WI, USA) in which the
Meu13 protein sequence was linked to a decahistidine and
hexahistidine tag, respectively. The sequence of S. pombe
Meu13þ was identical to accession number SPAC222.15.
Mcp7þ (herein Mnd1þ) was isolated from S. pombe
genomic DNA using JYM123 (50-gggaattccatatgcctcc-
caaaatcggtagttgcagatcgtc) bearing NdeI and BamHI
An intronless derivative was generated by removing a
94bp intron essentially as published by Wang and
Malcolm using primers JYM161 (50aggccattttccatgactc
gene was amplified by PCR with primers bearing NdeI
and BamHI sites. The PCR product was cloned into
pET16b (Novagen) to generate pET16b-Mcp7. To gen-
erate Myc-tagged Mcp7, Mcp7 was PCR amplified using
primers JYM580 (50-ccatggatggaggagcagaagctgatctcagag
gaggacctgatgcctcccaagggactatcg) and JYM581 (50-ggatcct
cacaaaatcggtagttgcag) and the PCR product was cloned in
pET28b using NcoI and BamHI sites. The sequence of
S. pombe mcp7þ was identical to accession number
Purification of S. pombe Hop2–Mnd1 complex
Recombinant spHop2–Mnd1 proteins were purified from
800ml of Escherichia coli BL21(DE3) RP (Stratagene,
La Jolla, CA, USA) carrying plasmids pET28b-Meu13
and pET16b-Mcp7 grown at 378C in tryptone phosphate
media supplemented with 100mg/ml ampicillin, 50mg/ml
kanamycin and 25mg/ml
OD600¼0.4, Hop2 and Mnd1 synthesis was induced by
the addition of 0.1mM IPTG, incubated at 308C, after 4h,
Nucleic Acids Research, 2007, Vol. 35, No. 8
the cells were harvested by centrifugation, frozen in dry ice
and stored at ?808C. The cell paste was resuspended in
80ml of P5 buffer (50mM sodium phosphate pH 7.0,
500mM NaCl, 10% glycerol, 0.02% Triton X-100)
containing 5mM imidazole and the protease inhibitors
PMSF (1mM), aprotinin (0.019 TIU/ml) and leupeptin
(1mg/ml). The suspension was divided into 40ml aliquots,
which were sonicated three times for 30s. Insoluble
material was removed by centrifugation at 35000r.p.m.
for 1h in a Sorvall Ultra Pro 80 T647.5 rotor. The
supernatant was loaded on a 5ml Talon column (BD
Biosciences, Palo Alto, CA, USA).
The column was washed successively with 50 and 10ml
of P buffer containing 30 and 40mM imidazole, respec-
tively before Hop2-Mnd1 were eluted with 25ml of
P buffer containing 500mM imidazole. Fractions of
Hop2-Mnd1, were identified by SDS-PAGE, pooled and
dialyzed against QS buffer (10mM Tris–HCl pH 8.0, 10%
glycerol, 1mM DTT) containing 100mM KCl (QS100)
and loaded on a 1ml MonoS column (HR 5/5) using a
Pharmacia FPLC system. The column was washed with
20ml of QS buffer containing 150mM NaCl before Hop2-
Mnd1 was eluted using a linear gradient of 10ml of
0.1–0.8M KCl in QS buffer. The protein eluted around
400mM KCl and was dialyzed against 1l of QS100 buffer
and stored in aliquots at ?808C. The concentration of
Hop2-Mnd1 was determined by SDS-PAGE using pur-
ified BSA as a standard and verified by Bradford assay.
This fraction did not contain monomers of Hop2-Mnd1.
Purification of Dmc1, Rad51, mouse Hop2,
mouse Hop2-Mnd1and E. coli SSB
Schizosaccharomyces pombe Rad51 and Dmc1 were
purified as described (31). Mouse Hop2 or Hop2-Mnd1
was purified as described previously (24). Human RAD51
and Dmc1 were purified as described (32,33). Purified
E. coli single-stranded binding protein (SSB) was pur-
chased from USB. Purified BSA was obtained from Sigma
(St. Louis, MO, USA).
Escherichia coli total and soluble extracts expressing
spMnd1, spHop2 or spMnd1 and spHop2 were prepared
as follows. Escherichia coli BL21(DE3) RP (Stratagene)
carrying either pET16b-spMnd1, pET28b-Hop2 or both
plasmids were grown at 308C in 50ml LB with antibiotics.
At OD600¼0.4, spHop2 and spMnd1 synthesis was
induced by the addition of 0.1mM IPTG, incubated at
308C, after 4h, the cells were harvested by centrifugation,
frozen in dry ice and stored at ?808C. The cell paste
was resuspended in 5ml of P5 buffer (50mM sodium
phosphatepH 7.0, 500mM
0.02% Triton X-100) containing 5mM imidazole and
the protease inhibitorsPMSF
(0.019 TIU/ml) and leupeptin (1mg/ml). The suspension
was divided into 40ml aliquots, which were sonicated
three times for 30s. An aliquot was collected and
insoluble material was removed by centrifugation at
13 000 r.p.m. for 20min. The supernatant was kept as
the soluble fraction.
Immunoprecipitations were conducted with soluble
extracts from E. coli BL21(DE3) RP (Stratagene) carrying
pET16b-spHop2 and pET28b-spMnd1-Myc prepared as
above. Protein complexes in the supernatant were pulled
down for 1.5h at 48C using anti-Myc antibody (Santa
Cruz, California, CA, USA) or beads also. Complexes
were washed four times in P5 buffer, and visualized by
western blotting using anti-mHop2 or anti-His antibody.
For affinity pull-downs, purified BSA, spDmc1 or
Sepharose (Amersham, Piscataway, NJ, USA) to generate
the affinity columns. spHop2-Mnd1 was incubated with
affinity matrix in 20ml buffer containing 20mM Tris–HCl,
pH 7.5, 150mM KCl, 10% glycerol, 1mM DTT and
protease inhibitors. After 15min of incubation at 378C,
the samples were washed four times with the same buffer.
The proteins were eluted with SDS-PAGE loading buffer
and visualized bywestern
bound to NHS-activated
blotting using anti-His
Molecular mass estimation of spHop2-Mnd1
The molecular mass of purified spHop2-Mnd1 (100mg)
was determined by comparison with gel filtration stan-
dards [250mg; bovine thyroglobulin (670kDa), bovine
gamma globulin (158kDa), chicken ovalbumin (44kDa),
horse myoglobin (17kDa) and vitamin B-12 (1.35kDa)].
Proteins were analyzed on a FPLC Explorer 10 system
fitted with a 24ml Superdex 200 PC 3.2/30 column
(Pharmacia, Peapack, NJ, USA) equilibrated in R150
buffer (20mM Tris–Cl pH 8.0, 150mM NaCl, 10%
glycerol, 1mM EDTA, 0.5mM DTT). Fractions (250ml)
were collected and analyzed by SDS-PAGE followed by
western blotting with a monoclonal anti-histidine anti-
body (BD Biosciences) or an antibody against mouse
Hop2 that recognize S. pombe Hop2. Native (non-
denaturing) electrophoresis was performed using the
NativePAGE Novex Bis–Tris gel system (Invitrogen).
The full-length S. pombe Meu13þ and Mcp7þ genes were
cloned into pGBK-T7 and pGAD-T7 (BD Biosciences), to
produce fusions to the Gal4 DNA-binding and activation
domains. Plasmids spHop2-NAD (amino acids 1–106),
spHop2-CAD(107–211) and spMnd1-NAD(amino acids
1–110), spMnd1-CAD (amino acids 111–216) were con-
structed by cloning the appropriate PCR products in
pGBK-T7. All fusions were confirmed by sequencing.
AH109 was transformed with the indicated plasmids.
Colony growth on media lacking tryptophan and leucine
was observed with all constructions tested herein.
Interactions between partners were assayed by growth
on synthetic media lacking tryptophan, leucine, adenine
and histidine supplemented with 2.5mM 3-aminotriazole.
Transformations were carried out according to the
matchmaker kit manual (BD Biosciences).
Reactions (10ml) contained 100 nM DNA in binding
buffer (20mM triethanolamine–HCl, pH 7.5, 2mM ATP,
1mM Mg (CH3COO)2, 1mM DTT and 100mg/ml BSA).
Nucleic Acids Research, 2007, Vol. 35, No. 82721
After 5min at 378C, the indicated amount of spHop2-
Mnd1 was added (2ml) and incubation was continued for
a further 10min. Complexes were fixed by addition of
0.2% glutaraldehyde followed by 15min incubation at
378C. Protein–DNA complexes were analyzed by 6%
PAGE using TBE buffer. The gels were dried on DE81
filter paper followed by autoradiography. DNA substrates
were prepared by annealing a32P-labeled oligonucleotide
(100nt in length)with appropriate
sequences. The 100-mer double-stranded, and the corre-
sponding 50-tailed and 30-tailed DNA (containing both
50nt of ssDNA) were purified by 10% PAGE. The
sequence of the 100-mer is 50-GGGCGAATTGGGCC
GTCTCCATTTAAAGGACAAG-30. DNA concentra-
tions are expressed in terms of moles of nucleotides.
Percentage of DNA binding were quantified on a Storm
860 phosphorimager (Molecular Dynamics, Piscataway,
The substrate was prepared as follows. pPB4.3 was
digested with NdeI and HindIII, dephosphorylated
with calf alkaline phosphatase (NEB), followed by gel
purification of the corresponding 400bp fragment using
Quiaquick gel purification kit (Qiagen). The 400bp
fragment was end-labeled with g-ATP and T4 polynucleo-
tide kinase (NEB). Reactions (10ml) contained denatured
50-end labeled 400bp fragment (450 nM) and spH2M1
(1.2mM) in HEPES buffer (20mM HEPES pH 7.5,
6.5mM Mg (CH3COO)2, 2mM ATP, 5mM DTT,
100mM NaCl and 100mg/ml BSA). Incubation was at
208C for 5min or the indicated time. The reaction
products were deproteinized by addition of one-tenth
volume of stop buffer (10% SDS and 10mg/ml proteinase
K) followed by 15min incubation at 208C. Labeled DNA
products were analyzed by electrophoresis through a 4%
TBE1X/PAGE gel run at 150V for 2h 15min, dried onto
DE81 filter paper and visualized by autoradiography.
Reactions were performed according to Lio et al. (34).
Reactions (10ml) contained a 63-mer single-stranded
oligonucleotide (0.7mM) with the indicated concentrations
of spHop2-Mnd1 and spDmc1 in PTUK buffer (20mM
Tris–HCl, pH 7.5, 2.5mM MgCl2, 2mM ATP, 1mM
DTT, 7.5mM creatine-phosphate and 30 U/ml creatine
kinase). After 5min at 378C, the corresponding32P-end
labeled 63bp dsDNA (0.7mM) was added and incubation
was continued for 90min. Reaction products were
deproteinized by addition of one-fifth volume of stop
buffer (0.1M Tris–HCl, pH 7.5, 0.1M MgCl2, 3% SDS,
5mg/ml ethidium bromide and 10mg/ml proteinase K)
followed by 30min incubation at 378C. Labeled DNA
products were analyzed by electrophoresis through 8%
TBE1X/PAGE. For DNA-melting assays, 13.1mM of a
79-mer ssDNA was included in the reaction stop buffer.
Strand exchange with longer substrates contained
purified single-stranded pPB4.3 DNA (15mM) with the
indicated concentrations of either human Dmc1 or Rad51
and/or mouse Hop2 or Hop2-Mnd1 in standard buffer
(50mM triethanolamine–HCl, pH 7.5, 1mM Mg (OAc)2,
2mM ATP, 1mM DTT and 100mg/ml BSA). Otherwise
indicated, hRad51 or hDmc1 was added before mHop2 or
mHop2-Mnd1 on the ssDNA. After 5min at 378C,
32P-end labeled pPB4.3 DNA (400bp fragment) was
Reaction products were deproteinized as above. Labeled
DNA products were analyzed by electrophoresis through
0.8% TAE agarose gels containing 1mg/ml ethidium
bromide, run at 4.3V/cm, dried onto filter paper and
visualized by autoradiography.
Strand invasion assays were performed as published
Rad51 reactions (10ml) contained 5mM ?X174 single-
stranded DNA, or pPB4.3 tailed DNA in 20mM
triethanolamine–HCl, pH 7.5, 1mM DTT, 2mM ATP,
2.5mM Mg (CH3COO)2. After 5min at 378C, the
indicated amount of protein was added and incubation
was continued for a further 10min. When fixation was
required, protein–DNA complexes were fixed by addition
of glutaraldehyde to 0.2% followed by 15min incubation
at 378C. Samples were diluted and washed in 5mM Mg
Complexes were visualized at a magnification of 22000?
using a Philips CM12 electron microscope.
Expression andpurification ofS. pombe Hop2and Mdn1
The expression of Meu13 (herein, S. pombe Hop2) during
meiosis was monitored in order to amplify the gene by
RT-PCR since it contains four introns. Meiosis was
induced and the expression of spHop2 was observed by
western blot analysis. Maximal expression of S. pombe
Hop2 protein was observed after 6h (Figure 1A), which is
consistent with published northern blotting experiments
(20). Hence, the Meu13þ gene was amplified by RT-PCR
from mRNA extracted 6h after the induction of meiosis.
The mcp7þ gene (Mnd1þ) was amplified from genomic
DNA and the intron was removed by a modified
Quickchange site-directed mutagenesis protocol (30).
S. pombe hop2þ was cloned into pET28b and S. pombe
mnd1þ was cloned in pET16b (Novagen) to add,
respectively, a 10- and 6-histidine tag at the N-terminus
of the proteins. Escherichia coli BL21 (DE3) RP carrying
both plasmids was induced by IPTG, the solubilized
proteins were then subjected to Talon affinity chromatog-
raphy. Individual fractions were subjected to SDS-PAGE
and Coomassie blue staining. By carefully selecting the
fractions, the Hop2–Mnd1 proteins were generally pure
after this step. When necessary, a Mono S chromatogra-
phy was performed and co-elution of spHop2-Mnd1 was
observed. The purified proteins were free of endo- and
Nucleic Acids Research, 2007, Vol. 35, No. 8
exonuclease activities (data not shown). The predicted
molecular weight of his-tagged Hop2 is 28 652 Da and his-
tagged Mnd1 is 26 761 Da. Hence, the two proteins
co-migrate on a SDS-PAGE (Figure 1C, SDS-PAGE).
Mass spectrometry was performed to confirm the purity of
our preparation and the presence of both Hop2 and Mnd1
(data not shown).
Gel filtration and interactions between S. pombe Hop2
The native molecular weight of the spHop2-Mnd1
protein preparation was determined by gel filtration
through Superdex 200. The bulk of spHop2 and
spMnd1 eluted in fractions 46–50 suggesting that these
proteins could form complexes containing a variable
number of molecules (Figure 1B). The corresponding
molecular mass of these fractions range from 47 to
80kDa with a peak observed at 60kDa (Figure 1B,
revealed that our preparation contained two complexes
of 40 and ?66kDa (Figure 1C, native). Given the
(28.6kDa) and his-tagged Mnd1 (26.7kDa), this is
indicative of multimeric forms composed of homo- or
Figure 1. Purification and interaction of fission yeast Hop2-Mnd1. (A) Expression of Hop2 during S. pombe meiosis. Meiosis was induced by
nitrogen starvation and the expression of spHop2 was observed by western blot analysis using anti-Hop2at the indicated times. (B) Native molecular
mass of purified spH2M1 as determined by gel filtration through Superdex 200. Top part: size standards, bottom part: western blotting of spHop2
and spH2M1 using anti-mHop2 and anti-His, respectively. (C) Left: SDS-PAGE of purified spHop2-Mnd1 (spH2M1). Lane a, molecular weight
markers; lane b, purified spHop2-Mnd1 (5mg). Proteins were loaded on a 10% SDS-PAGE and stained with Coomassie blue. Right: native
electrophoresis of purified spHop2-Mnd1. Lane a, NativeMark protein standard; lane b purified spHop2-Mnd1 (2mg). Proteins were loaded on a
4–16% NativePAGE Bis–Tris gel and stained with Coomassie blue. (D) Solubility of spMnd1, spHop2 and co-expressed spHop2 and spMnd1 in
E. coli. Thirty microgram of total (lanes a–c) or soluble extracts (lanes d–f) of E. coli BL21 (DE3) RP (Stratagene) expressing the indicated proteins
were prepared and subjected to western blotting of spHop2 and spH2M1 using anti-mHop2 and anti-His, respectively. (E) Co-immunoprecipitation
of spHop2 and spMnd1. SpMnd1-Myc and spHop2-His were co-expressed in E. coli and soluble extracts were prepared followed by
immunoprecipitation with anti-Myc (lane a) or beads alone (lane b). The proteins were detected by western blotting using anti-Myc and anti-His,
respectively. Lane c, purified H2M1 (100ng). (F) Fission yeast Hop2 interacts with Mnd1 by two-hybrid analysis. AH109 strain was co-transformed
with the indicated plasmids and positive interactions were monitored by growth and coloration on media lacking tryptophan, leucine, histidine,
adenine and supplemented with 2.5mM aminotriazole. The annotations DBD and AD design fusions to the Gal4 DNA-binding domain and Gal4
activation domain, respectively.
Nucleic Acids Research, 2007, Vol. 35, No. 82723
hetero-complexes. Monomeric Hop2 or Mnd1 were not
detected (Figure 1B, lane 56 and Figure 1D).
In order to characterize these complexes, three experi-
ments were conducted. First, we expressed spMnd1,
spHop2 or both proteins in E. coli and total and soluble
extracts were prepared (Figure 1D). When expressed on
their own, spMnd1 and spHop2 were not soluble
(lanes d–e). Solubility was only achieved if both proteins
were co-expressed (lane f). Similar results were obtained in
insect cells (data not shown). This result suggests that our
protein preparation is composed of an hetero-complex
of spHop2-Mnd1. Second, immunoprecipitations were
conducted using cells extracts expressing Myc-tagged
spMnd1 and His-tagged spHop2. Anti-Myc antibodies
pulled down a complex between spMnd1 and spHop2
(Figure 1E, lane a). Third, the interactions of Hop2 and
Mnd1 were further characterized by two-hybrid analysis.
The AH109 strain (BD Bioscience) was co-transfected
with plasmids encoding fusions to the Gal4 DNA binding
and activation domains, respectively, and spotted on
media lacking tryptophan, leucine, histidine and adenine
with 3-aminotriazole. Strong interactors are white in color
because of the expression of the ADE gene. Co-expression
of full-length spMnd1 fused to the Gal4 DNA binding and
full-length spHop2 fused to the activation domains in
yeast lead to growth of white colonies on omission media
(Figure 1F). Interactions were also observed when
spHop2 fused to the Gal4 DNA binding and Mnd1
fused to the activation domain were co-transformed.
The strength of interactions between spHop2 and Mnd1
was similar to spRad51 self-interactions, which indicates
a strong interaction between the proteins. Truncations
of spHop2 and spMnd1 in N-terminal and C-terminal
portions revealed that the C-terminus of spHop2 (amino
acids 111–216) interacts with the C-terminal portion
of spMnd1 (amino acids 107–211). The N-terminus
of spHop2 (amino acids 1–110) and spMnd1 (amino
acids 1–106) did not interact with each other or the
C-terminus of the proteins. Interestingly, spHop2 and
spMnd1 self-interactions were also observed suggesting
that these complexes can form in vivo although we were
unable to get these proteins soluble when expressed on
DNA binding by S.pombe Hop2-Mnd1
In the presence of ATP, RecA binds single-strand DNA
preferentially over double-strand DNA because of a
kinetic barrier to associate with dsDNA (36). This
difference in affinity is critical for strand exchange
reactions since pairing occurs between single-strand
Mobility Shift Assays were used to investigate the
ssDNA- and dsDNA-binding properties of spHop2-
Mnd1. spHop2-Mnd1 bound preferentially to ssDNA,
over 30- or 50-tailed DNA or dsDNA (Figure 2A–D). We
observed the formation of protein–DNA networks that
tended to smear up the gel, which is characteristic of the
formation of networks containing a variable number of
protein and DNA molecules. Quantification of the DNA–
protein complexes at 0.5mM Hop2-Mnd1 indicated that
31% of the input ssDNA was stably bound compared with
10% for 30-tailed and 50-tailed DNA or 3% for dsDNA.
The formation of protein–DNA complexes on ssDNA was
optimal at 50mM NaCl, supported in a wide range of Mg
(CH3COO)2concentrations (from 1 to 10mM; with an
optimal concentration at 2mM) and was gradually
reduced by inclusion of increasing concentrations of
potassium chloride over 150mM (data not shown).
Binding to ssDNA was observed both in the absence of
ATP or ATP analogs suggesting that DNA binding can
occur without ATP or ATP hydrolysis.
Strandannealing and strandexchange catalyzed
We next investigated their ability to promote single-strand
annealing (Figure 2E). Long single-strand DNA of 400
bases was used instead of oligonucleotides, which can be
prone to artifacts. Interestingly, spHop2-Mnd1 promotes
annealing of long complementary single stranded DNAs.
Single-strand annealing was observed rapidly, within
0.5min (6%) and increased to a level of 16% after
10min (Figure 2E, lanes g–k). Low levels of spontaneous
strand annealing were observed in the control alone (lanes
b–f). The renatured products were in the form of linear
duplex as opposed to high molecular weight networks.
Annealing by spHop2-Mnd1 could persist in the presence
of a single-strand binding protein (Figure 2F). The
functional analogy between RPA and SSB is supported
by results indicating that SSB can substitute for budding
yeast and human RPA in strand exchange mediated by
S. cerevisiae and human Rad51, respectively (37,38).
spHop2-Mnd1 annealing did not require ATP consistent
with the fact that spHop2 or spMnd1 do not contain any
known ATP-binding motif (data not shown).
a characteristic shared by the S. pombe Rad51 and
spHop2-Mnd1 possessed other recombination activities.
We then examined the ability of purified Hop2-Mnd1 to
promote strand exchange in vitro using a linear single-
stranded DNA and homologous linear duplex DNA.
spHop2-Mnd1 could perform strand exchange with
oligonucleotides (Figure 2G, lane c–l) but not with
longer substrates (data not shown) suggesting that it has
limited ability to do this reaction. The reaction did not
require ATP hydrolysis but homologous single-strand
DNA (Figure 2H, lanes b–f, g). DNA strand exchange
activity with oligonucleotides can be due to thermal
opening of the duplex followed by subsequent annealing,
as demonstrated for the RAD51C protein (34). In order to
eliminate this possibility, we performed experiments as
described by Lio et al. (Figure 3A–B). First, no released
ssDNA was produced when the DNA substrates were
heterologous (Figure 3B, compare lanes a–b). This result
demonstrates that spHop2-Mnd1 DNA strand exchange
is homology-dependent. Next, the standard homology-
dependent experiment was carried out, with an excess
of homologous ssDNA added to the stop buffer. The
oligonucleotide used is of a different length (79-mer).
DNA pairing was observed with homologous DNA but
Nucleic Acids Research, 2007, Vol. 35, No. 8
not heterologous DNA (compare lane c–d). The level of
pairing observed with heterologous DNA was similar to
spontaneous reannealing (lane e). This result shows that
spHop2-Mnd1 promotes strand exchange independently
of a DNA melting activity. The temperature dependence
of spHop2-Mnd1 strand exchange was also analyzed.
Strand exchange increased over a period of 90min
and was more efficient at 378C than 308C or and 208C
(Figure 3D). This result is inconsistent with DNA melting
which occurs within 5min (34). Since spHop2-Mnd1 are
expected to function in concert with the spRad51 and
spDmc1 recombinases during meiotic recombination we
investigated whether spHop2-Mnd1 was able to stimulate
spDmc1andspRad51 strandexchange. We have
previously shown that spRad51 and spDmc1 have
strand exchange abilities (31). When limiting concentra-
tions of spDmc1 were used, only 2% of displaced ssDNA
were produced (Figure 3C, lane d, 2% products).
exchange occurred at a higher level than with either
protein alone (compare Figure 3C, lanes g–j to Figure 2G,
lanes e–h). Quantification of the products at 5mM
spDmc1 and 4mM spHop2-Mnd1-Dmc1 revealed a
5-fold increase in activity compared to spHop2-Mnd1
alone. These results reveal that stimulation of spHop2-
Mnd1 was specific for spDmc1, as the effect was not
observed on Rad51 (data not shown).
Figure 2. DNA binding, single-strand annealing and strand exchange properties of S. pombe Hop2-Mnd1. (A–D) DNA binding of spH2M1. DNA-
binding reactions contained ssDNA (A), 50-tailed duplex DNA (B), dsDNA (C) and 30-tailed duplex DNA (D) and the indicated concentrations of
spH2M1. (E) Time course of single-strand annealing by spH2M1. Lane a, purified 400bp duplex DNA. Reactions contained denatured 400bp duplex
DNA and no protein (lanes b–f) or spH2M1 (1.2mM, lanes g–k). The reactions were stopped at the indicated times. (F) spH2M1 promote single-
strand annealing in the presence of a single-strand binding protein. Lane a, purified 400bp duplex DNA; lane b, spH2M1 alone (1.2mM); lanes c–g,
SSB alone; lanes h–l, spH2M1 (1.2mM) with the indicated concentrations of SSB. (G) spH2M1 promotes strand exchange. spH2M1 was mixed with
1.5mM 63-mer ssDNA. DNA strand exchange was initiated by addition of homologous 3mM of 63-bp dsDNA, in which the strand that would be
displaced during DNA strand exchange was32P-labeled.Lane a, duplex DNA; lane b, denatured duplex DNA; lanes c–l, DNA strand exchange as
a function of spH2M1 concentration. (H) Effect of cofactors on spH2M1 strand exchange. Reactions were carried out in buffer containing an
ATP regeneration system using 3mM spH2M1 (lane b); in buffer without ATP (lane c); in standard buffer in which ATP was replaced with ATPgS
(lane d); AMP-PNP (lane e); standard buffer lacking Mg2þ(lane f); reactions with heterologous ssDNA (lane g); in buffer without ATP regeneration
(lane h); lane a, without protein.
Nucleic Acids Research, 2007, Vol. 35, No. 8 2725
Dmc1strand invasion isstimulated by Hop2-Mnd1
spHop2-Mnd1 are also expected to help the spRad51 and
spDmc1 recombinases during strand invasion. First,
purified spHop2-Mnd1 was unable to perform strand
invasion on its own (Figure 4A, lane g). Strikingly,
spHop2-Mnd1 stimulated spDmc1 D-loop formation in a
buffer containing magnesium (Figure 4A, lanes b–f).
At 1mM Hop2-Mnd1a 8-fold increase was observed
(Figure 4E). This spDmc1-dependent spHop2-Mnd1
stimulatory effect was even greater in calcium buffer
(Figure 4B, lanes c–h) and quantification of the products
revealed a 17-fold increase in D-loop formation at 1mM
spHop2-Mnd1 (Figure 4F). D-loop formation is a
reversible reaction, known as the D-loop cycle (39).
Therefore, we studied the reversibility of the D-loop
assay using 1mM spHop2-Mnd1, which was optimal for
the magnesium and calcium reactions (Figure 4E and F).
In magnesium buffer, we observed a constant diminution
of the D-loop reaction from 5 to 60min (Figure 4C
and G). Apparent reversibility of the D-loops may be due
to nicking of the supercoiled plasmid DNA by contam-
inating nucleases. This possibility was ruled out since
4mM concentrations of spDmc1 or spH2M1 did not reveal
any increase in the nicked DNA (data not shown). D-loop
cycles were not observed in calcium buffer as D-loop
products increased over a period of 60min (Figure 4D
and H). The order of addition of the proteins to the DNA
in D-loop was assayed in a magnesium buffer. Stimulation
of spHop2-Mnd1 on spDmc1 was maximal when Dmc1
was added to the ssDNA and spHop2-Mnd1 to the
dsDNA and the two mixtures were combined (Figure 5A,
lanes d and h). Intermediate levels of stimulation were
Figure 3. spHop2-Mnd1-promoted DNA strand exchange is not due to a DNA-melting activity. (A) Strategy to detect DNA melting. If spH2M1
melts the dsDNA, the free ssDNA strands would quickly re-anneal after deproteinization. The standard strand exchange with 63-mer
oligonucleotides were carried out, but in addition, an excess amount of 79-mer homologous ssDNA was added to the stop buffer. Thus, pairing that
might occur during the deproteinization step would be distinguished from DNA strand exchange with the intended partner as diagramed. (B) Strand
exchange reactions were carried using homologous 63-mer ssDNA (lane a); heterologous 48-mer ssDNA (lane b); homologous 63-mer ssDNA and
79-mer ssDNA in the reaction stop buffer (lane c); heterologous ssDNA and 79-mer ssDNA in the reaction stop buffer (lane d); heterologous ssDNA
and 79-mer ssDNA in the reaction stop buffer without protein (lane e). (C) Stimulation of S. pombe Dmc1 strand exchange by spH2M1. Lane a,
duplex DNA; lane b, denatured duplex DNA; lane c, spDmc1 (15mM); lane d, spDmc1 (5mM); lanes e–n, effect of the indicated concentrations of
spH2M1 (0.5–18mM) on spDmc1-mediated strand exchange (5mM). (D) Time-course and temperature dependence of spH2M1 strand exchange.
spH2M1 (mM) strand exchange reactions were carried out at 208C (diamonds), 308C (squares) or 378C (triangles) over a period of 90min.
Nucleic Acids Research, 2007, Vol. 35, No. 8
observed when spDmc1 was loaded on the ssDNA,
followed by spHop2-Mnd1 and the dsDNA (Figure 5A,
lanes b and f). When the ssDNA and dsDNA were
premixed, followed by the addition of spDmc1 and
spHop2-Mnd1, stimulation occurred at lower efficiency
(Figure 5A, lanes c and g). Notably, spHop2-Mnd1
stimulated spDmc1 regardless of the order of addition in
calcium buffer (Figure 5A, lanes j–p). These results suggest
that calcium is an important regulator of spHop2-Mnd1
functions. As forstrand
enhanced the activity of spDmc1 specifically, as the
effect was not observed on spRad51 (Figure 5B). No
stimulation of the D-loop formation was observed at
concentrations of Rad51 and Hop2-Mnd1 up to 6 and
8mM, respectively. Negative results were also observed
when various order of addition was used as well as
different salt conditions and the use of non-hydrolysable
ATP analogs ATPgS and AMP-PNP (data not shown).
A number of different Rad51: Hop2-Mnd1 ratios were
tested and at least two time points (8 and 30min) were
taken for all reactions. Specific stimulation of Dmc1 but
not Rad51 by Hop2–Mnd1 complex was also observed in
budding yeast S. cerevisiae [(23) and Ting-Fang Wang,
personal communication]. Therefore, we investigated
whether spHop2-Mnd1 interacted with spDmc1 and
spRad51 (Figure 5C). Pull-down assays revealed an
interaction with both spRad51 and spDmc1. Hence, the
specificity of spHop2-Mnd1 for spDmc1 is not due to
protein–protein interactions in solution.
hRad51and hDmc1 strand exchange isstimulated
by mouseHop2 and Hop2-Mnd1
We were curious to verify whether this specificity was
conserved during evolution. Using mouse Hop2 and
Hop2-Mnd1, we next investigated its ability to promote
hRad51 and hDmc1 strand exchange in vitro in the
presence of larger substrates using a circular single-
stranded DNA (4300 nucleotides) and homologous
Figure 4. Fission yeast Hop2-Mnd1 stimulates D-loop formation by spDmc1. (A) Effect of spH2M1 in a buffer containing Mg2þ(2.5mM). Lane a,
spDmc1 alone (4mM); lanes b–f, effect of spH2M1 (0.25–4mM) on spDmc1-mediated D-loop formation (4mM); lane g, reaction with spH2M1 (4mM)
alone. (B) Effect of spH2M1 in a buffer containing Ca2þ(2.5mM). Lane a, DNA substrates (ssDNA and dsDNA) without proteins; lane b, spDmc1
alone (4mM); lanes c–h, effect of spH2M1 (0.125–4mM) on spDmc1-mediated D-loop formation (4mM). (C) Analysis of the reversibility of the
D-loop reaction by S. pombe Dmc1 (4mM) and spH2M1 (1mM) in buffer containing Mg2þ(2.5mM). (A) Lane a, no protein (5min reaction); lane b,
no protein (60-min reaction); lanes c–i, reactions with spDmc1 and spH2M1 stopped at 1, 2.5, 5, 10, 20, 30 and 60min, respectively; lanes j–p,
spDmc1-mediated D-loop formation stopped at 1, 2.5, 5, 10, 20, 30 and 60min, respectively. (D) Absence of reversibility of the D-loop reaction by
spDmc1 (4mM) and spH2M1 (1mM) in buffer containing Ca2þ(2.5mM). Lane a, no protein (reaction was stopped immediately); lanes b–h, spDmc1-
mediated D-loop formation stopped at 1, 2.5, 5, 10, 20, 30 and 60min, respectively; lane i, no protein (60min reaction); lanes j–p, reactions with
spDmc1 and spH2M1 stopped at 1, 2.5, 5, 10, 20, 30 and 60min, respectively. (E–F) Quantification of (A), lanes b–g and (B), lanes c–h, respectively.
(G) Quantification of (C), diamonds represent lanes c–i and squares correspond to lanes j–p. (H) Quantification of (D), diamonds signify lanes j–p
and squares correspond to lanes b–h.
Nucleic Acids Research, 2007, Vol. 35, No. 82727
linear duplex DNA (400bp). Although the proteins used
in this assay come from different species, it is important to
note that the human and mouse Rad51 and Dmc1
recombinases are homologous at 98% at the amino acid
level. When hDmc1 was used at subsaturating concentra-
tions (Figure 6A, lane c), we observed a strong stimulation
by both mHop2-Mnd1 (lanes d–e) and mHop2 (lane f).
hRad51 strand exchange was also enhanced by increasing
concentrations of mHop2 (Figure 6B, lanes d–i) or
mHop2-Mnd1 (Figure 6C, lanes d–i). Stimulation was
also observed either when hRad51 or mHop2 was added
to the ssDNA first, or when the proteins were premixed
and added to the ssDNA (Figure 6D, lanes d–i).
Enhancement of hRad51 or hDmc1 strand exchange by
mHop2 or mHop2-Mnd1 required the presence of ATP.
Strand transfer products were not observed when ATP
was replaced by the non-hydrolysable form AMP-PNP,
when Mg2þwas omitted or when heterologous single-
stranded DNA was used (data not shown). To ascertain
whether mHop2 or mHop2-Mnd1 was able to stimulate
hRad51 or hDmc1 activity specifically, and not any
recombinase, we tested whether mHop2 was able to
stimulate E. coli RecA. Very low level of strand exchange
was observed in the presence of both proteins (data nor
shown). Altogether, we conclude that mouse Hop2 or
Hop2-Mnd1 can work in concert specifically with hRad51
or hDmc1 and not a distant homolog such as RecA.
Visualization of fission yeast Hop2-Mnd1on DNA
When complexes formed between spHop2-Mnd1 and
DNA were visualized by electron microscopy, we observed
large protein complexes bound to single-strand DNA
(Figure 7A, right). We also observed less frequently
irregular filament-like structures possibly due to many
molecules bound to the ssDNA (Figure 7A, left).
Consistent with gel-retardation assays, spHop2-Mnd1
had high affinity for ssDNA since unbound protein was
not distinguished. Because meiotic recombination is
thought to be initiated by DNA that contains single-
stranded tails, the possibility that spHop2-Mnd1 may bind
specifically to tailed linear duplex molecules was investi-
gated. Following uranyl acetate staining, duplex DNA is
well visible while single-stranded DNA, if not bound by
proteins, is collapsed to very small blobs or bushy
structures. Consistent with a specific interaction with
ssDNA, spHop2-Mnd1 (5mM) selectively bound the
ssDNA of linear duplex DNA molecules with ssDNA at
both ends (Figure 7B). Very interestingly, the two ends of
the tails were often found entangled in one and other,
suggestive of homology search. Unbound spHop2-Mnd1
was also discerned (white arrow). In order to confirm
specific interactions with ssDNA, we also observed
binding of ssDNA ends at lower concentrations of
spHop2-Mnd1 (Figure 7C). About 70% of the molecules
observed had spHop2-Hop2at both extremities. These
results are consistent with specific roles for spHop2-Mnd1
The DNA strand exchange activity of both Rad51 and
Dmc1 is critical for proper meiotic DNA double-strand
break repair in lower eukaryotes. Many factors have been
found to stimulate Rad51, including RPA, Rad52 and
Rad54. On the other hand, human Dmc1 is stimulated by
Figure 5. (A) Effect of the order of addition on S. pombe Dmc1-Hop2-Mnd1 D-loop formation in Mg2þor Ca2þbuffer. Reactions with Dmc1
(4mM) and Hop2-Mnd1 (1mM) were performed for 5min (lanes a–d) or 30min in MgCl2(2.5mM) (lanes e–h) or 5min (lanes i–l) or 30min (lanes
m–p) in CaCl2buffer. (2.5mM). (B) SpH2M1 does not stimulate spRad51. Indicated concentrations of spRad51 and spH2M1 were used (lanes a–f)
and mammalian Dmc1 and Hop2-Mnd1 reaction is shown for comparison (lane g). (C) SpH2M1 interacts with both spDmc1 and spRad51. Purified
His-tagged spH2M1 was incubated with affinity beads containing crosslinked BSA (lane a), spRad51 (lane b), or spDmc1 (lane c). Eluted material
was probed by western blotting using anti-His antibody.
Nucleic Acids Research, 2007, Vol. 35, No. 8
the human Rad54B, which could be an ortholog of
budding yeast Rdh54 and Tid1 (40). Much attention is
now turned toward the Hop2 and Mnd1 proteins because
of their important roles in meiotic recombination.
S. pombe Hop2and Mnd1 form acomplexbinding
Sequence analysis revealed that Hop2 and Mnd1 possesses
a coiled-coil motif (41,42). Fission yeast Hop2 coiled-coil
region spans amino acids 128–159 whereas spMnd1
possess a long coiled-coil from amino acids 82 to 149.
Using two-hybrid analysis we showed that spHop2
interacts with spMnd1. Domain mapping experiments
revealed that the C-terminus of spHop2 (amino acids
111–216) interacts with the C-terminal portion of spMnd1
(amino acids 107–211). Hence, it is not the complete
coiled-coiled region of spMnd1 that is required for the
interaction with spHop2. These results are in accordance
with a recent report showing that mouse Hop2 and
Mnd1 interact through their coil–coil motif and that
the C-terminus of the protein is important for the
interaction (41). Gel filtration experiments revealed that
spHop2-Mnd1 eluted in a broad profile, suggesting that it
can form structures containing a variable number of
molecules. However, our results suggest that purified
spHop2-Mnd1 strictly forms a hetero-complex in solution.
Indeed, we were unsuccessful in detecting soluble spHop2
or spMnd1 alone suggesting that these proteins are
unstable without each other. In accordance with this,
the overexpression of Mnd1 suppressed a temperature-
sensitive mutant allele of Hop2 in budding yeast (19).
Instability of Hop2 without Mnd1 was also observed with
Figure 6. Mouse Hop2 or Hop2-Mnd1 stimulates hDmc1 or hRad51 strand exchange. (A) Stimulation of hDmc1 strand exchange by mHop2 or
mH2M1 using long DNA substrates (lanes a–f). Single-stranded DNA (15mM) was incubated with the indicated concentrations of hDmc1 and
mHop2 or mH2M1 proteins for 5min at 378C followed by addition of32P-end labeled double-stranded DNA (1.38mM) and incubation at 378C for
90min. hDmc1 was added before mHop2 or mHop2-Mnd1 to the ssDNA. (B) Stimulation of hRad51 strand exchange by the indicated amounts of
mouse Hop2 (lanes a–i) and (C) mH2M1 (lanes a–i). The reactions were carried out as described in (A). (D) Effect of the order of addition of the
proteins on hRad51-mHop2 DNA strand exchange. Lane a, no protein; lane b, hRad51 (5mM); hRad51 (2mM, lane c) was added to ssDNA first
followed by addition of the mHop2 protein (0.5 and 3mM, lanes d and e); mHop2 was added to ssDNA first followed by addition of the hRad51
protein (lanes f and g); or both of the proteins were added to ssDNA at the same time (lanes h and i). The reactions were then carried out as
described in (A). Joint molecules and Nicked circles (NC) are indicated.
Nucleic Acids Research, 2007, Vol. 35, No. 8 2729
the human homologs (26). The 40kDa complex observed
by native electrophoresis thus corresponds to spHop2-
Mnd1. The 66kDa complex most likely corresponds to a
dimer of Hop2 bound to Mnd1 (or a dimer of Mnd1
bound to Hop2) or spHop2-Mnd1 having a different
conformation. It is most likely that one or both of these
spHop2–Mnd1 complexes exist in vivo. Immunoprecipita-
tion analyses from S. cerevisiae meiotic cell extracts
revealed that Hop2-Myc and Mnd1-GFP interact (18).
Moreover, in pat1 fission yeast strain, Hop2-GFP
and Mnd1-HA show almost
profiles during meiosis and both proteins interact (21).
Co-immunoprecipitation, of mouse Hop2 and Mnd1 has
been reported (24). We do not exclude the possibility of
other spHop2–Mnd1 complexes in vivo. We observed
two-hybrid analysis. In this context, perhaps endogenous
S. cerevisiae proteins could stabilize spHop2–Hop2 or
spMnd1–Mnd1 complexes in vivo.
SpHop2-Mnd1 were purified with short histidine tags at
their N-terminus. The tags should not affect the function
of the heterodimer as tagging spMnd1 or Hop2 with large
proteins such as GFP did not affect spore viability [(20,21)
and Hiroshi Nojima and Takamune Saito, personal
communication]. When observed by electron microscopy,
purified spHop2-Mnd1 formed filament-like structures on
single-strand DNA. The nature of this structure remains
to be determined but it might be the result of random
binding of spHop2-Mnd1 to the ssDNA so the whole
DNA is covered. Our electron microscopic observations
support the concept of specific binding of spHop2-Mnd1
to ssDNA ends on 30-tailed DNA. The preferential
interaction with ssDNA tails may have important
biological consequences at the initiation of recombination
during meiosis since double-strand breaks are processed
into 30-tailed DNA molecules. Presumably, the resected
tails could act as a landing site for spHop2-Mnd1 leading
to the nucleation of a presynaptic filament of spDmc1.
In contrast, S. cerevisiae and human Hop2-Mnd1 was
found to bind dsDNA preferentially (23,26), whereas
mouse Hop2–Mnd1 binds DNA with no preference
toward single- or double-strand DNA (41). This is an
important issue since it influences the way we perceive the
roles of these proteins in vivo. Very importantly, we found
that the addition of calcium eliminates the order of
addition requirement on DNA in spDmc1-Hop2-Mnd1
strand invasion assays. Dependence of meiosis on Ca2þ
has been observed during meiosis I, when homologous
recombination occurs, and it has been shown that calcium
stimulates strand exchange by the human Rad51 (43) and
human Dmc1 proteins (44). Hence, although the prefer-
ential binding of Hop2–Mnd1 proteins on DNA differs
between species, calcium might be a key component in the
regulation of their interactions with DNA in vivo by
altering preferential binding.
Strand exchange with oligonucleotides can occur in an
ATP-independent fashion and also by a DNA melting and
strand separation activity (34). We show that SpHop2-
Mnd1 does not possess DNA melting activity. We have
shown previously that fission yeast Dmc1 and Rad51 can
both promote strand exchange (31). At a concentration of
one monomer per nucleotide, spDmc1 promotes weak
strand exchange unless a single-strand binding protein
such as SSB is included in the reaction (31). In support of
this, RPA is required for meiotic recombination in
budding yeast (45). Very interestingly, the addition of
spHop2-Mnd1 markedly increases strand exchange activ-
ity of the spDmc1 protein. SpDmc1 strand invasion, but
not spRad51, was stimulated by spHop2-Mnd1. Budding
yeast Hop2-Mnd1 and mouse Hop2 can also stimulate
Dmc1 specifically (23–25). However, it is not clear how
this stimulation occurs. It may be caused by changes in the
DNA structure resulting either from spHop2–Mnd1
binding. Second, although spHop2-Mnd1 interacts with
both spRad51 and spDmc1, protein–protein interactions
might only be beneficial for Dmc1 nucleoprotein fila-
ments. Since spHop2-Mnd1, spDmc1 and spRad51 bind
DNA, this might be difficult to assess experimentally.
D-loop products from Dmc1–spHop2–Mnd1 reactions
were about 3-fold more abundant in calcium buffer than
in magnesium buffer. Moreover, we observed that the
D-loop reaction was not reversible in the presence of
calcium. These results suggest that calcium might be an
important regulator of spDmc1-Hop2-Mnd1 strand inva-
sion in vivo. D-loop cycle was first described for RecA
protein and was proposed to protect from illegitimate
recombination (39). Only if the sequences are truly
homologous, successive D-loop cycles at many sites will
result in stable pairing. Ca2þwas shown to stimulate the
strand exchange activity of human Rad51 protein by
Figure 7. Electron microscopic visualization of fission yeast Hop2-
Mnd1. (A) Electron microscopic visualization of spH2M1 (1.65mM)
bound to ?X174 single-stranded DNA (5mM). (B) Close up view of
spH2M1 (0.75mM) bound to linear duplex with a single-stranded tail at
both ends (5mM). The white arrows designate unbound protein.
(C) spH2M1 (5mM) bound to linear duplex with a single-stranded tail
at both ends (5mM). The black arrows designate bound spH2M1 on
ssDNA. The magnification bars represents 50nm.
Nucleic Acids Research, 2007, Vol. 35, No. 8
inhibiting the ATPase activity of Rad51 thus preventing
the conversion of the Rad51-ATP-ssDNA filament to an
inactive Rad51-ADP-ssDNA form (43). Hence, activation
of DNA strand exchange appears to be correlated with
calcium-triggered transition from inactive ring-shaped
oligomers to active nucleoprotein filaments (40,46). It is
possible that a similar mechanism might be involved in the
S. pombe Dmc1–Hop2–Mnd1 reaction. Dmc1 filaments
might be more stable in the presence of calcium and
cycling of the D-loop reaction in Mg2þbuffer is observed
only in the presence of ATP, but not when ATP
is replaced by non-hydrolysable analog AMP-PNP
(our unpublished data).
Knowing that fission yeast Hop2-Mnd1 enhances Dmc1
recombinase activity specifically, we investigated whether
we could find the same stimulatory effect for mouse Hop2
or Hop2-Mnd1. As expected, the addition of mouse Hop2
or Hop2-Mnd1 markedly increases strand exchange
However, in order to achieve the same level of stimulation,
a smaller amount of mHop2-Mnd1 was required com-
pared to mHop2. Hence, mHop2-Mnd1 perhaps functions
in a catalytic manner. It is interesting to note that
mammalian Hop2 functions in transcription (47) and
interacts with the DNA-binding domains of nuclear
receptors (48). Our results suggest that Hop2, in the
absence of Mnd1, can also function in DNA recombina-
tion. We believe that the stimulation observed involves a
specific interplay between mHop2 or mHop2-Mnd1 and
hRad51/Dmc1 for three reasons. First, we could not
detect any stimulatory effect of mHop2 or mHop2/Mnd1
on RecA-promoted strand exchange. Second, the stimu-
latory activity was not severely affected by the order of
addition of proteins on the single-stranded DNA,
suggesting that there was no sequestration of ssDNA by
mHop2 or mHop2-Mnd1 alone. This is important as
sequestration of ssDNA would lead to a decrease in the
available concentration of ssDNA by hDmc1 or hRad51
therefore increasing the local concentration of the
recombinases per DNA molecule, which could explain
the strand exchange activity. If it was the case, mHop2 or
mHop2-Mnd1 would have also stimulated RecA. Third,
hRad51 and hDmc1 stimulation by mHop2 or mHop2-
Mnd1 was dependent on ATP hydrolysis, which suggests
that the stimulation required the activity of both
recombinases. Altogether, these results would argue that
mHop2 is not a non-specific factor capable of stimulating
any strand exchange reaction in general. Our results are in
accordance with recent studies showing that human
Hop2-Mnd1 stimulates hRad51 and hDmc1 strand
exchange (26). In addition, we show that mHop2 can
also stimulate hRad51 and hDmc1 and perhaps Mnd1 is
the catalytic subunit.
Roles ofHop2-Mnd1 inmeiotic recombination
Genetic studies have established that fission yeast Hop2 is
important during DSB-independent homolog pairing
when cells are transferred to sporulation media (20,49).
Our results also support roles for Hop2-Mnd1 later,
duringhomologous pairing andstrandexchange.
After resection of a meiotic double-strand break, the 30-
tailed DNA invades an homologous double-strand DNA,
a step known as homologous pairing without extensive
heteroduplex formation. In meiosis, this occurs between
homologs rather than sister chromatids. Then, the
heteroduplex region is expanded by strand exchange to
form joint molecules and Holliday junctions. The Holliday
junctions are resolved to produce at least one crossover
per chromosome. We observed that spHop2-Mnd1 can
promote strand exchange with oligonucleotides and
stimulate strand invasion by spDmc1. These activities
demonstrate a key role for spHop2-Mdn1 in strand
invasion. Also, mHop2-Mnd1, stimulates hRad51 and
hDmc1 strand exchange, which means that these proteins
can stabilize interactions between homologous sequences,
as suggested recently with genetic studies in budding yeast
(50). A subset of meiotic DSBs is also repaired by
synthesis-dependent strand annealing (51). It is thought
that following the initial strand invasion and repair
synthesis, the invading strand containing newly synthe-
sized DNA is displaced and reannealed to the other 30end.
Annealing by spHop2-Mnd1 might be very important in
this reaction. Indeed, spHop2-Mnd1 promoted strong
annealing with long 400 bases single-strand DNAs.
Moreover, this reaction persisted in the presence of a
single-strand binding protein. Altogether, our results
suggest important functions of Hop2–Mnd1 complex in
the various steps of homologous recombination. These
results are supported in vivo by the phenotypes of the hop2
knock-out mice which displays failure of DSB repair and
sterility, although the Rad51 and Dmc1 recombinases are
still present (16).
We are grateful to Isabelle Brodeur and Ame ´ lie Rodrigue
for helpful comments on the manuscript. We thank Drs
Ting-Fang Wang, Hiroshi Nojima and Takamune Saito
for communication of unpublished results, Dr Doug
Sauvageau and Pierre Plante for technical help, anon-
ymous reviewers for helpful comments and Jacques
Dubochet for support and interest. AS was supported by
Swiss National Science Foundation J.Y.M is a Canadian
Institutes of Health New Investigator and this research is
supported by funds from the National Cancer Institute of
Canada and the Natural Sciences and Engineering
Research Council of Canada. Funding to pay the Open
Access publication charges for this article was provided by
the National Cancer Institute of Canada.
Conflict of interest statement. None declared.
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