This article is a Plant Cell Advance Online Publication. The date of its first appearance online is the official date of publication. The article has beenedited and the authors have corrected proofs, but minor changes could be made before the final version is published. Posting this version online
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Sufﬁcient Amounts of Functional HOP2/MND1 Complex
Promote Interhomolog DNA Repair but Are Dispensablefor Intersister DNA Repair during Meiosis in Arabidopsis

W

Clemens Uanschou,a,1 Arnaud Ronceret,b,c,1,2 Mona Von Harder,a,1 Arnaud De Muyt,b,c,3 Daniel Vezon,b,c
Lucie Pereira,b,c Liudmila Chelysheva,b,c Wataru Kobayashi,d Hitoshi Kurumizaka,d Peter Schlögelhofer,a,4
and Mathilde Grelonb,ca Department of Chromosome Biology, Max F. Perutz Laboratories, University of Vienna, A-1030 Vienna, Austriab Institut National de la Recherche Agronomique, Unité Mixte de Recherche 1318, Institut Jean-Pierre Bourgin, F-78000 Versailles,

Francec AgroParisTech, Institut Jean-Pierre Bourgin, F-78000 Versailles, France
d Graduate School of Advanced Science and Engineering, Waseda University, Shinjuku-ku, Tokyo 162-8480, Japan

During meiosis, homologous recombination (HR) is essential to repair programmed DNA double-strand breaks (DSBs), anda dedicated protein machinery ensures that the homologous chromosome is favored over the nearby sister chromatid as a repair
template. The HOMOLOGOUS-PAIRING PROTEIN2/MEIOTIC NUCLEAR DIVISION PROTEIN1 (HOP2/MND1) protein complex has
been identiﬁed as a crucial factor of meiotic HR in Arabidopsis thaliana, since loss of either MND1 or HOP2 results in failure of
DNA repair. We isolated two mutant alleles of HOP2 (hop2-2 and hop2-3) that retained the capacity to repair meiotic DSBs via thesister chromatid but failed to use the homologous chromosome. We show that in these alleles, the recombinases RADIATIONSENSITIVE51 (RAD51) and DISRUPTED MEIOTIC cDNA1 (DMC1) are loaded, but only the intersister DNA repair pathway isactivated. The hop2-2 phenotype is correlated with a decrease in HOP2/MND1 complex abundance. In hop2-3, a truncated HOP2
protein is produced that retains its ability to bind to DMC1 and DNA but forms less stable complexes with MND1 and fails to
efﬁciently stimulate DMC1-driven D-loop formation. Genetic analyses demonstrated that in the absence of DMC1, HOP2/MND1 isdispensable for RAD51-mediated intersister DNA repair, while in the presence of DMC1, a minimal amount of functional HOP2/
MND1 is essential to drive intersister DNA repair.

INTRODUCTION
A specialized type of cell division, called meiosis, ensures the reduction of the genome prior to the formation of generative cells.
During meiosis, genetic information between maternal and paternal chromosomes is exchanged, leading to novel combinations ofgenetic traits in the following generation. The molecular basis of
this process is recombination between homologous chromosomes. Meiotic homologous recombination is initiated with theprogrammed induction of DNA double-strand breaks (DSBs) induced by the conserved Sporulation11 (Spo11) protein (Bergerat

1 These authors contributed equally to this work.
2 Current address: Group of Reproductive Development and Apomixis,

Laboratorio Nacional de Genómica para la Biodiversidad, Centro deInvestigación y de Estudios Avanzados del Instituto Politécnico Nacional,
Km 9.6 Libramiento Norte Carretera Irapuato-León C.P. 36821, Irapuato,
Guanajuato, Mexico.
3 Current address: Institut Curie/Centre de Recherche, Unité Mixte deRecherche 218, Pavillon Pasteur, F-75248 Paris, France.
4 Address correspondence to peter.schloegelhofer@univie.ac.at.
The authors responsible for distribution of materials integral to the ﬁndingspresented in this article in accordance with the policy described in theInstructions for Authors (www.plantcell.org) are: Peter Schlögelhofer (peter.
schloegelhofer@univie.ac.at) and Mathilde Grelon (mathilde.grelon@
versailles.inra.fr).
W
Online version contains Web-only data.
www.plantcell.org/cgi/doi/10.1105/tpc.113.118521

et al., 1997; Keeney et al., 1997; Malik et al., 2007). Following
meiotic DSB formation, Spo11 is removed from the DNA bya single-stranded DNA (ssDNA) nick next to the DSB site (Keeney
and Kleckner, 1995; Neale et al., 2005; Garcia et al., 2011; Panet al., 2011). Subsequent nucleolytic resection yields 39-primed,
single-stranded DNA, which serves as a probe for ﬁnding a repair
template. The ssDNA strand is associated with protein factors toform a nucleoprotein ﬁlament that then mediates strand invasion(single end invasion) into an intact DNA duplex (reviewed in Pâques
and Haber, 1999; reported in Hunter and Kleckner, 2001; Neale and
Keeney, 2006; Edlinger and Schlögelhofer, 2011).
In a diploid cell, after replication, the DNA template for repair
can either be one of the two chromatids of the homolog or sister
chromatid. During meiosis, only a selected number of Spo11catalyzed DSBs proceed to form crossovers (COs; reciprocal
exchange between chromosomes) (Schwacha and Kleckner,
1994, 1997; Allers and Lichten, 2001; Hunter and Kleckner, 2001).
These ensure subsequent regular chromosome disjunction atmeiosis I. The remaining DSBs are repaired as noncrossovers(with nonreciprocal exchange between homologous chromosomes) or from the sister chromatid (Bishop and Zickler, 2004;
Baudat and de Massy, 2007; Mancera et al., 2008). At least one
CO per homologous chromosome pair is needed for the correct
segregation of homologs during the ﬁrst meiotic division. Despite
the presence of a sister chromatid in close proximity, the meiotic
DNA repair machinery has to be directed, at least in some cases,

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2 of 17

The Plant Cellto the homologous chromosome as a repair template. The molecular mechanisms underlying this promotion of interhomologous (IH) and the possible correlated suppression of intersister(IS) recombination are still poorly understood (De Massy et al.,
1994; Pâques and Haber, 1999; Niu et al., 2005; Callender andHollingsworth, 2010; Goldfarb and Lichten, 2010).
Two proteins play a major role in establishing the nucleoprotein
ﬁlament and ﬁnding the target sequence: the RecA homologs
RADIATION SENSITIVE51 (Rad51) and DISRUPTED MEIOTIC
cDNA1 (Dmc1) (Bishop et al., 1992; Shinohara et al., 1992, 1997;
Ogawa et al., 1993; Bishop, 1994). Rad51 is involved in both mitotic
and meiotic recombination, whereas Dmc1 is exclusively a meiotic
player (Bishop et al., 1992; Tashiro et al., 1996, 2000; Yamamoto
et al., 1996; Klimyuk and Jones, 1997) (reviewed in Masson andWest, 2001; Li and Ma, 2006). It has been demonstrated that duringmeiosis, Rad51 and Dmc1 function in independent DNA repair
pathways and that their loading onto replication protein A–coated
ssDNA is supported by distinct sets of proteins (Bishop, 1994;
Dresser et al., 1997; Gasior et al., 2001; Hayase et al., 2004; Liet al., 2004; Lao et al., 2008).
During wild-type meiosis, DMC1-mediated IH DNA repair appears to be the predominant pathway, with RAD51 just having
a supportive role, as recently shown for yeast (Saccharomyces
cerevisiae) and plants (Garcia et al., 2011; Cloud et al., 2012). In theabsence of DMC1, RAD51 can promote efﬁcient meiotic IS DNA
repair in Arabidopsis thaliana (Couteau et al., 1999; Vignard et al.,2007; De Muyt et al., 2009) and in yeast when the Rad51 inhibitor
High copy suppressor of Red1 (Hed1) is not present (Busygina
et al., 2008). These data are in line with our recent ﬁndings thatduring meiosis in Arabidopsis, RAD51 and DMC1 form distinct
functional units, also represented in nonoverlapping nuclear foci(Kurzbauer et al., 2012). Puriﬁed DMC1 and RAD51 from humanand budding yeast seem not distinct from each other with respect
to their biochemical properties. Both proteins form right-handed
helical ﬁlaments on ssDNA in an ATP-dependent manner andcatalyze homologous DNA pairing and strand exchange reaction(Shinohara et al., 1992; Ogawa et al., 1993; Benson et al., 1994;
Sung, 1994; Baumann et al., 1996; Li et al., 1997; Masson et al.,
1999; Hong et al., 2001; Sehorn et al., 2004; Yu and Egelman,2010). By contrast, ﬁssion yeast (Schizosaccharomyces pombe)
Rad51 and Dmc1 proteins differ from each other in in vitro assays
with respect to the polarity of strand exchange reactions they
support (Murayama et al., 2011). Generally, it has been suggested
that a set of distinct accessory proteins that modulate the activityof Dmc1 or Rad51 is responsible for the observed differences intheir meiotic function (Sheridan and Bishop, 2006; Sheridan et al.,2008; Kagawa and Kurumizaka, 2010; Dray et al., 2011).
The proteins HOMOLOGOUS-PAIRING PROTEIN2 (Hop2) and
MEIOTIC NUCLEAR DIVISION PROTEIN1 (Mnd1) have been
identiﬁed in yeast as factors supporting homologous chromosome
pairing and meiotic DSB repair (Leu et al., 1998; Tsubouchi andRoeder, 2002; Chen et al., 2004). Analyses in yeast have shownthat both mnd1 and hop2 mutants initiate recombination, but do
not form heteroduplex DNA or double Holliday junctions, accumulate Dmc1 and Rad51, and lead to severe DNA repair defects,
suggesting that they are involved in strand invasion. These mutants arrest in prophase I due to DNA damage checkpoint activation (Leu et al., 1998; Gerton and DeRisi, 2002; Tsubouchi andRoeder, 2002; Chen et al., 2004; Zierhut et al., 2004). Furthermore,in yeast, Dmc1 directs meiotic repair to the homologous chromosome only in the presence of Mnd1 and Hop2 (Zierhut et al.,2004). Recent results demonstrate that the Hop2/Mnd1 protein
complex can efﬁciently condensate double-stranded DNA (dsDNA)
to support strand invasion as a key step in stimulating associationof the ssDNA-nucleoprotein ﬁlament with the dsDNA (Pezza et al.,2010). Mammalian HOP2/MND1 complexes can interact with
DMC1 but also with RAD51 in vitro, stimulating the strand exchange activities of both proteins, 35-fold or 10-fold, respectively,
but do not show strand exchange activity alone (Petukhova et al.,2005; Enomoto et al., 2006).
The involvement of the HOP2/MND1 complex in meiotic DSB
repair has also been observed in Arabidopsis thaliana, since nullmutants of HOP2 and MND1 lead to chromosome fragmentationand sterility. Nevertheless, the Arabidopsis HOP2/MND1 complex
has not been shown to be involved in the promotion of IH repair via
a DMC1-directed pathway since both IH and IS DNA repair are
dysfunctional in the absence of any of these two proteins(Schommer et al., 2003; Kerzendorfer et al., 2006; Stronghill et al.,2010). Recently, we identiﬁed two mutant alleles of Arabidopsis
HOP2 (De Muyt et al., 2009) that differ from the already characterized hop2-1 null mutant allele (previously named ahp2-1; renamed hop2-1 for nomenclature consistency; Schommer et al.,2003). hop2-1 mutants exhibit strong DNA fragmentation duringmeiosis I, while the two newly identiﬁed alleles show random
segregation of univalents during meiosis I (accompanied by only
limited chromosome fragmentation), reminiscent of the defects
observed in Arabidopsis dmc1 mutants. This led to the assumptionthat in these two mutants, IH bias is lost and the repair of meiotic
DSBs occurs exclusively via the sister chromatid. Here, we
present molecular and biochemical analyses demonstrating thatplant HOP2 protein and its heterodimeric partner MND1 need toform functional complexes at sufﬁcient levels to support IH DNA
repair. Genetic analyses revealed that the HOP2/MND1 complex
is dispensable for RAD51-mediated IS DNA repair in the absence of DMC1. In the presence of DMC1, HOP2/MND1 becomes an essential factor: At low levels, it indirectly allows
RAD51-mediated IS repair and only at high levels does it support DMC1 driven IH repair.
RESULTS
In a forward-genetic meiotic mutant screen in Arabidopsis, twomutant alleles of HOP2, hop2-2 (initially named eyu48) and hop2-3(initially named exi5), were previously identiﬁed (De Muyt et al.,2009). The mutants can be recognized by their reduced siliquelength and reduced seed set, while their overall appearance is
indistinguishable from the wild type. Only 1.6 6 0.75 (n = 1099
siliques) seeds per silique (3.8% of the wild type) were observed in
hop2-2 and 0.84 6 0.53 (n = 825 siliques) seeds per silique (2.03%of the wild type) were observed in hop2-3. This is in strong
contrast with the observations made in the previously publishedhop2-1 null mutant allele (Schommer et al., 2003), which produces only 0.005 6 0.01 (n = 886 siliques) seeds per silique(0.01% of the wild type). To understand these differences andtheir molecular basis, we ﬁrst analyzed the meiotic progressionof all the mutants in detail.

HOP2/MND1-Mediated Interhomologous Bias

hop2-2 and hop2-3 Show a High Incidence of Univalents
in Meiosis I
We could detect strong abnormalities in meiotic progression in all
Arabidopsis hop2 mutants investigated in this study. First, normal
chromosome pairing was not observed during prophase I of malemeiosis in any of the studied alleles (Figures 1Ae and 1Ai to 1Aa)
(Schommer et al., 2003; Stronghill et al., 2010). As described before (Schommer et al., 2003), severe chromosomal defects (chromosome bridges and DNA fragmentation) could be observed from
anaphase I to the end of meiosis in hop2-1 (Figures 1Af to 1Ah) ina large majority of the meiotic cells (Figure 1B, 82%, n = 39). In
clear contrast, hop2-2 meiocytes showed hardly any chromosome
fragmentation or chromosome bridges, but instead exhibited intact chromosomes (Figures 1Aj and 1Ak) in the vast majority of thecells observed (Figure 1B, 86%, n = 154,). Nevertheless, contrary
to the wild type (Figure 1Ab), very few bivalents but a majority of
univalents were observed among hop2-2 metaphase cells. On
average, we counted 0.5 bivalent/meiocytes (n = 133), while thewild type always shows ﬁve bivalents per cell. For the remaining
cells (21 out of 154), no major chromosomal defects were observed but chromosomes display poorly condensed chromatin or
occurrence of a reduced number of chromosome fragments andwere classiﬁed as intermediate class in Figure 1B.
In hop2-3 meiocytes, the situation is intermediate, with 55% of
the cells showing intact chromosomes (mean number of bivalent
per cell of 0.3 6 0.1; n = 95) and 41% showing mild chromosomal
defects (Figure 1B). For hop2-2 and hop2-3 mutants, random
segregation of the univalents at anaphase I and subsequent sister
chromatid segregation at meiosis II yielded unbalanced pools ofchromosomes at the end of meiosis (Figure 1Al), contrary to thesituation in the wild type where the balanced segregation of homologous chromosomes during meiosis I and the subsequent
sister chromatid segregation at meiosis II generates four haploiddaughter cells (Figures 1Ac and 1Ad).
Reciprocal Partial Complementation of hop2-2 and hop2-3
Interestingly, we found that in heteroallelic hop2-2/hop2-3 plants,
chromosome fragmentation and bridges were completely suppressed (see Supplemental Figure 1 online). In addition, the level ofbivalent formation was signiﬁcantly increased when comparedwith each single mutant line (P = 9.5 3 10212 and P = 3.8 3 10210).
While hop2-2 and hop2-3 have a mean number of 0.3 bivalents
per cell, the hop2-2/hop2-3 line shows 1 6 0.1 bivalents per cell(n = 91). This increase in bivalent formation is also followed by anincrease in fertility, with an average number of 8.1 seeds per silique(61.3, n = 29; 9.2% of the wild type). This reciprocal (partial)
complementation suggested that different aspects of gene regulation or protein functions are affected in the two mutant alleles.
hop2-2 and hop2-3 Represent Different Hypomorphic Allelesof HOP2
To understand the differences between hop2-1 and the alleles
hop2-2 and hop2-3, we characterized the mutant alleles in detail.
Sequencing of the HOP2 gene in these alleles revealed the natureof each mutation. The original hop2-1 null-mutant (in the Landsberg

3 of 17erecta [Ler] background) has a T-DNA insertion in exon 4, does notproduce functional mRNA, and lacks HOP2 protein (Figure 2A)(Schommer et al., 2003; Stronghill et al., 2010). The hop2-2 allele
has a 147-bp deletion 101 bp upstream of the start codon of the
HOP2 gene (Figure 2A). The hop2-3 allele has a deletion of 128 bp
between exon 4 and exon 5. The deletion in hop2-3 leads toa transcript lacking parts of sequences from exon 4 and 5 and all of
intron 4 but keeping the translational phase of the coding sequence(CDS; Figures 2A and 2D; see Supplemental Figure 2 online).
We analyzed the expression levels of HOP2 in the four mutants
using material from young ﬂower buds by RT-PCR using threedifferent primer sets (Figure 2B). As described previously, the 59
end and the 39 end of the HOP2 mRNA could be identiﬁed in thehop2-1 mutants, but a transcript spanning the T-DNA insertionsite could not (Schommer et al., 2003).
For the hop2-3 allele, we observed transcript levels as in thewild type. It should be noted that in hop2-3 mutants, the corresponding transcript is 42 nucleotides shorter due to the deletion
described above (Figure 2B). The nature of the hop2-3 mRNA has
been conﬁrmed by sequencing. We reasoned that the shortenedmRNA leads to the expression of a HOP2 protein variant thatlacks 14 amino acids (positions 123 to 136; UniProtKB Q9FX64) in
its putative coiled coil domain (Figure 2D) and that it may be thistruncated protein that causes the observed phenotype in hop2-3
mutant plants.
In hop2-2 mutants, we observed drastically reduced mRNAlevels of HOP2 (Figures 2B and 2C). Quantitative RT-PCR showedthat the HOP2 mRNA is at least ﬁvefold decreased when evaluating
the 59 part of mRNA and ;30-fold decreased when evaluating the
39 part of the mRNA in hop2-2 mutants. The low abundance of the
39 part of the mRNA in hop2-2 mutants may indicate mRNA
truncation or instability. We reasoned that the observed phenotype
in hop2-2 has to be attributed to the severely reduced HOP2
transcript levels and the anticipated lower HOP2 protein levels.
The High Incidence of Univalents in hop2-2 and hop2-3 Is
Not Due to a Failure to Generate DSBs
In order to understand if the high incidence of univalents and thedecrease of chiasma numbers observed in hop2-2 and hop2-3
meiocytes was due to reduction of DNA DSBs, we generated
double mutants of hop2-2 and hop2-3 with mre11-3 (Puizina
et al., 2004) and rad51 (Li et al., 2004) mutants. Both MEIOTIC
RECOMBINATION11 (MRE11) and RAD51 are well-studied proteins, essential for meiotic progression in plants and other organisms (Cao et al., 1990; Shinohara et al., 1992; Haaf et al.,
1995; Muris et al., 1997; Li et al., 2004; Puizina et al., 2004).
MRE11 is involved in early steps of SPO11-generated DSB processing, and RAD51, as discussed above, is needed in the strand
invasion process during DNA repair. Since DSB formation is notaffected in Arabidopsis mre11 and rad51 mutants, the respective
DNA repair defects lead to chromosome fragmentation and sterility. If the hop2-2 and hop2-3 alleles affect DSB formation, we
reasoned that the DNA fragmentation observed in mre11-3 andrad51 mutants should be alleviated in the hop2-2 mre11, hop2-3
mre11, hop2-2 rad51, and hop2-3 rad51 double mutants. Since
this is not the case (see Supplemental Figure 3 online), we can
exclude major defects in DSB formation in both hop2 mutant

4 of 17

The Plant Cell

Figure 1. Meiotic Progression in hop2 Mutants.
(A) 49,6-Diamidino-2-phenylindole staining of chromosomes of wild-type (Wt; [a] to [d]), hop2-1 ([e] to [h]), and hop2-2 ([i] to [l]) pollen mother cells. (a),(e), and (i), Pachytene/pachytene-like stage; (b), (f), (j), and (k), metaphase I–anaphase I transition; (c) and (g), metaphase II; (d), (h), and (l), end ofanaphase II. While full synapsis was achieved at pachytene in the wild type (a), no evidence of synapsis could be observed in any of the three hop2
mutant lines (shown here for hop2-1 in [e] and hop2-2 in [i]). At metaphase I in the wild type, ﬁve bivalents were always observed aligned on themetaphase plate (b). Homologous chromosomes segregated during anaphase I, generating two pools of ﬁve chromosomes (c). Then, during thesecond meiotic division, sister chromatids were separated, producing four haploid cells (d). By contrast, during hop2-1 meiosis (f), severe chromosomal
defects (fragmentation and chromosome bridges) were observed at anaphase I (f) and anaphase II (g), yielding aberrant meiotic products (h). In hop2-2([j] to [l]) and hop2-3, hardly any chromosomal defects could be observed; instead, a mixture of intact univalents and bivalents could be seen ([j], 10
univalents; [k], six univalents and two bivalents). Random segregation of these univalents at anaphase I ([j] and [k]) and the subsequent sister chromatid
segregation at meiosis II (data not shown) yielded unbalanced pools of chromosomes at the end of meiosis (l) instead of the four pools of ﬁve
chromosomes observed in the wild type (d). u, univalent; b, bivalent. Bars = 10 mm.
(B) dmc1 suppresses the DNA repair defects observed in hop2-1, hop2-2, and hop2-3 mutants. In hop2-1, a large majority of meiocytes showed drastic
chromosomal defects (fragmentation and chromosome bridges) at metaphase I–anaphase I transition. By contrast, hop2-2 cells predominantly present
intact chromosomes (86%). In hop2-3 meiocytes, the situation is intermediate with 55% of the cells showing no DNA fragmentation and 41% showing
mild chromosomal defects (“intermediate” class). The severe chromosome fragmentation observed in hop2-1 mutants is largely suppressed by thedmc1 mutation. The limited chromosome fragmentation observed in hop2-2 and hop2-3 mutants is nearly completely suppressed by the dmc1
mutation.

HOP2/MND1-Mediated Interhomologous Bias

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Figure 2. Molecular Analysis of HOP2 Alleles.
(A) Schematic representation of the HOP2(AHP2) gene and three mutant alleles. Open boxes represent exons, ATG and TAA encompass the open
reading frame. The inverted triangle in orange shows the position of the T-DNA insertion in hop2-1 (the renamed ahp2-1 allele described in Schommer
et al., 2003). Text and symbols in light blue indicate the 147-bp deletion in the promoter region of the hypomorphic hop2-2 allele. Text and symbols inturquoise indicate the 128-bp deletion in the hypomorphic hop2-3 allele.
(B) RT-PCR analysis of HOP2 and hop2 mutant allele expression in young ﬂower buds. A HOP2 transcript spanning the T-DNA insertion site was notdetected in hop2-1 (primers AHP2_P7 and AHP2_B), while sequences upstream (AHP2_C and AHP2_D primers) and downstream (AHP2_A and
AHP2_B primers) of the insertion could be ampliﬁed. In hop2-2, reduced levels of mRNA can be detected with all primer combinations. In hop2-3,
normal levels of mRNA can be detected with all primer combinations. Nevertheless, an expected shorter transcript is ampliﬁed when using primers
AHP2_P7 and AHP2_B, which span the genomic deletion of hop2-3. The mRNA/cDNA of the phosphoribosyltransferase (APT) gene was used fornormalization. The corresponding wild-type controls were from ecotypes Ws and Ler.
(C) Quantitative RT-PCR of HOP2 and hop2-2 mutant expression in young ﬂower buds. Primers were designed to determine expression levels of the 59
part of the mRNA (blue arrows in [A]; blue bar) and the 39 part of mRNA (red arrows in [A]; red bar). All values were normalized to Actin2/7 gene
expression. The mRNA levels detected in hop2-2 mutants are strongly decreased compared with the wild type: approximately ﬁvefold decreased whenevaluating the 59 part of mRNA (green bars) and ;30-fold decreased when evaluating the 39 part of the mRNA. The corresponding wild-type control wasfrom ecotype Ws.
(D) Schematic representation of the anticipated HOP2 protein and its hypomorphic variant. The HOP2 wild-type protein is structured into an N-terminal
domain, a putative coiled coil region, and a C-terminal domain. hop2-1 plants do not express the protein (Stronghill et al., 2010). Plants carrying the
hypomorphic hop2-2 allele produce very low levels of the mRNA and anticipated very low levels of HOP2 protein. The deletion in hop2-3 leads toexpression of an mRNA variant, anticipated to encode a protein variant that lacks 14 amino acids (position 123 to 136 of the wild-type protein) within the
coiled coil region of the protein.

alleles. In addition, these results suggested that RAD51 is instrumental for DNA repair in hop2-2 and hop2-3 mutants.
dmc1 Suppresses the DNA Repair Defects Observed in
hop2-1, hop2-2, and hop2-3 Mutants
The observed phenotype in hop2-2 and hop2-3 mutants is reminiscent of the defects observed in the Arabidopsis dmc1 mutants(Couteau et al., 1999), in which meiotic DSBs are formed but repaired
without CO outcome. It has been proposed earlier that in dmc1
mutants, meiotic DNA repair depends on RAD51 and that the sister
chromatid is used as a repair template (Zenvirth et al., 1997; Couteau
et al., 1999; Kurzbauer et al., 2012; Wijnker et al., 2012). Accordingly,
dmc1 rad51 mutants show severe chromosome fragmentation
during meiosis (Siaud et al., 2004; Vignard et al., 2007). To understand the impact of DMC1 on meiotic DNA repair in hop2 mutants, various double mutants have been generated and analyzed.

While in hop2-2 and hop2-3, some chromosomal defects areobserved, dmc1 mutants display 100% intact chromosomes(Figure 1B). Double mutants of hop2-2 dmc1 (n = 67 cells) andhop2-3 dmc1 (n = 67 cells) resemble dmc1 single mutants witha nearly complete suppression of the chromosomal defects
observed in the hop2-2 and hop2-3 single mutants (Figure 1B).
By contrast, hop2-1 single mutants show, as outlined above,
severe chromosome fragmentation. Interestingly, this severe fragmentation could largely be suppressed by the introduction of thedmc1 mutant allele (Figure 1B). Accordingly, 79% (n = 87) of allobserved cells do not show any DNA fragmentation (with only intact univalents present) in hop2-1 dmc1 double mutants, as opposed to the occurrence of DNA fragmentation in 82% of thehop2-1 cells observed (Figure 1B). Moreover, in hop2-1 dmc1
double mutants, 2.08% of seeds per silique were generated(compared with the wild type; n = 1028 siliques), in clear contrast
with hop2-1 single mutants (0.01% seeds per silique compared6 of 17

The Plant Cellwith the wild type; see above) and similar to dmc1 mutant plants(3.2% seeds per silique compared with the wild type; n = 791
siliques). Furthermore, in hop2-1 mnd1 dmc1 triple mutants,
2.35% of seeds per silique were generated (compared with thewild type; n = 312 siliques), similar to dmc1 single and hop2-1
dmc1 double mutants. This implies that HOP2 (and the HOP2/
MND1 complex) is largely dispensable for RAD51-mediated meiotic
DNA repair in the dmc1 mutant background. It furthermore implies that a lack of HOP2 protein (hop2-1 null mutant allele)
impedes DNA repair only in the presence of DMC1 protein. The
HOP2 alleles hop2-2 and hop2-3, while anticipated to be compromised in optimally supporting DMC1-mediated DNA repair,
may allow DNA repair to take place in a RAD51-dependent
manner using the sister chromatid as a repair template, even in
the presence of functional DMC1 protein.

allele (Figure 3E; n = 159), while in hop2-2 mutants, the MND1
signal was severely reduced (Figure 3D; n = 131). This latter
result further corroborated that different functional aspects of
HOP2 are compromised in the hop2-2 and hop2-3 mutants.
To conﬁrm the presence and importance of MND1 in hop2-2
and hop2-3 mutant plants genetically, we introgressed the mnd1
mutation (Kerzendorfer et al., 2006) into the hop2 mutants (Figures
3F to 3J). In the absence of the MND1 protein, meiotic DSB repair
is highly perturbed and mnd1 mutant meiosis is similar to hop2-1
meiosis, showing strong chromosome fragmentation and entanglements (cf. Figures 3G and 1Af). We found that mnd1 is epistatic to hop2-2 and hop2-3 (Figures 3I and 3J), indicating that the
DSB repair observed in the two hop2-2 and hop2-3 alleles depends on the presence of MND1. This underlines the importanceof the MND1 protein and its protein function in combination withthe hop2-2 and hop2-3 alleles.

MND1 Is Required for DNA Repair in hop2-2 and hop2-3
Mutant Alleles but Its Loading on Meiotic Chromatin Is
Reduced in hop2-2

DMC1 and RAD51 Foci Numbers in hop2 Mutant Alleles Are
Comparable to the Wild TypeIn order to gain more insight into the origin of the meiotic defects
observed in the hop2 allelic series, we performed immunohistochemical detection of MND1 on meiotic chromatin spreads
together with ASYNAPTIC1 (ASY1), a protein associated withthe axial elements of meiotic chromosomes that allows meiotic
progression to be followed (Armstrong et al., 2002). In wild-type
Arabidopsis meiosis, MND1 can be detected on chromatin loops
during meiotic prophase, as shown in Figure 3A. Besides, MND1
loading onto chromatin was shown to be dependent on HOP2
(Vignard et al., 2007). We conﬁrmed this observation, since no
MND1 signal could be detected above background in the nullallele hop2-1 (Figure 3C). In clear contrast, we detected an
MND1 signal indistinguishable from the wild type in the hop2-3

Since MND1 is less abundant in the hop2-2 mutant allele, wereasoned that this may lead to altered stability of DMC1-coatednucleoprotein ﬁlaments, manifested by altered numbers of DMC1
recombinase foci counted on meiotic spreads. Previously we, andothers, established that loading of DMC1 requires RAD51 (Bishop,
1994; Cloud et al., 2012; Kurzbauer et al., 2012) and that in an
Arabidopsis mnd1 mutant, DMC1 foci accumulate in a RAD51dependent manner (324 6 103 foci in mnd1 [n = 22] versus 234
foci 6 89 [n = 28] in the wild type; P = 0.0001) (Vignard et al., 2007).
At that time, we inferred that DMC1-coated nucleoprotein ﬁlaments
will not be supported in strand invasion reactions in mnd1 mutants;
therefore, their turnover may be decreased, resulting in more DMC1
foci on meiotic chromatin. In this sense, we anticipated a similar

Figure 3. The Formation of MND1 Foci Is Normal in hop2-3, Reduced in hop2-2, and Abolished in hop2-1 Mutants.
(A) to (E) Coimmunolocalization of ASY1 (red) and MND1 (green) in wild-type (Wt; [A]), mnd1 (B), and hop2 mutant meiocytes ([C] to [E]). In accordancewith our previous studies (Vignard et al., 2007), no MND1 foci were detected in mnd1 or hop2-1 meiocytes ([B] and [C]). By contrast, MND1 signal
appeared normal in hop2-3 mutant meiocytes (E) and reduced in hop2-2 meiocytes (D). Bars = 5 mm.
(F) to (J) 49,6-Diamidino-2-phenylindole staining of metaphase I pollen mother cells in the wild type (F), mnd1 (G), hop2-1 mnd1 (H), hop2-2 mnd1 (I),and hop2-3 mnd1 (J) mutants. Bars = 10 mm.

HOP2/MND1-Mediated Interhomologous Bias

increase in DMC1 foci numbers in hop2-1 and to a certain extent in
hop2-2 mutants. To follow DMC1 focus formation throughout
meiosis, coimmunolocalization was performed with antibodies thatrecognize the meiotic proteins ASY1 and DMC1 (Armstrong et al.,
2002; Chelysheva et al., 2007). DMC1 foci appear at late leptotene/
early zygotene, reaching an average of 234 foci (689, n = 28) per
nucleus and disappear by pachytene. The mean number of DMC1
foci is not statistically different between the wild type and hop2-2(205 6 49, n = 17) (P = 0.4533) or between the wild type and hop2-3(227 6 89, n = 21) (P = 0.6312). In the case of the null allele hop2-1,
we counted 182 (647, n = 17) DMC1 foci (Figure 4; see Supplemental
Figure 4A online). This number is slightly but signiﬁcantly smaller(P = 0.0316) when compared with foci counts in the wild type(234 foci 6 89; n = 28).
RAD51 foci were counted in leptotene/early zygotene andreached an average number of 224 (678, n = 35) in wild-type
meiocytes. The mean number of RAD51 foci is not statisticallydifferent between the wild type and hop2-1 (188 6 41, n = 9) (P =0.2801), mnd1 (210 6 41, n = 20) (P = 0.5157), and hop2-3 (216 6
56, n = 25) (P = 0.9442). In the case of hop2-2, we counted 169(639, n = 14) RAD51 foci, a value signiﬁcantly different from thewild type (P = 0.015) (Figure 4; see Supplemental Figure 4B online).
Despite the slight reduction of RAD51 and DMC1 foci, in somehop2 alleles, RAD51 and DMC1 loading or turnover does notappear to be strongly affected. Following our observations of thedifferential effects of the mnd1 and hop2-1 null mutant alleles on
DMC1 foci numbers (but not on RAD51 foci numbers), we inferthat MND1 and HOP2 proteins may have some different molecular functions, even though they may form a functional unit formost of their tasks.
HOP2-3/MND1 Complex Has a Reduced Thermal Stability
We were interested in determining whether the anticipated,
truncated HOP2-3 protein has retained its full functionality or,
alternatively, has lost parts of its function and may therefore be
impaired in supporting IH DNA repair processes. To test

Figure 4. No Major Differences in DMC1 and RAD51 Foci Numbers in All
hop2 Mutant Alleles.
Chromosome spreads of Arabidopsis meiocytes from the wild type, mnd1,
hop2-1, hop2-2, and hop2-3 mutants were stained with an a-ASY1 antibody
and an a-RAD51 or a-DMC1 antibody (see Supplemental Figure 4 online).
Foci numbers were determined and the counts blotted. While DMC1 andRAD51 foci numbers are not dramatically changed in all hop2 mutant alleles,
the DMC1 foci number in mnd1 is signiﬁcantly higher than in the wild type andall other mutant lines (Vignard et al., 2007). Please refer to text for more details.

7 of 17protein function directly, we heterologously expressed andpuriﬁed protein complexes comprising either MND1 and thefull-length HOP2 or MND1 and the internally truncated HOP2-3.
We furthermore characterized the molecular function of otherdomains of the HOP2 and MND1 proteins, using different truncated protein variants.
First, we coexpressed untagged MND1 and (6xHis-) tagged
HOP2 and HOP2 variants in Escherichia coli cells and copuriﬁedproteins with columns, exclusively retaining His-tagged proteins.
MND1 and HOP2 as well as MND1 and the HOP2-3 protein variant
showed tight association and were coeluted (Figure 5A). While theinteraction between MND1 and HOP2 could be demonstrated
directly by staining proteins in gels, proving the interaction between MND1 and HOP2-3 was complicated due to their similar
size. We therefore blotted the proteins on a membrane following
separation on a protein gel and thereafter probed the membranewith speciﬁc antibodies directed against either MND1 or HOP2
(see Supplemental Figure 5 online). Further experiments withtruncated protein versions of MND1 and HOP2 demonstrated thatthe coiled coil domains of both partners are sufﬁcient for formationof the protein complex (see Supplemental Figure 6 online). Since
the HOP2-3 protein has a 20% shortened coiled coil domainlacking two complete heptad repeats, we reasoned that the interaction between HOP2-3 and MND1 may be weakened compared with that of the wild-type proteins.
We therefore tested the thermal stability of the recombinant
protein complexes by electronic circular dichroism (ECD) spectroscopy. Figure 5B depicts ECD spectra in the far-UV region for
HOP2/MND1 and HOP2-3/MND1 in their native state, denatured
state, and partially refolded state. The overall secondary structure
according to the ECD spectra for both complexes is predominantly
a-helical, with typical minima at 208 and 222 nm in their ellipticity(Kelly et al., 2005; Greenﬁeld, 2006). Upon unfolding of both complexes (T = 85°C), some residual ellipticity remained. In the unfolded
state, spectra show ellipticity minima at around 200 nm (characteristic minimum for random coil conformation) and a shoulder ataround 220 nm (residual ellipticity from a-helices). ECD spectra
recorded at 20°C after heating and cooling of the protein complexes were indicative of partially refolded protein complexes.
We followed temperature-mediated unfolding at 222 nm(reﬂecting melting of secondary structure, mainly a-helices) (Figure
5C). In HOP2/MND1, a-helices melted between 30 and 40°C
(calculated Tm = 34°C) in a simple two-state transition. There wasa clear linear relationship between the equilibrium constants andthe reciprocal temperature, allowing the calculation of the van’t
Hoff enthalpy for this transition, which was 212 6 9 kJ mol21.
HOP2-3/MND1 melted at signiﬁcantly lower temperatures between
20 and 30°C (calculated Tm = 25°C). The melting of the secondary
structures followed a two-state transition and allowed the calculation of the van’t Hoff enthalpy, which was 272 6 15 kJ mol21.
These results demonstrate that the HOP2-3/MND1 protein complex is less stable than the wild-type complex.
The N Termini of Both HOP2 and MND1 Are Required for
DNA Binding
Following the tests above, we were interested in determining
whether the altered stability of the HOP2-3/MND1 complex has

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Figure 5. HOP2-3/MND1 Protein Complex Is Less Stable Than the HOP2/MND1 Complex.
(A) HOP2 and HOP2-3 form complexes with MND1. SDS-PAGE and Coomassie blue staining of the coexpressed and copuriﬁed recombinant HOP2/
MND1 and the HOP2-3/MND1 complexes. HOP2 or HOP2-3 has been fused to a 6xHis-tag, which was used to purify HOP2 or HOP2-3 and to copurify
MND1. Subsequent immunoblotting was performed to demonstrate the presence of both proteins (see Supplemental Figure 5 online).
(B) Temperature-mediated unfolding of the HOP2/MND1 and HOP2-3/MND1 protein complexes followed by ECD in the far UV. Conditions: 5 mM
phosphate buffer, pH 7.7. Comparison of far-UV ECD spectra of HOP2/MND1 (black) and HOP2-3/MND1 (red) at 20°C (solid lines), 85°C (dashed lines),and 20°C after heating and cooling (dotted lines).
(C) Thermal unfolding of HOP2/MND1 (solid line) and HOP2-3/MND1 (dashed line) followed at 222 nm. The insets show the corresponding van’t Hoffplots.
(D) DNA binding activities of the recombinant HOP2/MND1 and HOP2-3/MND1 protein complexes. Linearized PhiX174 Replication form I dsDNA andcircular PhiX174 virion ssDNA were incubated with the following amounts of protein: 0, 1, 2.5, 5, 10 µM HOP2/MND1 for lanes 1 to 5 and 1, 2.5, 5, and10 µM HOP2-3/MND1 for lanes 6 to 9. Reactions were loaded and separated on a 0.8% agarose gel and stained with ethidium bromide.
Diagrams below the gel pictures ([A] and [D]) indicate the recombinant proteins: red, MND1; blue, HOP2 or HOP2-3; ellipsoids represent the putative
coiled coil domains; the turquoise rectangle indicates the 14–amino acid deletion in the second half of the putative coiled coil region in HOP2-3. M,
Fermentas prestained protein ladder (numbers indicate molecular mass in kilodaltons). The calculated molecular weight for MND1 is 26.5 kD, for 6xHIS/
HOP2-3 is 26.3 kD, and for 6xHIS/HOP2 is 28 kD.

an impact on its function. First, we tested DNA binding and, furthermore, which domains of the HOP2/MND1 complex are important for DNA binding. As demonstrated before (Chen et al., 2004;
Pezza et al., 2006; Ploquin et al., 2007) for puriﬁed HOP2/MND1
proteins from other organisms, the Arabidopsis HOP2/MND1
protein complex binds ssDNA and dsDNA. The internally truncated

HOP2-3 protein, in conjunction with MND1, binds to DNA as efﬁciently as the wild-type protein (Figure 5D). By contrast, the N
termini of both HOP2 and MND1 are essential for ssDNA anddsDNA binding (see Supplemental Figures 7A and 7B online).
Further experiments demonstrated that the Lys residue 58 in the
N-terminal domain of HOP2 is crucial for DNA binding (seeHOP2/MND1-Mediated Interhomologous Bias

Supplemental Figures 7F and 7G online and Supplemental
References 1 online), thereby corroborating the results above.
Furthermore, it is interesting to mention that neither the
C termini of HOP2 nor MND1 are needed for the DNA interaction (see Supplemental Figures 7D and 7E online) and thatthe interacting coiled coil domains of HOP2 and MND1 cannot
bind to DNA (see Supplemental Figure 7C online). The N-terminal
domain of HOP2 alone, as well as the full-length HOP2, are not
sufﬁcient to mediate DNA binding efﬁciently (see Supplemental
Figures 8 and 9 online). We furthermore determined that the
HOP2/MND1 complex in plants forms a heterodimer, as shown
before for other organisms (see Supplemental Figure 10 online)(Chen et al., 2004; Enomoto et al., 2006; Pezza et al., 2006;
Ploquin et al., 2007). We therefore infer that HOP2 and MND1
operate cooperatively to build an efﬁcient DNA binding platform consisting of their N-terminal domains.
HOP2-3/MND1 Fails to Activate DMC1-Mediated D-Loop
Formation Efﬁciently
Next, we were interested in determining whether the interaction
between the HOP2/MND1 complex variants and DMC1 wasmaintained, as this interaction was reported for several organisms(Petukhova et al., 2005; Ploquin et al., 2007). To this end, we performed afﬁnity pull-down assays with HOP2/MND1 or HOP2-3/

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MND1, which were chemically cross-linked to N- hydroxysuccinimide
beads. The resulting afﬁnity resins were used to pull down recombinant DMC1 proteins (puriﬁed from Hi-5 insect cells). Alternatively, we coexpressed DMC1, MND1, and the respective HOP2
protein variants in E. coli and precipitated the complexes via theHis tag on the HOP2 (variant) proteins. All three, the wild-type
HOP2/MND1, the N-terminally truncated DN-HOP2/MND1, andthe internally truncated HOP2-3/MND1 complexes, interacted with
DMC1 (see Supplemental Figure 11 online and Supplemental
References 1 online).
We were further interested in knowing whether the altered
HOP2-3/MND1 complex would stimulate DMC1 activity as efﬁciently as wild-type HOP2/MND1. Recombinases like DMC1 polymerize on ssDNA to form nucleoprotein ﬁlaments that canlocate a homologous dsDNA molecule, catalyze invasion, andform a DNA displacement loop called the D-loop. This activity canbe measured in vitro with so-called D-loop assays. After several
failed attempts to obtain sufﬁcient amounts of soluble and active
Arabidopsis DMC1 protein, we decided to use the two DMC1
protein variants from rice (Oryza sativa), which have been shown
to be active in in vitro strand invasion assays (Sakane et al., 2008),
to perform D-loop assays with puriﬁed rice DMC1A, DMC1B,
HOP2/MND1, and HOP2-3/MND1 proteins (Figure 6). The OsHOP2-3 protein variant exactly copies the anticipated Arabidopsis HOP2-3 protein variant (see Supplemental Methods

Figure 6. HOP2-3/MND1 Fails to Activate DMC1-Mediated D-Loop Formation Efﬁciently.
Principle of the D-loop assay (A). A labeled ssDNA is ﬁrst incubated with DMC1, followed by addition of HOP2/MND1 and then the supercoiled plasmid
DNA to allow formation of D-loops. Reactions are stopped, proteins are removed, and DNA structures are separated by gel electrophoresis. Gel
pictures showing D-loop assays employing rice DMC1A (B) or DMC1B (C) proteins with either the addition of wild-type HOP2/MND1 complex or HOP23/MND1 complex in increasing concentrations. All reactions were performed in triplicate and quantiﬁcations are shown below the gel pictures aspercentages of ssDNA incorporated into D-loops.

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online). The assays were performed in triplicate and show very
robust results. While rice HOP2/MND1 stimulates Os-DMC1A
1.9- to 2.0-fold (depending on the concentration of HOP2/MND1)
and Os-DMC1B 12.3- to 14.1-fold, the hypomorphic Os-HOP2-3
protein variant, in conjunction with Os-MND1, fails to do so efﬁciently and stimulates Os-DMC1A only 1.2- to 1.5-fold and OsDMC1B only 2.6- to 10.0-fold. We therefore conclude that the
suboptimal stability of the HOP2-3/MND1 complex compromises
its ability to efﬁciently support DMC1-mediated strand invasion.
DISCUSSION
Meiotic RAD51 Function Is Independent of HOP2/MND1
and Is Negatively Regulated by DMC1
In vitro experiments with mammalian and yeast (Saccharomyces
cerevisiae and Schizosaccharomyces pombe) proteins establishedthat puriﬁed HOP2/MND1 stabilizes DMC1 nucleoprotein ﬁlaments and stimulates DMC1-mediated D-loop formation in vitro(Chen et al., 2004; Enomoto et al., 2006; Pezza et al., 2007; Ploquin
et al., 2007). The mammalian complex can also stimulate RAD51
activity, but to a lesser extent than DMC1 activity (Petukhova
et al., 2005), leading to the idea that the HOP2/MND1 complex isan accessory factor of DMC1 and, therefore, a crucial determinant of interhomolog DNA repair during meiosis. In plants,
it was therefore surprising to ﬁnd that in mnd1 or hop2 mutants,
meiotic DNA repair would fail completely, while RAD51-mediated DNA repair via the sister chromatid was not compromisedin a dmc1 mutant (Schommer et al., 2003; Kerzendorfer et al.,2006). These results suggested that in plants, RAD51 needs tobe supported by HOP2/MND1 during meiotic DNA repair.
However, our analysis revealed that the DNA repair defectobserved in hop2-1 null mutants can be largely suppressed by

the introgression of a dmc1 null mutant allele (Figure 1B). This
result is intriguing as it suggests that in the presence of DMC1
and in the absence of HOP2, RAD51-mediated repair is suppressed. Consistent with our data, we suggest a model (Figure 7)
in which HOP2/MND1 is needed to overcome constraints imposed on RAD51-mediated repair in the presence of DMC1.
These constraints may either be of mechanical nature or embedded in a signal pathway. In this sense, DMC1 may block
access of RAD51 to ssDNA, while not being sufﬁciently activated for repair reactions in the absence of functional HOP2/
MND1. Alternatively, HOP2/MND1 may be part of a signaling
pathway in which DMC1-coated nucleoprotein ﬁlaments elicit
a signal to (transiently) inhibit RAD51-mediated IS repair, which
is only alleviated after DMC1-nucleoprotein ﬁlament interaction
with HOP2. In both scenarios, HOP2/MND1 would be dispensable in the absence of DMC1 for RAD51-mediated IS repair. In the presence of DMC1, high levels of HOP2/MND1
would sufﬁciently activate DMC1 for meiotic DNA repair and, as
shown before (Kurzbauer et al., 2012), RAD51 would only havea limited back-up function (Figure 7). This backup function is
anticipated to be needed in case DMC1-coated nucleoprotein
ﬁlaments fail to repair via the homologous chromosome (forexample, because of sequence variation). We speculate that the
inhibition of RAD51 has to be transient and eventually alleviatedduring progression of wild-type meiosis.
Such a negative regulatory mechanism suppressing RAD51
function during meiosis has not yet been described in plants, while
in S. cerevisiae, several factors that indirectly promote IH bias bysuppressing meiotic IS repair have been found. Suppression of IS
repair is mediated by the activation of the Thr/Ser DNA damage
checkpoint kinase Mek1 (for Meiosis-speciﬁc serine/threonine
protein kinase1) in response to activated Red1 and Hop1, twostructural components of the meiotic chromosome axis (Schwacha

Figure 7. Model of the Functional Relationship between HOP2/MND1, DMC1, and RAD51.
In a wild-type context, high levels of HOP2/MND1 stimulate DMC1 activity, thereby promoting IH DNA repair. We anticipate that RAD51-mediated IS
DNA repair is transiently repressed, limiting IS repair to a backup function. In the absence of DMC1, RAD51 efﬁciently repairs meiotic DSBs via sister
chromatids. We anticipate that this repair is independent of HOP2/MND1, since in hop2 mnd1 dmc1 triple mutants, IS DNA repair is in place.
Importantly, in the absence of HOP2 and/or MND1, DMC1 is not stimulated and permanently inhibits RAD51-mediated IS DNA repair. Therefore, in theabsence of HOP2/MND1, RAD51-mediated DNA repair can only take place if DMC1 is removed. Last, in conditions with only limited amounts offunctional HOP2/MND1 complex available (hop2-2 and hop2-3 mutant alleles), DMC1 is not optimally supported and only limited IH DNA repair takes
place. The limited stimulation of DMC1 is sufﬁcient to indirectly promote RAD51-mediated IS repair. Possible mechanisms are discussed in the text.

HOP2/MND1-Mediated Interhomologous Biasand Kleckner, 1997; Wan et al., 2004; Niu et al., 2005). In brief,
Mek1-mediated phosphorylation of Rad54, a protein promoting
Rad51 function, (Niu et al., 2009), and binding of Hed1 to Rad51(Tsubouchi and Roeder, 2006; Busygina et al., 2008) limit Rad51’s
repair function in the absence of Dmc1.
While no plant homologs of Mek1 or Hed1 are known yet, the
HORMA domain (for Hop1p, Rev7p, Mad2) protein ASY1 is conserved among plant species and shares homology with Hop1 fromyeast (de los Santos and Hollingsworth, 1999). Arabidopsis mutants lacking ASY1 preferentially repair meiotic DSBs via the sister
chromatids (Sanchez-Moran et al., 2007). Even though a protein
related to yeast’s Red1, ASY3, has recently been described in
Arabidopsis (Ferdous et al., 2012), genetic evidence and the lack ofa clear Mek1 homolog suggest that IH repair in plants is rather
directly promoted via ASY1, HOP2, MND1, and DMC1 and notindirectly by suppression of RAD51-mediated IS repair. Recent
data demonstrated that plant DMC1 is a promiscuous recombinase, proﬁcient at repair from either the sister or the homolog, and its IH speciﬁcity is mediated by factors like ASY1
(Sanchez-Moran et al., 2007; Kurzbauer et al., 2012).
DMC1-Driven IH Repair Is Dependent on the Levelof Functional HOP2/MND1
The most prominent feature of the hypomorphic hop2-2 and hop2-3
mutants is their deﬁciency in IH bias, while retaining their capacity
for IS DNA repair (Figure 1). This is in clear contrast with theobservations made in the hop2-1 null allele (Schommer et al.,
2003; Stronghill et al., 2010), which exhibits impaired meiotic DNA
repair. Genetic crosses revealed that the IS repair observed in the
hypomorphic hop2-2 and hop2-3 alleles depends on the presence of RAD51, MND1, but not on DMC1. It therefore appears
that in the presence of either low amounts (hop2-2) or less stable(hop2-3) HOP2/MND1 complex, DMC1 is not sufﬁciently activated to promote IH repair but is sufﬁciently activated to allow IS
DNA repair, potentially mediated via RAD51. It is important to notethat RAD51-mediated IS repair does not directly depend on
HOP2/MND1, since a hop2-1 mnd1 dmc1 triple mutant appears
like a dmc1 single mutant in which DSBs are formed but repaired
in a RAD51-dependent manner via the sister chromatid.
The Plant HOP2/MND1 Complex Binds to DNA andStimulates DMC1-Mediated D-Loop Formation
We further performed a systematic analysis of DNA binding
characteristics of HOP2 and MND1. We found that both proteins,
HOP2 and MND1, are important for ssDNA and dsDNA binding invitro. In more detail, we could show that the N termini of both
HOP2 and MND1 are important for binding DNA in vitro, supporting the idea that they cooperate to form a DNA binding platform. It is interesting to note that both the N-terminal domains of
HOP2 and MND1 are rich in conserved Lys and Arg residues,
which are thought to be involved in DNA binding. We substituted
all conserved Lys residues in the N-terminal domain of HOP2 from
Arabidopsis individually with Ala (Lys21Ala, Lys39Ala, Lys44Ala,
Lys58Ala, and Lys62Ala) and observed that only the substitution
Lys58Ala exhibits a prominent effect on DNA binding in the HOP2/
MND1 complex of Arabidopsis. Individual Lys-to-Ala (Lys30Ala,

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Lys34Ala, Lys47Ala, and Lys63Ala) substitutions of residues in the
N-terminal domain of MND1 had no impact on DNA binding, indicating that only substitutions of multiple residues may have an
impact on DNA binding, as also shown recently for mouse HOP2/
MND1 (Zhao et al., 2013).
The performed D-loop assay clearly demonstrates that in plants,
the HOP2/MND1 complex also stimulates DMC1, as has beenshown earlier for heterologously expressed and puriﬁed proteinsfrom human, mouse, and budding yeast, and the extent of DMC1
stimulation is also comparable (Chen et al., 2004; Petukhova et al.,2005; Enomoto et al., 2006). It is interesting to note that OsDMC1A has higher basic activity but is less responsive to theaddition of Os-HOP2/Os-MND1 (twofold stimulation), while OsDMC1B has very low D-loop activity that can strongly be activatedby the addition of Os-HOP2/Os-MND1 (14-fold). Importantly, ourexperiments also demonstrate that the plant HOP2/MND1 complex has no intrinsic D-loop-forming activity (Figure 6).
Taken together, the results outlined above provide explanations
for the observed phenotypes in hop2-2 and hop2-3 mutant plants.
Plant HOP2/MND1 is a crucial cofactor of DMC1-mediated D-loop
formation and, therefore, of DNA repair in meiosis. Our analysis
revealed negative regulation of RAD51 by DMC1 during meiosis.
We believe that this negative regulation is of a transient nature andis important for the establishment of DMC1-mediated interhomolog connections during meiotic DNA repair. Reduced availability of functional HOP2/MND1 complex leads to suboptimal
support of DMC1 and, therefore, to a failure to repair meiotic DSBs
via the homologous chromosome but is sufﬁcient to allow RAD51mediated IS repair.
METHODS
Plant Material
Arabidopsis thaliana wild-type plant material used in this study is Wassilewskija-4 (Ws-4) or Ler. The hop2-1 (ahp2-1) mutant line is in the Ler ecotype
and has been described previously (Schommer et al., 2003). hop2-2 andhop2-3 mutant lines were obtained from the Versailles collection (Ws-4
accession; http://www-ijpb.versailles.inra.fr/en/sgap/equipes/ﬁchiers.
old-christine/T-DNA_information.htm). (Bechtold et al., 1993; De Muyt
et al., 2009). All other mutant lines were described earlier: asy1-3 (Institut
National de la Recherche Agronomique line EJZ8, Ws-4 ecotype; De Muyt
et al., 2009), mre11-3 (SALK_054418, Columbia ecotype; Puizina et al.,2004), mnd1-1 (SALK_110052, Columbia ecotype; Kerzendorfer et al., 2006),
rad51-1 (Columbia ecotype; Li et al., 2004), and dmc1-1 (Wassilewskija [Ws]ecotype; Couteau et al., 1999; Li et al., 2004).
Growth Conditions
Arabidopsis plants were cultivated in a greenhouse or growth chambers
under the following conditions: photoperiod 16 h/day and 8 h/night;
temperature 20°C day and night; humidity 70%.
Genetic Analyses
Allelism tests were performed by crossing plants heterozygous for eachmutation. Heteroallelic plants were identiﬁed by PCR using speciﬁc primers (see below). Double mutants were obtained by crossing plants
heterozygous for each mutation. The resulting hybrids were obtained after
self-pollination. PCR screening was then used to identify plants homozygous for both mutations in the F2 progeny.

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Genotyping
The hop2-1 and hop2-2 mutants have been described earlier (Schommer
et al., 2003; De Muyt et al., 2009). In case of hop2-3 mutant alleles, thealtered genomic regions at the HOP2 gene locus were PCR ampliﬁedusing speciﬁc primer sets. Ampliﬁcation products obtained from sterile
plants were analyzed initially by gel electrophoresis and sequencing, aspreviously described (De Muyt et al., 2009), and subsequently by gelelectrophoresis only.
To determine the genotype in hop2-1 mutant plants by PCR, the primerset AHP2_P7 (59-GAAAACTATCAGTGATGTGG-39) and AHP2_B (59CCTCGAGGCCTCTTTTTACC-39) was used to amplify the wild-type sequence(632 bp) and primer set AHP2_P7 and GABIo8409 (59-ATATTGACCATCATACTCATTGC-39) to amplify a 572-bp fragment present in hop2-1
mutant plants. The deletion present in hop2-2 mutants was determined by
PCR using the primer set N6360002U (59-CACCATCAATACAAAAGACG-39)
and N6360002L (59-CCAAGTGATTAAATTCCCAG-39), yielding a fragmentof 900 bp in case of presence of the wild-type allele and of 750 bp in case ofpresence of the hop2-2 mutant allele. The deletion found in hop2-3
mutants was determined by PCR using the primers set AHP2_P7 and Hop2-1L(59-TTGTACAGTTGCATATGTGTG-39) to amplify a 700-bp fragment in caseof the presence of the wild-type allele and a 572-bp fragment in case ofpresence of the hop2-3 mutant allele.
cDNA Characterization
For RT-PCR experiments, mRNA was obtained from ﬂower buds, roots, orleaf tissues as described (De Muyt et al., 2007) and reverse transcribed into
cDNA, and the amounts were normalized to the expression of the adenine
phosphoribosyltransferase-encoding gene (APT; Moffat et al. 1994) using
primers APT_RT1 (59-TCCCAGAATCGCTAAGATTGCC-39) and APT_RT2-1(59-CTCAATTACGCAAGCAC-39) for 30 PCR cycles (annealing temperatureof 60°C).
The HOP2 CDS was ampliﬁed using different sets of primers: AHP2_C(59-TGATTTCTGATTCCACATCACTG-39) and AHP2_D (59-AAATCGGATAACACCGAAGC-39), AHP2_7 (59-GAAAACTATCAGTGATGTGG-39)
and AHP2_B (59-CCTCGAGGCCTCTTTTTACC-39), or AHP2_A (59GGTGAGGCCAGAAGACAAAA-39) and AHP2_B for 35 cycles (annealing
temperature of 60°C).
For hop2-3, the internal deletion was determined using primers
AHP2_P7 and AHP2_B, yielding a 384-bp fragment in wild-type plants
and a 342-bp fragment for mRNA coming from hop2-3 mutant plants. The
RT-PCR products were cloned into vector Zero Blunt (Invitrogen) andsequenced by LGC Genomics (see Supplemental Figure 2 online).
Quantitative RT-PCR
Total RNA from hop2-2 and wild-type (Ws-4) Arabidopsis ﬂower buds fromseveral different individual plants was extracted using the SV Total RNA
isolation system (Promega) according to the manufacturer’s instructions.
Subsequently, cDNA was synthesized using the iScript cDNA synthesis kit(Bio-Rad) and quantitative PCR was performed with a Bio-Rad iQ5 Cyclerusing the iQ SYBR green supermix and the following program: step 1, 95°C, 3min; step 2, 95°C, 15 s; step 3, 60°C, 30 s; step 4, 72°C, 30 s; repeat steps 2 to
4 50 times; step 5, 94°C, 1 min; step 6, 60°C, 1 min; step 7, 60°C, 30 s withincreasing the temperature over 71 cycles (0.5°C/cycle, end temperature95°C). Primers for the amplicon adjacent to the ATG were HOP2_59ATG_fw(59-TGCTCTCGCTTCAGGTTTTT-39) and HOP2_59ATG_rv (59-CGAAATAATTGGCGGGAAAT-39) and for the amplicon bridging exons 4 and 5
were HOP2_39hop2-1_fw (59-AAGAGAAAGATGCCAAACTAAGG-39) and
HOP2_39hop2-1_rv (59-AGTTTCTCTTCCATTTCCTTAAC-39). Two technicalrepeats were performed. All values were normalized to Actin2/7 gene expression using primers actin_ampl3_dn (59-TTGCTGACCGTATGAGCAAAGA-39) and actin_ampl3_up (59-TCGATGGACCTGACTCATCGT-39). mRNA

quality, lack of contaminating genomic DNA, and concentration were determined prior to each experiment. Additionally, melting point analysis was
performed with each amplicon. Data analysis and calculation of expression
proﬁles were performed with iQ5 optical system software.
Antibodies
Primary antibodies for ﬂuorescent immunolocalization of proteins were used
as follows: aASY1 raised in rabbit (1:500 diluted in blocking buffer; PBS +0.1% Triton + 3% BSA) (Armstrong et al., 2002), aASY1 raised in rat (1:500;
Higgins et al., 2004), aMND1 raised in rat (1:200; Vignard et al., 2007),
aRAD51 raised in rat (1:500; Kurzbauer et al., 2012), and aDMC1 raised in
rabbit (1:20; Chelysheva et al., 2007). Secondary antibodies were used asfollows: goat-anti-rabbit conjugated to ﬂuorescein isothiocyanate (1:300;
Sigma-Aldrich), goat-anti-rat conjugated to Cy3 (1:300; Chemicon), goatanti-rabbit conjugated to Alexa 488 (1:100; Invitrogen), goat-anti-rat conjugated to Alexa 488 (1:100; Invitrogen), and goat-anti-rat conjugated toAlexa 568 (1:100; Invitrogen).
Microscopy
Comparison of early stages of microsporogenesis and the development ofpollen mother cells was performed as described (Grelon et al., 2001).
Preparation of prophase stage spreads for immunocytology was performed
according to Armstrong et al. (2002) with the modiﬁcations described(Chelysheva et al., 2005, 2010; Kurzbauer et al., 2012).
Parts of the observations were made using a Leica DM RXA2 microscope or a Zeiss Axio Imager 2 microscope; photographs were taken usinga CoolSNAP HQ camera (Roper Scientiﬁc) driven by OpenLAB 4.0.4
software or a Zeiss camera AxioCam MR driven by Axiovision 4.7. All of theimages acquired were further processed with OpenLAB 4.0.4, Axiovision
4.7, or AdobePhotoshop 7.0 (Adobe). Other parts of the analysis were
performed on a Zeiss Axioplan microscope, where Z-stacks with 100-nm
intervals were acquired with MetaMorph software. Z-stacks were deconvolved using AutoQuant software and are presented as projections donewith HeliconFocus software. Recombinase foci were counted manually withthe help of the “count” tool in Adobe Photoshop CS4.
Generation of Protein Expression Constructs for Heterologous
Expression in Escherichia coli
In order to coexpress Arabidopsis HOP2 and MND1 in E. coli, the CDSs of
both HOP2 and MND1 were cloned into the vector pRSFDuet-1 (Merck).
First, the CDS of MND1 (Kerzendorfer et al., 2006) was ampliﬁed by PCR
using KOD DNA polymerase (Merck) and the primer pair MND_FWD_duet(59-TATCATATGTCTAAGAAACGGGGAC-39) and MND_REV_duet (59ATACTCGAGCTAAGCTTCATCTTGTACT-39). Each primer included either an NdeI or an XhoI restriction site, which were later used to clone
MND1 in frame into the second multiple cloning site (MCS) of the plasmidpRSFDuet-1. Second, the CDS of HOP2 (Schommer et al., 2003) was
ampliﬁed by PCR using Thermococcus kodakaraensis KOD1 DNA
Polymerase and the primer pair AHP2_FWD_duet (59-AACGGATCCTATGGCTCCTAAATCGGATAACAC-39) and AHP2_REV_duet (59-ATACTGCAGTTACTGTCCTCGAGGCCTC-39). Each primer included eithera BamHI or a PstI restriction site, which were later used to insert HOP2 inframe into the ﬁrst MCS of the plasmid pRSFDuet-1/MND1. As a result,
the recombinant HOP2 protein is expressed with an N-terminal 6xHIS-tag
and the MND1 protein is expressed without any additional amino acids
from the vector pRSFDuet-1/HOP2/MND1.
To construct the expression plasmid for the N-terminal truncatedversion of the recombinant HOP2 protein (Met84-Gln226), the primer pair
AHP2-84Met-fwd (59-AGGATCCTATGAAAGAAGACAATGCCAAAC-39)
and AHP2_REV_duet (59-ATACTGCAGTTACTGTCCTCGAGGCCTC-39)
was used for PCR (KOD DNA polymerase) and pRSFDuet-1/HOP2/MND1

HOP2/MND1-Mediated Interhomologous Bias

as a template. Each primer included either a BamHI or a PstI restriction
site, which were later used to clone the sequence of DN-HOP2 in frame
into the ﬁrst MCS of the plasmid pRSFDuet-1/MND1.
The small deletion (Glu-123–Glu-136) in the HOP2 sequence that corresponds to the hop2-3 hypermorphic allele was generated by deletion
PCR. To this end, the outlined above vector pRSFDuet-1/HOP2/MND1 wasused as a template for PCR ampliﬁcation using KOD DNA polymerasewith a forward primer that was phosphorylated at the 59 end (Phos/59AAGAGAAACTGGTCAAACTAC-39) and a reverse primer (59-GTATCTCTTCTAGTGTCAAGT-39). These two primers were designed to anneal
exactly to the border sequences adjacent but outside of the deletion in thehop2-3 hypomorphic allele with their 39 ends pointing away from the deletion. After ampliﬁcation, the PCR product was gel puriﬁed and ligated with
T4 DNA ligase in green buffer (Fermentas). Subsequently the ligase washeat inactivated at 70°C for 20’, and the restriction enzyme DpnI was added
to the reaction mixture for 2 h at 37°C. Finally, the solution was transformed
into chemical competent XL-1 Blue E. coli cells, and the success of thedeletion PCR was veriﬁed by restriction analysis using DraI together with
EcoRI and subsequent sequencing. All PCR products were conﬁrmed bysequencing. Further information on truncated HOP2 and MND1 protein
variants can be found in Supplemental Methods 1 online. All primers
employed to construct the various truncated versions are compiled in
Supplemental Table 1 online.
In order to coexpress rice (Oryza sativa spp japonica) HOP2 and MND1 in
E. coli, the protein CDSs of both were synthesized (see Supplemental Table 2
online for sequence details) (GeneArt, Invitrogen) and subsequently cloned
into the vector pRSFDuet-1 (Merck) using the BamHI and XhoI restriction
sites. As a result, recombinant Os-HOP2 protein is expressed with an
N-terminal 6xHis-tag and the Os-MND1 protein is expressed without any
additional amino acids from the vector pRSFDuet-1/OsHOP2/OsMND1.
The small deletion (Ser-123–Glu-136) in the Os-HOP2 sequence, corresponding to the Arabidopsis hop2-3 hypomorphic allele, was generated bydeletion PCR (see Supplemental Table 3 online for multiple protein sequencealignment of At-HOP2, At-HOP2-3, and Os-HOP2). To this end, the vector
pRSFDuet-1/OsHOP2/OsMND1 served as a template in PCR ampliﬁcation(Phusion high-ﬁdelity DNA polymerase; New England Biolabs,) witha phosphorylated forward primer (Phos/59-GAGAAACTGAATAAACTGCGC-39)
and a nonmodiﬁed reverse primer (59-TTTGATTTCTGCCAGGGTCAG-39).
These two primers were designed to anneal exactly to the border sequences
adjacent but outside of the deletion with their 39 ends pointing away from thedeletion. After ampliﬁcation, the PCR product was gel puriﬁed and ligated (T4DNA ligase; Fermentas). Subsequently, the ligase was heat inactivated at
70°C for 20’ and the restriction enzyme DpnI was added to the reaction
mixture for 2 h at 37°C. Finally, the solution was transformed into chemicalcompetent XL-1 Blue E. coli cells and the obtained plasmid veriﬁed bysequencing.
Expression and Puriﬁcation of Recombinant Proteins from E. coli
The plasmids for expression of the protein complexes (from Arabidopsis
and rice) HOP2/MND1 as well as the truncated versions DN-HOP2/MND1
and HOP2-3/MND1 were transformed into chemically competent Rosetta(DE3)pLysS E. coli cells (Merck), and expression was started by autoinduction (Studier, 2005). To this end, a single colony was inoculated in 3 mL
of ZYP-0.8G liquid medium [1 mM MgSO4, 0.05 M KH2PO4, 0.05 M
Na2HPO4, 0.025 M (NH4)2SO4, 1% peptone, 0.5% yeast extract, 0.8% Glc,
and 0.01% kanamycin], and after several hours of incubation at 37°C with
200 rpm, 1 mL was added to two liters of ZYP-5052 liquid medium [1 mM
MgSO4, 0.05 M KH2PO4, 0.05 M Na2HPO4, 0.025 M (NH4)2SO4, 1% peptone, 0.5% yeast extract, 0.5% glycerol, 0.05% Glc, 0.2% lactose, and
0.01% kanamycin]. On the next day, the cells were harvested by centrifugation (5000g, 30’, 4°C). The cell pellet was frozen at 220°C and thawedon ice. Subsequent resuspension of the pellet in ice cold buffer A (1 g pellet
per 5 mL of 50 mM phosphate buffer, pH 8.0, with 500 mM NaCl, 2 mM

13 of 17b-mercaptoethanol, 0.05% Nonidet P-40, and one tablet of Complete-Mini
EDTA-free [Roche] per 50 mL of buffer) was followed by sonication on ice.
After that, an additional centrifugation (20,000g, 30’, 4°C) was conducted,
and the resulting precleared lysate was incubated with 5 mL of Ni-beads(Proﬁnity IMAC Ni-charged resin; Bio-Rad) for 1 h at 4°C. The Ni-beads were
washed four times with buffer A, and the recombinant 6xHis-tagged proteins were eluted with 1 M imidazole, pH 8.0, in buffer A. After elution, theprotein solution was concentrated with the help of Vivaspin 20 10,000
MWCO polyethersulfone membrane centrifugal concentrators (Sartorius
Stedim Biotech), and gel ﬁltration was performed using an ÄKTA fast protein
liquid chromatography system, a Superdex 200 16/60 column (GEHealthcare Life Sciences), and buffer A. The fractions that contained theprotein complex were pooled and aliquots were shock frozen in liquid nitrogen and stored at 280°C. The purity and concentration of the recombinant proteins was analyzed by SDS-PAGE using Coomassie Brilliant
Blue staining and Bradford assay according to standard techniques. Finally,
the identities of the Coomassie Brilliant Blue–stained bands for the HOP2/
MND1 complex were conﬁrmed by mass spectrometry (see Supplemental
Table 4 online). Information on At-DMC1 protein expression in Hi-5 insect
cells can be found in the Supplemental Methods 1 and in Supplemental
Table 5 online.
EMSA
Afﬁnity-puriﬁed HOP2/MND1, DN-HOP2/MND1, HOP2-3/MND1, and
HOP2/MND1 protein complex variants (protein concentrations are denotedin the ﬁgures) were incubated with circular ssDNA FX174 virion (20 µM/bp;
NEB) and/or linearized dsDNA FX174 Replication form I (20 µM/bp; NEB) for
10’ at room temperature. Subsequently, the DNA-protein complex was
mixed with 63 Luria-Bertani (15% Ficoll-400, 50 mM EDTA, pH 8, and
0.15% Orange G), separated on a 0.8% agarose gel (13 TAE: 40 mM Tris,
20 mM acetic acid, and 1 mM EDTA, pH 8) at 4°C, and visualized by staining
with ethidium bromide.
Temperature-Mediated Unfolding Followed by ECD Spectroscopy
Thermal unfolding was followed by ECD spectroscopy (Chirascan, Applied
Photophysics). The instrument was ﬂushed with nitrogen at a ﬂow rate of 5
liters min21 and was equipped with a Peltier element for temperature
control. Temperature-mediated denaturation was monitored between 20
and 85°C. The temperature was increased stepwise at 1.0°C min21. Singlewavelength scans were performed with instrumental parameters set as
follows: for far-UV ECD at 222 nm, 5 mM HOP2/MND1 or HOP2-3/MND1 in
5 mM phosphate buffer, pH 7.7; path length, 1 mm; spectral bandwidth,
3 nm; scan time per point, 10 s. ECD spectra in the far-UV region (190 to
280 nm) were recorded for 5 µM HOP2/MND1 and 5 µM HOP2-3/MND1 at
20°C, at 85°C after thermal unfolding, and at 20°C again after heating andcooling. The fraction a of unfolded protein was calculated according to a =(uN 2 u)/(uN 2 uU) with uN being the ellipticity (in mdeg) at 222 nm of theprotein in the native folded state, u the ellipticity at deﬁned temperature (T),and uU being the ellipticity at 222 nm of the completely unfolded state.
D-Loop Assay

DNA Substrates
For the D-loop formation assay, HPLC-puriﬁed DNA oligonucleotides
were purchased from Nihon Gene Research Laboratory: G5E4 90mer,
59-CCTGCCTAGGCAAAATAGCACCCTCCCGCTCCAGAACAACATACAGCGCTTCCACAGCGGCAGCCATAACAGTCAGCCTTACCAGTAAAA-39.
The superhelical dsDNAs were prepared as described (Kagawa et al.,2001). To prevent the superhelical dsDNA from undergoing irreversible
denaturation, alkaline treatment of the cells harboring the plasmid DNA
was avoided.

14 of 17

The Plant Cell

D-Loop Formation Assay
Os-DMC1A or Os-DMC1B (Sakane et al., 2008) (400 nM) was incubatedwith the 32P-labeled G5E4 90-mer single-stranded oligonucleotide (1 mM) at
37°C for 5 min, in 8 mL of reaction buffer, containing 22 mM HEPES, pH 7.5,
50 mM KCl, 0.025 mM EDTA, 1 mM DTT, 0.2 mM 2-mercaptoethanol, 1%
glycerol, 1 mM MgCl2, 1 mM ATP, 20 mM creatine phosphate, 75 mg/mL
creatine kinase, and 100 mg/mL BSA. After this incubation, 1 mL of rice
HOP2/MND1 or HOP2-3/MND1 was added, and the samples were further
incubated at 37°C for 5 min. The HOP2/OsMND1 or HOP2-3/MND1
concentrations were 12.5, 25, and 50 nM. Subsequently, 1 mL of the CP943
superhelical dsDNA (30 mM) was added, and the reactions were continued
at 37°C for 10 min. The reactions were terminated by the addition of 2 mL ofstop solution containing SDS (0.2%) and proteinase K (1.4 mg/mL; Roche
Applied Science), and the deproteinized DNA products were separated by
1% agarose gel electrophoresis in 13 TAE buffer at 4 V/cm for 2 h. The gelswere dried and exposed to an imaging plate. The gel images were visualizedusing an FLA-7000 imaging analyzer (Fujiﬁlm), and the band intensities were
quantitated with Multi Gauge software (Fujiﬁlm).

Supplemental Table 1. Primer Sequences for Construction of
Truncated HOP2/MND1 Complexes.
Supplemental Table 2. Sequence of Rice HOP2/MND1 Synthesizedby Geneart.
Supplemental Table 3. Multiple Sequence Alignment of Arabidopsis
HOP2 and HOP2-3 and Rice HOP2.
Supplemental Table 4. Mass Spectrometric Determination of theProtein Identity of Heterologously Expressed At-HOP2 and At-MND1
Proteins.
Supplemental Table 5. Codon-Optimized At-DMC1 Sequence for
Expression in Hi5 Insect Cells
Supplemental Methods 1. Description of Experimental Procedures
Related to Supplemental Data.
Supplemental References 1. References Related to Experimental
Procedures Described in Supplemental Methods 1.

ACKNOWLEDGMENTS
Accession Numbers
Sequence data for the genes from this article can be found in the
GenBank/EMBL data libraries under the following accession numbers:
AT1G13330 (At-HOP2), AT4G29170 (At-MND1), AT5G20850 (At-RAD51),
AT3G22880 (At-DMC1), AT1G67370 (At-ASY1), AT5G54260 (At-MRE11),
Os12g0143800 (Os-DMC1A), Os11g0146800 (Os-DMC1B), Os09g0280600(Os-MND1), Os03g0710100 (Os-HOP2). Sequence data for the proteins fromthis article are as follows: Q9FX64 (Swiss-Prot) (At-HOP2), CAJ44238 (EMBL)(At-MND1), AAC49617 (GenBank) (At-DMC1), ABF98498 (GenBank) (OsHOP2), BAF24680 (DDBJ) (Os-MND1), AAM76791 (GenBank) (Os-DMC1A),
and AAM76792 (GenBank) (Os-DMC1B).
Supplemental Data

We thank Stefan Hofbauer from the Obinger Lab at the University of
Natural Resources and Life Sciences (Vienna, Austria) for technicalassistance with ECD. We also thank Dorothea Anrather (Max F. Perutz
Laboratories, University of Vienna, Austria) for mass spectrometry analysisof the recombinant protein complex HOP2/MND1, Peggy Stolt-Bergner(Campus Support Facility, Vienna, Austria) for expression of the DMC1 in
Hi5 insect cells, and Aurélie Chambon (Institut Jean-Pierre Bourgin) for
technical assistance. We thank Pascale Doutriaux (Université Paris-Sud
11, Orsay, France) for generously supplying the At-DMC1 cDNAs. We
thank the European Union (FP7 program Meiosys-KBBE-2009-222883),
Institut National de la Recherche Agronomique (Department of Biologie etAmélioration des Plantes), Agence Nationale pour la Recherche (ANR07BLAN-0215), and Rijk Zwaan for funding our research.

The following materials are available in the online version of this article.
Supplemental Figure 1. Epistasis Analysis.
Supplemental Figure 2. Molecular Characterization of hop2-2 andhop2-3 Alleles.

AUTHOR CONTRIBUTIONS

Supplemental Figure 6. Putative Coiled Coil Domains of Both HOP2
and MND1 Are Sufﬁcient for Complex Formation.

C.U., A.R., and M.V.H. performed most of the experiments, evaluated thedata, and wrote parts of the article. C.U. performed all biochemical
experiments (apart from Os-DMC1A/B puriﬁcation and the D-loop assay).
M.V.H. performed parts of the cytology and the molecular characterization.
A.R. performed parts of the cytology and the molecular characterization.
A.D.M. and D.V. were involved in the initial screen and mutant mapping. L.P.
performed some of the experiments for the molecular characterization. L.C.
performed some of the cytological characterization. W.K. and H.K. puriﬁed
OsDMC1A/B and performed the D-loop assay. P.S. and M.G. designed theexperiments, evaluated the data, and wrote the article.

Supplemental Figure 7. The N-Terminal Domains of Both MND1 and
HOP2 Are Needed for DNA Binding of the HOP2/MND1 Protein
Complex.

Received September 25, 2013; revised November 24, 2013; accepted
December 5, 2013; published December 20, 2013.

Supplemental Figure 3. DSBs Are Formed in the hop2-2 and hop2-3
Mutants.
Supplemental Figure 4. No Major Differences in DMC1 and RAD51
Foci Numbers Are Observed among All hop2 Mutant Alleles.
Supplemental Figure 5. HOP2 and HOP2-3 Form Protein Complexes
with MND1.

Supplemental Figure 8. The N-Terminal Domain of HOP2 Is Not
Sufﬁcient for DNA Binding.
Supplemental Figure 9. The HOP2/MND1 Complex Is More Proﬁcientat DNA Binding Than HOP2 Alone.
Supplemental Figure 10. The HOP2/MND1 protein complex isheterodimeric
Supplemental Figure 11. The Recombinase DMC1 Binds to WildType HOP2/MND1 and the Variant DN-HOP2/MND1 and HOP2-3/
MND1 Protein Complexes in Vitro.

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Sufficient Amounts of Functional HOP2/MND1 Complex Promote Interhomolog DNA Repair but
Are Dispensable for Intersister DNA Repair during Meiosis in Arabidopsis
Clemens Uanschou, Arnaud Ronceret, Mona Von Harder, Arnaud De Muyt, Daniel Vezon, Lucie
Pereira, Liudmila Chelysheva, Wataru Kobayashi, Hitoshi Kurumizaka, Peter Schlögelhofer andMathilde Grelon
Plant Cell; originally published online December 20, 2013;
DOI 10.1105/tpc.113.118521
This information is current as of December 20, 2013
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