The Plant Journal (2005) 41, 533–545
doi: 10.1111/j.1365-313X.2004.02318.x
Differing requirements for the Arabidopsis Rad51 paralogs in
meiosis and DNA repair
Jean-Yves Bleuyard, Maria E. Gallego, Florence Savigny and Charles I. White*
CNRS UMR6547, Université Blaise Pascal, 24, avenue des Landais, 63177 Aubière, France
Received 8 October 2004; revised 12 November 2004; accepted 16 November 2004.
*
For correspondence (fax þ33 473 407 777; e-mail [email protected]).
Summary
In addition to the recombinase Rad51, vertebrates have five paralogs of Rad51, all members of the Rad51dependent recombination pathway. These paralogs form two complexes (Rad51C/Xrcc3 and Rad51B/C/D/
Xrcc2), which play roles in somatic recombination, DNA repair and chromosome stability. However, little is
known of their possible involvement in meiosis, due to the inviability of the corresponding knockout mice. We
have recently reported that the Arabidopsis homolog of one of these Rad51 paralogs (AtXrcc3) is involved in
DNA repair and meiotic recombination and present here Arabidopsis lines carrying mutations in three other
Rad51 paralogs (AtRad51B, AtRad51C and AtXrcc2). Disruption of any one of these paralogs confers
hypersensitivity to the DNA cross-linking agent Mitomycin C, but not to c-irradiation. Moreover, the atrad51c-1
mutant is the only one of these to show meiotic defects similar to those of the atxrcc3 mutant, and thus only
the Rad51C/Xrcc3 complex is required to achieve meiosis. These results support conservation of functions of
the Rad51 paralogs between vertebrates and plants and differing requirements for the Rad51 paralogs in
meiosis and DNA repair.
Keywords: DNA repair, meiosis, recombination, RAD51B, RAD51C, XRCC2.
Introduction
Homologous recombination (HR) and non-homologous
end-joining (NHEJ) are the two major pathways for DNA
double-strand break (DSB) repair (reviews by Dudas and
Chovanec, 2004; Lees-Miller and Meek, 2003). While NHEJ is
an error-prone pathway, frequently leading to deletions and/
or insertions, repair of DNA DSBs through HR generally
preserves the integrity of the genomic material. In addition,
HR events play an essential role in assuring proper meiotic
chromosomal disjunction and represent an essential mechanism to create genetic diversity. The proteins involved in
HR mechanisms in eukaryotic cells have been extensively
studied and mainly belong to the RAD52 epistasis group,
first identified in budding yeast (for review see Dudas and
Chovanec, 2004). Within this group, the sub-family of the
Rad51-like proteins has been the object of a considerable
interest as the finding that Rad51 is the homolog of the
bacterial recombinase RecA (Aboussekhra et al., 1992;
Shinohara et al., 1992). In addition to the highly conserved
recombinase Rad51, three Rad51-like proteins have been
identified in budding yeast: Dmc1 is a meiosis-specific
Rad51-like protein and is conserved in many eukaryotes,
ª 2004 Blackwell Publishing Ltd
while Rad55 and Rad57 are expressed ubiquitously and
seem to be specific to yeasts. All these Rad51-like proteins
play roles in DSBs repair and/or HR (for review see Dudas
and Chovanec, 2004).
As well as Rad51 and Dmc1, the genomes of vertebrates
encode five additional Rad51-like proteins, referred to as the
Rad51 paralogs: Rad51B (Albala et al., 1997; Cartwright
et al., 1998b; Rice et al., 1997), Rad51C (Dosanjh et al.,
1998), Rad51D (Cartwright et al., 1998b; Pittman et al.,
1998), Xrcc2 (Cartwright et al., 1998a; Liu et al., 1998) and
Xrcc3 (Liu et al., 1998; Tebbs et al., 1995). Two-hybrid and
immunoprecipitation experiments have shown that these
five Rad51 paralogs form two complexes: an heterodimer
composed of Rad51C and Xrcc3 (CX3) and an heterotetramer composed of Rad51B, Rad51C, Rad51D and Xrcc2
(BCDX2) (Liu et al., 2002; Masson et al., 2001; Miller et al.,
2002, 2004; Schild et al., 2000; Wiese et al., 2002). The
embryonic lethality observed in individual knockouts of
mouse RAD51B, RAD51D and XRCC2 shows that, as is the
case for Rad51, the Rad51 paralogs are required for animals
viability (Deans et al., 2000; Pittman and Schimenti, 2000;
533
534 Jean-Yves Bleuyard et al.
Shu et al., 1999; Tsuzuki et al., 1996). However, in contrast to
Rad51, knockouts of the Rad51 paralogs in chicken DT-40
cells do not lead to cell death, indicating that these proteins
are not required for vertebrate cells viability (Sonoda et al.,
1998; Takata et al., 2000, 2001). The numerous experiments
performed with mutant cells defective in the different Rad51
paralogs have shown that they all play roles in somatic
recombination, DNA repair and chromosome stability
(Brenneman et al., 2000; Cui et al., 1999; Deans et al., 2000,
2003; French et al., 2002; Godthelp et al., 2002; Johnson
et al., 1999; Liu et al., 1998; Mohindra et al., 2004; Pierce
et al., 1999; Takata et al., 2000, 2001; Tebbs et al., 1995).
However, the embryonic lethality of mutants defective in any
one of the Rad51 paralogs has complicated investigation of
their possible involvement in meiosis (Deans et al., 2000;
Pittman and Schimenti, 2000; Shu et al., 1999).
The absence of Rad51 foci following DNA damaging
treatment in cells defective in any one of the Rad51 paralogs
suggests a role in the initial steps of HR process (Bishop
et al., 1998; Godthelp et al., 2002; Liu, 2002; O’Regan et al.,
2001; Takata et al., 2000, 2001; Tarsounas et al., 2004). This
hypothesis is supported by several biochemical studies. In
vitro Rad51-dependent strand exchange reactions have
shown that a sub-complex composed of Rad51B and Rad51C
facilitates the assembly of the Rad51-ssDNA nucleoprotein
filament in the presence of RPA and thus has a mediator role
similar to that of the Rad55-Rad57 heterodimer in yeast (Lio
et al., 2003; Sigurdsson et al., 2001; Sung, 1997). The
Rad51C protein, the CX3 complex and a Rad51D/Xrcc2 subcomplex possess homologous pairing activities similar to
that of Rad51 (Kurumizaka et al., 2001, 2002; Lio et al., 2003).
Several recent studies however support a later role for the
Rad51 paralogs. In the absence of the Xrcc3 protein, both
conversion tract lengths and the frequency of discontinuous
tracts are increased (Brenneman et al., 2002). In vitro, the
Rad51B protein and BCDX2 complex preferentially bind
branched DNA strands, such as Holliday junctions (HJ)
(Yokoyama et al., 2003, 2004), and Rad51C and Xrcc3 play
roles in HJ resolution (Liu et al., 2004). Finally, the meiotic
defects observed in an Arabidopsis xrcc3 mutant suggest
that the Xrcc3 protein plays a post-synaptic role (Bleuyard
and White, 2004).
The genome of Arabidopsis thaliana codes for seven
Rad51-like proteins. In addition to the previously identified
Rad51, Dmc1, Rad51C and Xrcc3 Arabidopsis homologs
(Doutriaux et al., 1998; Klimyuk and Jones, 1997; Osakabe
et al., 2002; Sato et al., 1995; Urban et al., 1996), the
construction of a phylogenetic tree has allowed us to identify
the Arabidopsis homologs of Rad51B, Rad51D and Xrcc2,
showing that Arabidopsis has the same family of Rad51-like
proteins as vertebrates. To investigate the conservation of
functions of the Rad51 paralogs between vertebrates and
plants, we identified mutants defective for three of the four
other Arabidopsis Rad51 paralogs. Absence of any one of
the Rad51B (AtRad51B), Rad51C (AtRad51C) and Xrcc2
(AtXrcc2) Arabidopsis homologs confers hypersensitivity
to the DNA cross-linking agent Mitomycin C (MMC), but not
to ionizing radiation. Furthermore, only mutants impaired
for the AtRAD51C gene show meiotic defects similar to those
of atxrcc3 mutants. These results clearly show that the role
of the Rad51 paralogs in DNA repair is conserved between
vertebrates and plants and that only AtRad51C and AtXrcc3
(Bleuyard and White, 2004), which together form the CX3
complex, play essential roles in meiosis in Arabidopsis, and
very probably in other higher eukaryotes.
Results
Identification and molecular characterization of Arabidopsis
mutants defective for the Rad51 paralogs
The genome of the model plant A. thaliana codes for seven
proteins of the Rad51 recombinase family. Previous studies
have reported the identification of Rad51 (AT5G20850), Dmc1
(AT3G22880), Rad51C (AT2G45280) and Xrcc3 (AT5G57450)
homologs (Doutriaux et al., 1998; Klimyuk and Jones, 1997;
Osakabe et al., 2002; Sato et al., 1995; Urban et al., 1996). To
define the relationships existing between Arabidopsis
Rad51-like proteins and Rad51-like proteins from other model
species, we performed a phylogenetic analysis with 23
Rad51-like proteins from Arabidopsis, Drosophila, human
and Saccharomyces (Figure 1a). The resulting phylogenetic
tree clearly shows that Arabidopsis has a single homolog for
each of the five Rad51 paralogs first identified in vertebrates
(AT2G28560, AT1G07745 and AT5G64520 products correspond respectively to the AtRad51B, AtRad51D and AtXrcc2
proteins). The A-type nucleotide binding consensus amino
acid sequence [G/A]XXXXGK[S/T] (Walker A motif) is
Figure 1. Arabidopsis genome encodes homologs of the five Rad51 paralogs.
(a) The dendrogram illustrates the sequence relationships among 23 Rad51-like proteins in Saccharomyces cerevisiae (Sc), Drosophila melanogaster (Dm), Human
(Hs) and Arabidopsis (At). The branch lengths are proportional to the sequence divergence. Numbers along branches are bootstrap values (1000 replicates)
calculated using the PHYLIP package. The scale represents 0.1 substitutions per site.
(b) The core-conserved sequences were aligned using the ClustalX program. The amino acid sequences of the proteins are shown in the single-letter code. Gaps are
indicated by dashes. Conserved amino acids are black shaded and similar amino acids are gray shaded. The positions of the amino acids in each protein are shown
at left and right. Black boxes indicate the positions of Walker motifs A and B.
Accession numbers for 23 deduced amino acids sequences used in this analysis are as follows: AtDmc1 (AAC49617), AtRad51 (CAA04529), AtRad51B (NP_180423),
AtRad51Ca (BAB64343), AtRad51D (NP_172254), AtXrcc2 (NP_851268), AtXrcc3 (BAB64342), DmCG2412 (NP_610466), DmCG6318 (NP_573302), DmRad51
(BAA04580), DmSpn-B (NP_476740), DmSpn-D (AAP13056), HsDmc1(BAA10970), HsRad51 (BAA02962), HsRad51B (AAC39723), HsRad51C (AAC39604), HsRad51D
(AAC39719), HsXrcc2 (CAA70065), HsXrcc3 (AAC05368), ScDmc1 (AAA34571), ScRad51 (BAA00913), ScRad55 (AAA19688), ScRad57 (AAA34950).
ª Blackwell Publishing Ltd, The Plant Journal, (2004), 41, 533–545
Roles of Arabidopsis Rad51 paralogs 535
(a)
(b)
Walker A motif
(G/A)XXXXGK(S/T)
ª Blackwell Publishing Ltd, The Plant Journal, (2004), 41, 533–545
536 Jean-Yves Bleuyard et al.
Stock Centre. A sterility phenotype was observed in
plants homozygous for atrad51c-1, thus the atrad51c-1
T-DNA insertion was kept at the heterozygous state and
atrad51c-1 mutant plants were identified by PCR in the
progeny of ATRAD51C-1þ/) plants.
To characterize the insertions molecularly, the T-DNA
junctions were amplified and the PCR products sequenced
(Figure 2). The atrad51b-1 allele T-DNA is inserted in intron 4
and is associated with a deletion of 14 bp, including the first
4 bp of exon 5 (Figure 2a). The atrad51c-1 allele T-DNA is
inserted in exon 3 and is associated with a deletion of 47 bp,
containing the end of exon 3 and the beginning of intron 3
(Figure 2b). The atxrcc2-1 allele T-DNA is inserted in intron 5,
3 bp after the end of exon 5, and is associated with a deletion
of 1 bp (Figure 2c). These three T-DNA insertions are
surrounded by two left borders in opposite orientations,
designated as LB1 and LB2 (Figure 2 diagrams). In the case
conserved in the Arabidopsis Rad51 paralogs, except for
AtXrcc2, while the B-type binding consensus amino acids
sequence hhhhD (Walker B motif) is conserved in the five
Arabidopsis Rad51 paralogs (Figure 1b) (Higgins et al., 1985;
Walker et al., 1982).
In order to investigate the roles of the Arabidopsis
Rad51 paralogs in meiosis and the cellular responses to
DNA damage, we searched for mutants in public T-DNA
insertion line collections. Three lines carrying T-DNA
insertions in the ATRAD51B (Salk_024755), ATRAD51C
(Salk_021960) and ATXRCC2 (Salk_029106) coding
sequences were found in the SIGnAL T-DNA express
database (Alonso et al., 2003) and we have named the
corresponding alleles atrad51b-1, atrad51c-1 and atxrcc2-1
respectively. Plants homozygous for atrad51b-1, atrad51c-1
and atxrcc2-1 T-DNA insertions were identified by PCR in
the T3 seeds provided by the Nottingham Arabidopsis
T-DNA
AtRAD51B
Filler DNA 70 b
T-DNA
LB1
LB2
(+949)
(+964)
ATG (+1)
AtRAD51B LB1
LB2
Filler DNA
+945
+965
STOP (+2244)
∆ 14 b
Wild Type
{
(b)
3′-UTR
o548 o547
o549
500 b
o548/o549
o546/o547
Control
AtRAD51C
T-DNA
LB1
AtRAD51C
LB2
(+688)
LB1
T-DNA
LB2
AtRAD51C
AGGGATGT aaacaa...aagcgt CAATTTGAGAT
(+734)
ATG (+1)
atrad51b-1
STOP (+1939) A(n)
∆ 47 b
+685
+740
Wild Type
{
α
β
atrad51c-1
{
5′-UTR
o546
AtRAD51B
TTCTTTC aactta...tcc aag...tactgc CAAGAAC
{
(a)
o453/o454
5′-UTR
o450
3′-UTR
o454 STOP
o554 o453
A(n)
(+1553)
o450/o554
Control
500 b
(c)
AtXRCC2
LB1
T-DNA
AtXRCC2
LB2
(+1499)
(+1501)
LB1
T-DNA
LB2
+1495
∆1b
1
+1505
STOP (+2136)
Wild Type
2
{
3′-UTR
atxrcc2-1
{
ATG (+1)
AtXRCC2
AATCGGTTA aacaaa...gtcaat TTGGTTAAT
o552/o553
o550
o551
o552
o553
STOP (+2174)
500 b
o550/o551
Control
Figure 2. Molecular characterization of Arabidopsis Rad51 paralog T-DNA insertion mutants.
The diagrams show the genomic structure of ATRAD51B (a), ATRAD51C (b) and ATXRCC2 (c) loci, with the sites of T-DNA integrations. The gray boxes represent the
exons and the white boxes indicate 5¢ and 3¢ UTRs. The position of the T-DNA insertions is indicated by triangles. Sequences of the T-DNA junctions and RT-PCR
detection of the ATRAD51B (a), ATRAD51C (b) and ATXRCC2 (c) transcripts are presented at the right of each panel. The white box indicates the T-DNA insertion. LB1
and LB2 indicate the two left borders surrounding the T-DNA insertions, and arrows indicate their respective orientation. Micro-homologies between the T-DNA
ends and the genomic sequence are indicated in gray. The positions of the primers used for the RT-PCR detection of ATRAD51B (a), ATRAD51C (b) and ATXRCC2 (c)
transcripts are represented on the diagrams. Amplification of the adenosin phosphoribosyl transferase (APT1) transcript has been used as a control for reverse
transcription. The numbers represent positions relative to the start codon.
ª Blackwell Publishing Ltd, The Plant Journal, (2004), 41, 533–545
Roles of Arabidopsis Rad51 paralogs 537
of atrad51b-1, sequencing showed that the T-DNA insertion
is followed by an insertion of 70 bp of filler DNA (Figure 2b).
Plants homozygous for the T-DNA insertions were selected by PCR, and semiquantitative RT-PCR analysis performed to assess the presence of ATRAD51B, ATRAD51C
and ATXRCC2 transcripts in total RNA isolated from wild
type and mutant flower buds. ATRAD51B, ATRAD51C and
ATXRCC2 transcripts were detected in the wild type. In
contrast, the ATRAD51C mRNA was not detectable in
atrad51C-1 plants, and only truncated ATRAD51B and
ATXRCC2 mRNAs were detected in atrad51b-1 and atxrcc21 plants respectively (Figure 2). The T-DNA insertions in the
atrad51b-1, atrad51c-1 and atxrcc2-1 mutants thus prevent
the production of the full-length mRNAs of ATRAD51B,
ATRAD51C and ATXRCC2 respectively. Furthermore, the
complete absence of ATRAD51C transcript in atrad51c-1
plants shows that it is a null allele, while atrad51b-1 and
atxrcc2-1 may potentially encode truncated proteins.
for atrad51b-1, atrad51c-1 and atxrcc2-1 mutant plants, no
significant difference was found when compared with the
wild type (unpublished data).
Examples of non-sensitive and sensitive plants and dose–
response curves for the percentage of sensitive plants are
shown in Figure 3. atrad51b-1 and atxrcc2-1 mutant plants
clearly show hypersensitivity to MMC. Due to the sterility of
the atrad51c-1 mutant plants, the MMC hypersensitivity
of atrad51c-1 plants was assayed on the progeny of
ATRAD51C-1þ/) plants, one quarter of which are mutants
(Figure 3c). That the sensitive plants were the atrad51c-1
mutants was verified by PCR genotyping in one experiment
and all 27 sensitive plants scored were mutants. The
AtRad51B, AtRad51C and AtXrcc2 proteins are thus required
to repair DNA cross-links but are not essential for the repair
of DSBs, presumably due to the repair of DSBs by NHEJ.
atrad51b-1, atrad51c-1 and atxrcc2-1 mutant plants are
hypersensitive to Mitomycin C, but not to c-irradiation
In the progeny of self-fertilized heterozygous ATRAD51C-1þ/)
plants (41 plants screened): 10 (24.4%) were sterile and 31
(75.6%) were fertile, corresponding well to the 3:1 segregation expected for a single Mendelian locus (chi-squared, 1
d.f. ¼ 0.008). Genotyping confirmed that the sterile plants
were exclusively homozygous atrad51c-1 mutants. atrad51c1 plants produce atrophied siliques, which are devoid of any
seed, while heterozygotes for atrad51c-1 and homozygotes
for atrad51b-1 or atxrcc2-1 do not show any fertility defects
and all mutant plants grew normally with normal vegetative
development (data not shown).
We investigated the origin of the sterility of atrad51c-1
plants and confirmed the absence of such defects in
atrad51b-1 and atxrcc2-1 mutant plants (Figure 4). Anthers
were dissected from wild type and atrad51b-1, atrad51c-1
and atxrcc2-1 mutant flower buds and stained as described
by Alexander (1969) to assess pollen grain viability (Figure 4a–d). While none of the observed atrad51c-1 anthers
contained any viable (red-purple) pollen grains, anthers
from atrad51b-1 and atxrcc2-1 mutant plants could not be
differentiated from those of the wild type in terms of the
number of viable pollen grains produced. To assess female
gametophytic defects, we monitored post-meiotic nuclear
divisions during embryo sac development. In the wild type, a
single megaspore mother cell differentiates in each ovule
and undergoes meiosis (Figure 4e). Meiosis in female
tissues is followed by degeneration of three of the four
meiotic products, to preserve a single functional megaspore
(Figure 4f). The functional megaspore nucleus then undergoes three divisions to produce the eight-nuclei embryo sac,
which is the mature female gametophyte (Figure 4g,h). In
atrad51c-1 ovules, the megaspore mother cell could not be
differentiated from the wild type (Figure 4i), but gametophytic development is blocked after meiosis. The presence
Studies performed with Chinese Hamster Ovary and DT40
(Chicken B-Lymphocyte) cell lines have shown that mutations of the different Rad51 paralogs confer moderate sensitivity to DNA DSB inducing agents such as c-rays and
hypersensitivity to DNA cross-linking agents such as MMC
(Godthelp et al., 2002; Liu et al., 1998; Takata et al., 2001).
Our recent work with an Arabidopsis atxrcc3 mutant showed
that plants lacking the AtXrcc3 protein were slightly sensitive to bleomycin, a c-ray mimetic agent, and much more
sensitive to MMC, suggesting a conservation of the role of
Arabidopsis Rad51 paralogs in response to DNA damage
(Bleuyard and White, 2004).
To confirm the involvement of AtRad51B, AtRad51C and
AtXrcc2 proteins in the cellular response to DNA damage,
seeds from wild type, atrad51b-1 and atxrcc2-1 plants and
self-fertilized heterozygous ATRAD51C-1þ/) plants were
either irradiated with c-rays and sown on germination
medium, or sown on plates containing germination medium
and increasing doses of MMC. After 2 weeks, plants were
scored for c-irradiation or MMC sensitivity. In the absence of
treatment, most plants developed at least four true leaves (in
addition to the cotyledons), thus plants with three true
leaves or less were considered to be sensitive to c-rays or
MMC. Previous studies on DNA repair defective mutants
have shown that a dose of 100 Grays (Gy) allow discrimination between atku80, atlig4 and atatm radiosensitive
mutants and wild-type plants (Friesner and Britt, 2003;
Garcia et al., 2003). In our experiments, no significant
difference was found between wild type and atrad51b-1,
atrad51c-1 and atxrcc2-1 mutant plants, even at a dose of
200 Gy (data not shown). Similar c-irradiation assays were
performed on the seedlings of an ATXRCC3þ/) plant and, as
ª Blackwell Publishing Ltd, The Plant Journal, (2004), 41, 533–545
atrad51c-1 mutants, but not atrad51b-1 or atxrcc2-1, are
sterile
(b)
Wild Type
atrad51b-1
atxrcc2-1
90
80
% of sensitive plants
70
60
50
40
30
20
atrad51b-1
Wild Type
(a)
}
538 Jean-Yves Bleuyard et al.
10
0
0 µM
10 µM
20 µM
40 µM
(c)
30
Wild Type
AtRad51C-1+/-
25
% of sensitive plants
atxrad51c-1
atxrcc2-1
Mitomycin C concentration
20
15
10
5
0
0 µM
10 µM
20 µM
40 µM
Mitomycin C concentration
Figure 3. Mutations in Arabidopsis Rad51 paralogs confer hypersensitivity to the DNA cross-linking agent, Mitomycin C (MMC).
(a) Comparison of non-sensitive and sensitive plants at 40 lm MMC. In the absence of MMC, all plants develop at least four true leaves (excluding the cotyledons),
thus plants with three leaves or less were considered as sensitive. Scale bars ¼ 1 cm.
(b, c) The percentage of sensitive plants was used to produce an MMC dose–response curve. Values represent three replicates, each replicate containing an average
of 100 plants per dose. Error bars are # 1 standard deviation.
of a single degenerative cell, which persists throughout
embryo sac development, suggests that the megaspore
mother cell is unable to properly achieve meiosis in
atrad51c-1 ovules (Figure 4j–l). In some cases, one of the
meiotic products is preserved, but such products were never
able to proceed further than the first post-meiotic division
(data not shown). The AtRad51C protein is thus required to
complete both male and female gametogenesis, as is the
case for AtXrcc3. In contrast, AtRad51B and AtXrcc2 are not
required to achieve gametogenesis.
The AtRad51C protein is required to ensure chromosome
stability during meiosis
Meiotic progression in wild type, atrad51b-1, atrad51c-1 and
atxrcc2-1 pollen mother cells (PMCs) was examined by
fluorescence microscopy after DAPI staining of chromosomes. In the wild type, the 10 Arabidopsis chromosomes
condense during meiotic prophase I (Figure 5a) and can be
seen as five bivalents (corresponding to paired homologous
chromosomes) in metaphase I (Figure 5b). Homologous
chromosomes then separate from each other and migrate to
the opposite poles of the cell in anaphase I (Figure 5c). The
second meiotic division starts with the alignment of chromosomes in metaphase II (Figure 5d), followed by separation of sister chromatids in anaphase II (Figure 5e).
Telophase II ensues, chromosomes decondense (Figure 5f)
and cytoplasm is partitioned to produce a tetrad containing
four haploid microspores, which will differentiate into
mature pollen grains.
In atrad51c-1 mutant PMCs, prophase I proceeds normally
up to pachytene (Figure 6a), but in place of five expected
bivalents, a varying number of entangled chromosome
fragments can be seen in metaphase I figures (Figure 6b,c).
This chromosome fragmentation becomes more apparent in
anaphase I, with random segregation of chromosome
fragments and the presence of bridges, indicating chromosome fusion events and the presence of dicentric chromosomes (Figure 6d,e). In metaphase II, most of the visible
chromosome fragments are aligned on the spindle,
with some fragments scattered throughout the cytoplasm
(Figure 6f,g). Anaphase II separates several groups of
chromosome fragments (Figure 6h,i) and is followed by
chromosome decondensation in telophase II (Figure 6j).
ª Blackwell Publishing Ltd, The Plant Journal, (2004), 41, 533–545
Roles of Arabidopsis Rad51 paralogs 539
(a)
(b)
(c)
(d)
(e)
(f)
(g)
(h)
(i)
(j)
(k)
(l)
Figure 4. AtRad51C, but not AtRad51B and AtXrcc2, is required for gametophytic development.
(a–d) Alexander staining was applied to anthers from wild type (a), atrad51b-1 (b), atrad51c-1 (c) and atxrcc2-1 (d) plants to discriminate viable (stained in red-purple)
and dead (stained in green) pollen grains.
Wild type (e–h) and atrad51c-1 (i, j) ovules were cleared to observed embryo sac development. (e, i) megaspore mother cell; (f) functional megaspore; (g) two-nuclei
stage; (h) four-nuclei stage; (j–l) degenerative cell observed at different developmental stages.
The observation of bridges in anaphase II figures suggests
that fused chromosome fragments are still present at this
stage and that chromosome fragmentation continues to the
end of anaphase II. Meiosis in atrad51c-1 PMCs finally gives
rise to ‘polyads’, containing variable numbers of products
with variable DNA contents. In contrast, meiotic progression
in the atrad51b-1 and atxrcc2-1 mutants is normal. These
two Rad51 paralogs are thus not required to ensure
chromosome stability during meiosis (data not shown).
Taken together, these results indicate that, as previously
shown for AtXrcc3, the AtRad51C protein is required to
achieve meiosis. The chromosome fragmentation observed
in atrad51c-1 meiosis presumably resulting from mis- or
un-repaired meiotic double-strand breaks, in agreement
with a role of AtRad51C in HJ resolution (Liu et al., 2004;
Symington and Holloman, 2004).
Discussion
We report here the identification and characterization of three
Arabidopsis mutants, defective for the Rad51 paralogs
ª Blackwell Publishing Ltd, The Plant Journal, (2004), 41, 533–545
AtRad51B, AtRad51C and AtXrcc2 respectively. The first
striking result from this study is that, in contrast to vertebrates, mutations in any one of these Arabidopsis Rad51
paralogs do not impair plant viability (Deans et al., 2000;
Pittman and Schimenti, 2000; Shu et al., 1999). Studies carried out with vertebrate cell lines defective for the Rad51
paralogs have shown that these proteins are involved in DNA
repair, with mutant cell lines showing relatively moderate
sensitivity to DSB inducing agents such as c-rays and high
sensitivity to chemicals inducing the formation of interstrand
cross-links (ICLs) (Godthelp et al., 2002; Liu et al., 1998;
Takata et al., 2000, 2001). However, Drosophila xrcc3 (spn-B)
and rad51c (spn-D) mutants do not present DNA repair
defects and this role of the Rad51 paralogs is thus not selfevident (Abdu et al., 2003). In previous work, we have shown
that cultured cells defective for the Arabidopsis XRCC3
homologue have increased sensitivity to the radiomimetic
agent Bleomycin (Bleuyard and White, 2004). Here we report
that mutations in either ATRAD51B, ATRAD51C or ATXRCC2
genes did not increase sensitivity of plants to c-rays. Similar
results were also obtained with ATXRCC3-defective plants
540 Jean-Yves Bleuyard et al.
(a)
(b)
(a)
(b)
(c)
(d)
(c)
(d)
(e)
(f)
(e)
(f)
(g)
(h)
(i)
(j)
Figure 5. DAPI staining of wild-type meiotic chromosomes.
(a) Prophase I, (b) metaphase I, (c) anaphase I, (d) metaphase II, (e) anaphase II
and (f) telophase II. Arrowheads indicate bivalents (b) or chromosomes (c–e).
Scale bars ¼ 10 lm. White dotted lines have been added to clearly indicate
the separate sets of chromosomes.
(unpublished data), and we ascribe this difference to the different effects of chronic exposure of cultured cells to Bleomycin compared with the DNA breakage produced by the
acute c-irradiation. Our data thus indicate that mutations in
four different Arabidopsis Rad51 paralogs confer little or no
sensitivity to DSB-inducing agents. In contrast, the hypersensitivity to MMC observed in atrad51b-1, atrad51c-1 and
atrad51-1 mutant plants confirms the important role of Arabidopsis Rad51 paralogs in the repair of ICLs. Taken together,
our data strongly support functional conservation of the
Rad51 paralogs in DNA repair between vertebrates and plants
(this study; Bleuyard and White, 2004).
Meiotic function of the CX3 complex
Yeast rad51 mutants are unable to repair meiosis-specific
DSBs and their ability to produce viable spore is dramatically
reduced (Cao et al., 1990; Shinohara et al., 1992). Similarly,
Drosophila and Caenorhabditis mutants defective for Rad51
show defects in chromosome morphology during oogenesis
Figure 6. Meiosis is severely disturbed in atrad51c-1 pollen mother cells.
(a) Prophase I, (b, c) metaphase I, (d, e) anaphase I, (f, g) metaphase II, (h, i)
anaphase II and (j) telophase II. Arrowheads indicate bridges. Scale
bars ¼ 10 lm.
and thus, reduced fertility (Rinaldo et al., 2002; Staeva-Vieira
et al., 2003). In yeast and Drosophila, the Rad51 paralogs
share the meiotic defects observed in rad51 mutants. Yeast
ª Blackwell Publishing Ltd, The Plant Journal, (2004), 41, 533–545
Roles of Arabidopsis Rad51 paralogs 541
rad55 and rad57 mutants are unable to repair meiotic DSBs
and have reduced spore viability and oogenesis and fertility
are severely disturbed in Drosophila spnB (xrcc3) and spnD
(rad51c) mutants (Abdu et al., 2003; Game and Mortimer,
1974; Ghabrial and Schupbach, 1999; Ghabrial et al., 1998;
Schwacha and Kleckner, 1997).
Similarly, Arabidopsis AtRad51 and AtXrcc3 proteins are
both required to achieve meiosis and repair meiotic DSBs
(Bleuyard and White, 2004; Li et al., 2004). In contrast to
atxrcc3 mutants, the absence of meiotic homologous chromosome synapsis in atrad51-1 mutants shows that AtRad51
and AtXrcc3 proteins have distinct roles in meiosis, AtRad51
acting prior to AtXrcc3. Osakabe et al. (2002) have shown
that AtXrcc3 and AtRad51C can interact together and thus
presumably form a heterodimer in vivo, as is the case in
vertebrates. In this study, we show that atrad51c-1 mutant
plants present meiotic defects similar to those observed in
the atxrcc3 mutant plants (Figure 6), confirming the meiotic
role of the Arabidopsis CX3 complex. Mutations in the
ATSPO11-1 gene, the Arabidopsis SPO11 homolog, dramatically reduce meiotic HR and homologous chromosome
synapsis (Grelon et al., 2001). Absence of Spo11 activity in
the atxrcc3 mutant suppresses the chromosome fragmentation in half the meiotic cells and delays fragmentation to
the second meiotic division in the other half (Bleuyard et al.,
2004), raising the possibility that the meiosis II defects derive
from unresolved sister chromatid HR events. In addition, Liu
et al. (2004) reported that Rad51C plays a major role in HJ
branch migration and resolution activities, while Xrcc3 is
involved in HJ resolution. Taken together, these findings
strongly suggest that the CX3 complex is involved in the
Spo11 meiotic recombination pathway, presumably in the
HJ resolution.
Meiotic requirement for the Rad51 paralog proteins
Neither atrad51b-1 nor atxrcc2-1 mutants present visible
meiotic defects. A trivial explanation for this would be that
putative truncated proteins produced from incomplete
transcripts of these alleles are able to carry out the meiotic
functions of the native proteins. However, the atrad51b-1
and atxrcc2-1 mutant plants are hypersensitive to DNA
cross-linking agents, indicating defects in homologous
recombinational repair of ICLs (Figure 3). Furthermore, the
studies performed to identify interaction domains within
the Rad51 paralogs have shown that any deletion in either
the N-terminal or the C-terminal parts of the proteins
eliminate protein–protein interactions (Dosanjh et al., 1998;
Kurumizaka et al., 2003; Miller et al., 2004). This finding led
the authors to suggest that even a very short deletion can
severely disturb the folding of the Rad51 paralogs (Miller
et al., 2004). It thus appears very unlikely that putative
truncated proteins produced in either atrad51b-1 or
atxrcc2-1 would be functional.
ª Blackwell Publishing Ltd, The Plant Journal, (2004), 41, 533–545
Our finding that the AtRad51B and AtXrcc2 proteins are
not required to achieve meiosis shows that only the CX3
complex plays an essential role for the repair of AtSpo11-1
induced DSBs. A recent study by Liu et al. (2004) has shown
that the mammalian Rad51C and Xrcc3 proteins are both
involved in HJ resolution, while the other Rad51 paralogs
are implicated in branch migration processes. These results
strongly support the idea that the CX3 and BCDX2 (or at least
the Rad51B, Rad51C and Xrcc2 proteins) complexes have
distinct roles in HR mechanisms and hence in meiotic
recombination.
In the absence of the AtXrcc3 protein, meiosis is severely
disturbed (Bleuyard and White, 2004), indicating that the CX3
complex has an essential function during meiosis and that
this function cannot be complemented by the BCDX2 complex. In addition, one might expect that mutations in the
ATRAD51C gene lead to more critical defects, due to the
disruption of both CX3 and BCDX2 complexes. However,
atrad51c-1 and atxrcc3 mutants present very similar defects
(this study; Bleuyard and White, 2004), supporting the
existence of an essential role for the CX3 complex during
meiosis, while the BCDX2 complex is dispensable. At this
point we cannot however exclude the possibility that the
BCDX2 complex plays a non-essential role in meiotic recombination processes in contrast to the essential role of the CX3
complex (resolvase activity?), absence of which leads to
chromosome fragmentation in the first meiotic prophase. We
note that, although very probable, we cannot be certain that
the Arabidopsis AtRad51B, AtRad51C, AtRad51D and AtXrcc2
proteins form a BCDX2 complex in vivo, as this has not yet
been formally tested. Our results show the absence of
essential meiotic roles for the AtRad51B and AtXrcc2 proteins
in Arabidopsis, but that this conclusion also applies to the
BCDX2 complex must remain tentative until formal demonstration of the existence of the complex in this plant.
In vertebrates, the embryonic lethality of knockout
animals has greatly complicated studies of the meiotic
roles of Rad51-like proteins (Deans et al., 2000; Pittman
and Schimenti, 2000; Shu et al., 1999; Tsuzuki et al., 1996).
In contrast to other model organisms, Arabidopsis carries
the same range of Rad51-like proteins as vertebrates and
mutants defective for Rad51 or any of the Rad51 paralogs
are viable (this study; Bleuyard and White, 2004; Li et al.,
2004). With the recent availability of public, sequencetagged mutant collections, Arabidopsis thus shows great
promise as a model to study the meiotic functions of
proteins involved in recombination.
Experimental procedures
Phylogenetic analysis
Sequence alignments were carried out using the ClustalX software
package (Version 1.83, Thompson et al., 1997). Evolutionary
542 Jean-Yves Bleuyard et al.
distances were calculated using the Henifoff/Tillier PMB (Probability
Matrix from Blocks, Veerassamy et al., 2003) distance method of the
Protdist program (PHYLIP package version 3.6, Felsenstein, 1989). The
coefficient of variation of the c-distribution (to incorporate rate
heterogeneity) was obtained by pre-analyzing the data with the TreePuzzle program (Version 5.0, Strimmer and von Haeseler, 1997). The
phylogenetic tree was inferred using the unweighted pair group
method with arithmetic mean method in the neighbor program
(PHYLIP package version 3.6, Felsenstein, 1989). The tree was displayed using TreeView program (version 1.6.6). Consensus trees
were inferred using the Consense program (PHYLIP package version
3.6, Felsenstein, 1989) and the significance of the various phylogenetic lineages was assessed by bootstrap analyses (Hedges, 1992).
Plant material, growth conditions and mutant screening
All Arabidopsis plants used in this work were of ecotype Columbia
(Col0). A. thaliana seeds were sown directly into damp compost or
solid germination medium and under white light (16 h light/8 h
dark) as previously described by Gallego et al. (2001).
The atrad51b-1 (Salk_024755), atrad51c-1 (Salk_021960) and
atxrcc2-1 (Salk_029106) T-DNA insertion lines were found in the
public T-DNA Express database established by the Salk Institute
Genomic Analysis Laboratory accessible from the SIGnAL website
at http://signal.salk.edu (Alonso et al., 2003).
Plants heterozygous and/or homozygous for the atrad51b-1,
atrad51c-1 or atxrcc2-1 T-DNA insertion loci were identified by a
PCR genotyping assay. The following primer combinations were
used to amplify the different loci: the wild type ATRAD51B locus,
o519 (5¢-GAGTTAGTTGGTCCTCCTGG-3¢) and o520 (5¢-AAATTCAGCAAGCGATCTGG-3¢); the atrad51b-1 mutant locus, o519
and o405 (5¢-TGGTTCACGTAGTGGGCCATCG-3¢); the wild type
ATRAD51C locus, o527 (5¢-TTTTGTGACTAAACAAAGGAGC-3¢)
and o528 (5¢-ACCTCCACTTAAGCTAGTCAAGG-3¢); the atrad51c-1
mutant locus, o527 and o405; the wild type ATXRCC2 locus, o523
(5¢-TAGTCCAATGTAACTTTCGCAG-3¢) and o524 (5¢-GTCACGAGACAATGACAATACC-3¢); the atxrcc2-1 mutant locus, o523 and o405.
atrad51c-1 mutant plants identification was confirmed based on
their sterility phenotype.
Sequencing of T-DNA insertion sites
The following primer combinations were used to amplify DNA
flanking the T-DNA: atrad51b-1 LB1 left border, o519 and o405;
atrad51b-1 LB2 left border, o520 and o405; atrad51c-1 LB1 left border, o527 and o405; atrad51c-1 LB2 left border, o528 and o405; the
atxrcc2-1 LB1 left border, o523 and o405; the atxrcc2-1 LB2 left
border, o524 and o405.
The PCR products were then purified on a QIAquick column
(Qiagen, Courtaboeuf, France) and directly sequenced. Sequence
reaction were performed using one of the primers used for
amplification and the CEQ DTCS Quick Start Kit (Beckman Coulter,
Fullerton, CA, USA), and analyzed on a CEQ 2000 DNA Analysis
System (Beckman Coulter).
mix (Roche), 1 mM of each deoxyribonucleotide triphosphate, and
20 units of RNasin ribonuclease inhibitor (Promega, Charbonnieres, France). PCR was performed in 25 ll reaction mixtures
containing 2 ll of RT reaction mixture, 1 unit of HotStarTaq DNA
polymerase (Qiagen), 2.5 mM MgCl2, 100 lM of each deoxyribonucleotide triphosphate, and 0.4 lM of gene-specific primers.
The gene-specific primers were: o548 (5¢-TTTCCAGTAGCTTATGGAGG-3¢) and o549 (5¢-ATATGCCAACCCAACTGAGG-3¢), or o546
(5¢-AGTGAAGCTACTTCTCCACC-3¢) and o547 (5¢-CCGGAAAGCTTTCCAGTCCC-3¢) for ATRAD51B; o453 (5¢-CTTGATAACATTTTGGGCGG-3¢) and o454 (5¢-CAAGATGATTGACCAATGCG-3¢), or
o450 (5¢-ATGATTTCATTTGGGCGGCG-3¢) and o554 (5¢-TAATACGCGGCAAAGACTCC-3¢) for ATRAD51C; o552 (5¢-GCATTGGTGCTTTTCACTGG-3¢) and o553 (5¢-ATTCACGAAATGGAGGTTGC-3¢), or
o550 (5¢-GAAGCAGATGTTATCAAGGG-3¢) and o551 (5¢-CCATGCTCCATTTCCTAACC-3¢) for ATXRCC2. The initial denaturation was
performed at 95!C for 15 min, then amplification was performed for
45 cycles with a denaturation time of 30 secec at 94!C, followed by
annealing for 30 sec at 58!C and extension for 45 sec at 72!C. The
APT1 (adenine phosphorybosyl transferase) transcript has been
used as a control for reverse transcription (Moffatt et al., 1994). The
gene-specific primers were apt1 (5¢-TCCCAGAATCGCTAAGATTGC3¢) and apt2 (5¢-CCTTTCCCTTAA-GCTCTG-3¢). The initial denaturation was performed at 95!C for 15 min, then amplification was
performed for 35 cycles with a denaturation time of 30 sec at 94!C,
followed by annealing for 30 sec at 52!C and extension for 45 sec at
72!C.
Mitomycin C and c-irradiation assays
Col0, atrad51b-1, atrad51c-1 and atxrcc2-1 seeds were surfacesterilized with 7% calcium hypochlorite solution (w/v).
For MMC assays, seeds were sown on plates containing
fresh solid germination medium with different concentrations of
MMC (Sigma no. M-0503, Sigma, Lyon, France). The plates were
then incubated for 2 weeks (23!C, 16 h light), and resistance or
sensitivity was scored by the number of true leaves (excluding
the cotyledons) per plant.
For c-irradiation, surface-sterilized seeds were kept in sterile
water at 4!C for approximately 24 h. Then seeds were exposed to
50, 100 or 200 Gy (9.12 Gy min)1) from a 137Cs source (CIS Bio
International, Gif sur Yvette, France) and sown on plates containing
fresh solid germination medium. The plates were then incubated for
2 weeks (23!C, 16 h light), and resistance or sensitivity was scored
as for the MMS treatment.
Light and fluorescence microscopy
Cytological observations of Alexander-stained anthers, embryo sac
development and meiotic chromosomes were conducted as previously described (Bleuyard and White, 2004). Images were captured
on a Zeiss Axioplan 2 Imaging microscope with a Zeiss Axiocam
HRc video camera (Zeiss, Le Pecq, France) and enhanced using
Adobe Photoshop 6 software.
Semiquantitative RT-PCR
Acknowledgements
For semiquantitative RT-PCR, total RNAs extracted from flower
buds were treated with RNase-free DNase I (Roche, Meylan,
France). One microgram of DNA-free total RNA was reverse
transcribed in 20 ll of reaction mixture containing 50 units of
Expand Reverse Transcriptase (Roche), 1X random hexanucleotide
We thank members of BIOMOVE for their help and discussions and
the Salk Institute Genomic Analysis Laboratory for providing the
sequence-indexed Arabidopsis T-DNA insertion mutants. We also
thank Hong Ma and Bernd Reiss for communicating their data to us
before publication.
ª Blackwell Publishing Ltd, The Plant Journal, (2004), 41, 533–545
Roles of Arabidopsis Rad51 paralogs 543
This work was partly financed by a European Union research
grant (QLG2-CT-2001-01397), the Centre National de la Recherche
Scientifique and the Université Blaise Pascal.
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