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MOLECULAR AND CELLULAR BIOLOGY, Nov. 1998, p. 6423–6429
0270-7306/98/$04.0010
Copyright © 1998, American Society for Microbiology. All Rights Reserved.
Vol. 18, No. 11
Targeted Inactivation of Mouse RAD52 Reduces Homologous
Recombination but Not Resistance to Ionizing Radiation
TONNIE RIJKERS,1 JODY VAN DEN OUWELAND,1† BRUNO MOROLLI,1 ANTON G. ROLINK,2
WILLY M. BAARENDS,3 PETRA P. H. VAN SLOUN,1 PAUL H. M. LOHMAN,1 AND
ALBERT PASTINK1*
MGC-Department of Radiation Genetics and Chemical Mutagenesis, Leiden University Medical Center, Leiden,1 and
Department of Endocrinology and Reproduction, Faculty of Medicine and Health Sciences, Erasmus University,
Rotterdam,3 The Netherlands, and Basel Institute for Immunology, Basel, Switzerland2
The RAD52 epistasis group is required for recombinational repair of double-strand breaks (DSBs) and
shows strong evolutionary conservation. In Saccharomyces cerevisiae, RAD52 is one of the key members in this
pathway. Strains with mutations in this gene show strong hypersensitivity to DNA-damaging agents and defects
in recombination. Inactivation of the mouse homologue of RAD52 in embryonic stem (ES) cells resulted in a
reduced frequency of homologous recombination. Unlike the yeast Scrad52 mutant, MmRAD522/2 ES cells
were not hypersensitive to agents that induce DSBs. MmRAD52 null mutant mice showed no abnormalities in
viability, fertility, and the immune system. These results show that, as in S. cerevisiae, MmRAD52 is involved
in recombination, although the repair of DNA damage is not affected upon inactivation, indicating that
MmRAD52 may be involved in certain types of DSB repair processes and not in others. The effect of inactivating
MmRAD52 suggests the presence of genes functionally related to MmRAD52, which can partly compensate for
the absence of MmRad52 protein.
tants display the most severe radiation sensitivity and defects in
recombination.
Biochemical experiments with S. cerevisiae have shown that
the ScRad51 protein forms nucleoprotein filaments with single-stranded DNA and promotes pairing and limited strand
exchange (51). The ScRad52 protein alone or a heterodimer of
ScRad55 and ScRad57 functions as a cofactor in this reaction,
probably by overcoming the inhibitory effect of replication
protein A (32, 45, 49, 50). Recently, ScRad54 has been shown
to stimulate the pairing reaction (36).
Homologues of most of the RAD52 group genes in S. cerevisiae have been identified in other yeast strains as well as in
higher eukaryotes (1, 15, 26, 30, 35, 53). As in yeast, physical
interactions between HsRad51 and HsRad52 and between
HsRad51 and HsRad54 proteins have been observed in mammals (17, 42). Moreover, in humans, HsRad51 mediates pairing and strand exchange, which is stimulated by HsRad52 (3,
6). Phenotypic studies of eukaryotic null mutants also suggest
that recombination plays a role in the repair of DSBs. Inactivation of the RAD54 homologues in mouse embryonic stem
(ES) cells and chicken DT40 B cells increases their sensitivity
to ionizing radiation and leads to a decrease in homologous
recombination (7, 14). In a Drosophila strain with mutations in
both RAD54 alleles, larval survival was severely affected after
X-ray treatment. In addition, the mutant flies were almost
completely defective in X-ray-induced mitotic recombination
(23). These results imply that the RAD54 homologue in higher
eukaryotes plays a role in homologous recombination and in
the repair of induced DSBs. A RAD51 null mutation cannot be
obtained in chicken DT40 cells or in mouse ES cells, and
MmRAD512/2 mouse embryos arrest early in development
due to decreased cell proliferation rates and extensive chromosome loss (24, 48, 56). Furthermore, MmRAD51 antisense
oligonucleotides significantly inhibit cell growth and increase
radiation sensitivity in mouse cells, indicating that the gene is
essential for proliferation in vertebrate cells and is involved in
the repair of X-ray-induced DNA damage (52).
Double-strand breaks (DSBs) in the DNA of living organisms occur during several physiological processes including
meiotic recombination, mating-type switching in yeast, and
V(D)J rearrangement in developing B and T lymphocytes.
Agents such as ionizing radiation and certain chemicals also
lead to the induction of DSBs in the genome. If left unrepaired, DSBs result in broken chromosomes and cell death, as
has been shown convincingly in yeast (5). Alternatively, incorrect repair of DSBs may generate deletions, chromosome rearrangements, and cell transformation and eventually lead to
the formation of tumors.
Two main pathways are known to be involved in the repair
of DSBs in eukaryotes: end-to-end rejoining, a homology-independent but error-prone process, and error-free repair via
(homologous) recombination. Repair of DSBs in the yeast
Saccharomyces cerevisiae occurs predominantly via recombination, whereas a contribution of end-to-end rejoining can be
observed only in a recombination-deficient background (9, 27,
47). Recombinational repair in S. cerevisiae involves the genes
of the RAD52 epistasis group, of which nine members have
been identified thus far (ScRAD50, ScRAD51, ScRAD52,
ScRAD54, ScRAD55, ScRAD57, ScRAD59, ScMRE11, and
ScXRS2) (2, 11, 15, 16, 44). Interestingly, it has been shown
that ScRAD50, ScMRE11, and ScXRS2 are also involved in
end-to-end rejoining (10, 28, 55). Mutations in genes of the
RAD52 group result in an increased sensitivity to ionizing
radiation and defects in one or more types of recombination.
Among these mutants, the Scrad51, Scrad52, and Scrad54 mu* Corresponding author. Mailing address: MGC-Department of Radiation Genetics and Chemical Mutagenesis, Leiden University Medical Center, Sylvius Laboratory, Wassenaarseweg 72, 2333 AL Leiden,
The Netherlands. Phone: 31-71-5271603. Fax: 31-71-5221615. E-mail:
[email protected]
† Present address: MGC-Department of Medical Biochemistry and
Chemical Mutagenesis, Leiden University Medical Center, Leiden,
The Netherlands.
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Received 16 April 1998/Returned for modification 1 June 1998/Accepted 27 July 1998
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RIJKERS ET AL.
MATERIALS AND METHODS
Targeting vectors. A targeting vector was designed to replace MmRAD52 exon
3 with a positive selection marker, the neomycin gene driven by the phosphoglycerate kinase (PGK) promoter, and an upstream mouse sequence (UMS),
which functions as a transcription terminator. The 59 homology arm containing
MmRAD52 exon 2 and its surrounding intronic regions was isolated as a 3.9-kb
XbaI-BamHI fragment from lambda clone 52A, which contained genomic DNA
derived from a 129/Ola strain (57). This fragment was blunted and ligated into
the blunted ClaI site of the targeting vector pTKNeoUMS (a gift of M. Gassmann, Institute of Physiology, University of Zürich, Zürich, Switzerland) (37).
Plasmid TV5-2 contained the insert in the correct orientation. The 39 homology
arm was generated in two parts. First, an oligonucleotide (O52-29) (59-TATAA
GCGGCCGCCGTGGGCATGTATCTAGTTGTTGACAGAAG-39) that hybridizes just downstream of exon 3 and contains an artificial NotI site at the 59
end was synthesized. PCR was performed on DNA from lambda clone 52A with
primers O52-29 and O52-8 (59-TCAGTCACAGCCTCCTTCCT-39), resulting in
a 2.45-kb fragment. This fragment was digested with NotI and XbaI, and the
resulting 1.4-kb NotI-XbaI fragment was used as the first part of the 39 arm. The
second part consisted of an 8.1-kb XbaI-BamHI fragment from clone 52A. A
three-point ligation was carried out with pBluescript SK digested with NotI and
BamHI, the 1.4-kb NotI-XbaI fragment, and the 8.1-kb XbaI-BamHI fragment.
The resulting 9.5-kb 39 homologous arm was recloned as a NotI-BamHI (blunted) fragment into the NotI-EcoRV sites of pGEM-5Z. It was released from this
plasmid by digestion with NotI and SacII and ligated into the corresponding sites
of plasmid TV5-2. The resulting targeting vector TV5-3, containing the selection
markers herpes simplex virus thymidine kinase (HSV-TK) and UMS-PGK-Neo
flanked by the 3.9-kb 59 arm and the 9.5-kb 39 arm, was linearized by SacII
digestion and electroporated into ES cells. A second targeting vector was generated by first introducing a linker containing a SfiI site into the unique SacII site
of TV5-3. Then the UMS-PGK-Neo cassette was removed by digestion with
BamHI and NotI and replaced with a PGK-hygromycin-phosphotransferase cassette. The resulting plasmid, TV7-11, was linearized with SfiI prior to electroporation into ES cells. The 129/Sv-derived Rb-puromycin targeting construct
pHA268 was kindly provided by H. te Riele (The Netherlands Cancer Institute,
Amsterdam, The Netherlands) (54). The 129/Ola-derived targeting construct
CSB-pur was a gift of R. Kanaar (Erasmus University, Rotterdam, The Netherlands) (58). ES cells electroporated with these two targeting constructs were
selected with puromycin only.
ES cells and electroporation. 129/Ola-derived IB10 ES cells (a subclone from
E14 ES cells; kindly provided by E. Robanus-Maandag, The Netherlands Cancer
Institute) were cultured on lethally irradiated mouse embryonic fibroblasts
(MEFs) in Dulbecco modified Eagle medium supplemented with 10% fetal calf
serum, 0.1 M nonessential amino acids, 50 mM b-mercaptoethanol (all ES cell
grade; Gibco BRL), and 3.3 mM nucleosides (Sigma) in the presence of murine
leukemia inhibitory factor (ESGRO; Gibco BRL). Then 2 3 107 ES cells were
electroporated at 400 V and 250 mF with 20 to 30 mg of the linearized targeting
vector. G418 (0.3 mg/ml), hygromycin B (150 mg/ml), or puromycin (1.0 mg/ml,
selection on gelatinized plates without MEFs) was added 24 h after electroporation, and ganciclovir (2 mM) was added 48 h after electroporation. Resistant
colonies were picked after 10 days of selection, and their genomic DNA was
analyzed on Southern blots with a unique probe (probe 1) outside the targeting
construct (see Fig. 1).
Western blotting. Confluent cultures of ES cells were grown on gelatinized
plates without MEFs and lysed in Laemmli sample buffer. Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (10% polyacrylamide) and transferred to a polyvinylidene difluoride membrane (Immo-
bilon-P; Millipore). The filters were blocked with 5% bovine serum albumin and
probed with a 1:10,000 dilution of a crude rabbit antiserum generated against
full-length MmRad52 protein (in the presence of 1% bovine serum albumin). A
horseradish peroxidase-conjugated anti-rabbit second antibody was used in a
1:10,000 dilution and detected with enhanced chemiluminescence detection reagents (Sigma).
Cell survival assay. For X-ray exposure, ES cells were trypsinized, counted,
and irradiated at a dose rate of 0.2 Gy/min with a 225 SMART X-ray apparatus
(Andrex SA, Copenhagen, Denmark) at 200 kV and 4 mA with a 1-mm Al filter.
Dose and dose rate were monitored with a PTW (Freiburg, Germany) Dosimentor system. For treatment with MMS or mitomycin C, ES cells were incubated for
1 h in ES medium (described above) and in the presence of different mutagen
concentrations (see Fig. 2). After treatment, the cells were trypsinized, diluted,
and plated. For UV exposure, the cells were seeded at low densities on gelatinized plates and irradiated with UV light (254 nm) 16 h later. Medium was
aspirated for the duration of the UV exposure. All treated cells were plated in
triplicate at low density in Buffalo rat liver (BRL) cell-conditioned medium (20)
on gelatinized tissue culture plates without MEFs. The cells were grown for 7
days, fixed, and stained, and colonies were counted.
Mouse strains and detection of the MmRAD52 genotype. C57BL/6 female
mice for harvesting blastocysts and mating were obtained from Charles River
Laboratories. Other strains and the MmRAD522/2 mice were bred and kept at
the Transgenic Facility, Leiden University Medical Center, Leiden, The Netherlands. Individual mice were genotyped for MmRAD52 by PCR analysis of DNA
isolated from tail tips, using a primer specific for the neomycin gene (Neo-FB;
59-CGCATCGCCTTCTATCGCCT-39), a primer specific for MmRAD52 exon 3
(R52-45; 59-AGCCAGTATACAGCGGATG-39), and a primer complementary
to intronic sequences downstream of exon 3 (R52-46; 59-CAACTAGATACAT
GCCCACG-39). The PCR conditions were as follows: 1 min at 93°C, 1 min at
55°C, and 3 min at 72°C for 35 cycles. The product of the wild-type allele is 120
bp, and the targeted allele yields a 320-bp product.
Histology and nuclear DNA fragmentation labeling (TUNEL assay). Animals
were killed by cervical dislocation. The ovaries and uterus were removed from
female mice, weighed, and fixed in Bouin’s fixative for 24 h at room temperature.
The testes and epididymides were removed from males and weighed, and one
testis and epididymis from each animal were fixed in phosphate-buffered formalin at 4°C for 24 h. Subsequently, all the organs were embedded in paraffin.
Mounted sections were deparaffinized, rehydrated, and stained with hematoxylin
and eosin. Formalin-fixed sections were mounted on glass slides coated with a
2% solution of 3-aminopropyltriethoxysilane in acetone, and the terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL)
assay was performed as described previously (38).
Analysis of lymphocytes. Lymphocytes from spleen and bone marrow were
isolated from two wild-type and two MmRAD522/2 mice and analyzed for expression of surface markers (flow cytometry) as well as class switching as described previously (39).
RESULTS
Inactivation of MmRAD52 in mouse ES cells. The
MmRAD52 gene spans approximately 18 kb on chromosome 6
and consists of 12 exons (57). The N-terminal region of the
MmRad52 protein is significantly conserved among several
yeast species, chickens, mice, and humans and is encoded by
exons 3 to 7 (8, 29). To inactivate MmRAD52, a targeting
vector, TV5-3, was constructed in which exon 3 was replaced by
a positive selection marker, PGK-neomycin. To enable counterselection against random integration, an HSV-TK selection
marker was integrated at the 59 end of the 3.9-kb homologous
arm (Fig. 1A). After linearization, the construct was electroporated into IB10 ES cells and clones resistant to both the
neomycin analog G418 and ganciclovir were isolated and analyzed. Correctly targeted clones were identified by Southern
blot analysis with a unique probe outside the targeting construct (Fig. 1, probe 1). About one-third of the clones analyzed
contained a disrupted MmRAD52 allele. Two targeted clones
that had retained their normal karyotype, clone 6 and clone 29,
were used for further experiments.
Disruption of the second MmRAD52 allele was performed
with a similar targeting vector, in which the UMS-PGK-neo
cassette was replaced by the positive selection marker PGKhygromycin. This construct, TV7-11, was linearized and electroporated into the previously targeted clones 6 and 29. Clones
resistant to both hygromycin B and ganciclovir were again
analyzed by Southern blot hybridization (Fig. 1, probe 1).
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In this paper, inactivation of the homologue of ScRAD52 in
the mouse is described. Yeast Scrad52 mutant cells are extremely sensitive to X rays and methyl methanesulfonate
(MMS). Repair of DSBs is hardly detectable in these mutants
(13). Moreover, Scrad52 strains are defective in spontaneous
and induced mitotic recombination and mating-type switching.
The formation of viable spores in meiotically dividing cells is
almost completely blocked in a Scrad52-deficient background
(15, 16, 44). Using degenerate primers, homologues of
ScRAD52 in higher eukaryotes have been identified. The human and mouse proteins, HsRad52 and MmRad52, display
about 30% identity to their counterpart from S. cerevisiae (4,
29, 43). The homology is concentrated primarily in the Nterminal region, suggesting an important role in the function of
this protein. The highest levels of expression of MmRAD52
were seen in lymphoid organs and testes, suggesting a possible
role in recombination and/or cell proliferation (29). In this
paper, we describe the effects of inactivation of MmRAD52 on
recombination and repair of radiation-induced damage.
MOL. CELL. BIOL.
VOL. 18, 1998
TARGETED INACTIVATION OF THE MOUSE RAD52 GENE
6425
Clones that were targeted with TV5-3 on one MmRAD52 allele
and with TV7-11 on the other allele were identified and analyzed for the absence of random integration of the targeting
vector (probe 3; results not shown). In addition, hybridization
with a probe for the neomycin phosphotransferase gene (probe
23) showed that the predicted genomic fragment was present
in all of these clones (results not shown). To confirm that the
integration of the selection marker genes produced a null mutation, MmRad52 protein was measured in crude extracts of
ES cells by Western blotting with a polyclonal antiserum
against full-length MmRad52. MmRad52 protein was undetectable in MmRAD522/2 ES cells, whereas cells heterozygous
for MmRAD52 contained reduced levels of the protein compared to wild-type ES cells (Fig. 1C). In the targeted cells, no
truncated MmRad52 proteins were observed. Correctly targeted ES cell clones were used for the analysis of homologous
recombination events and survival studies.
Homologous recombination in MmRAD522/2 ES cells. In S.
cerevisiae, the ScRad52 protein is essential for homologous
recombination. To investigate whether the MmRAD52 gene is
also involved in homologous recombination in the mouse,
gene-targeting experiments were performed on two independent loci. Wild-type and MmRAD522/2 ES cells were electroporated with a retinoblastoma (Rb)-puromycin targeting vector. Genomic DNA from individual puromycin-resistant clones
was analyzed by Southern blot hybridization, and clones correctly targeted at the Rb locus were identified. A similar ex-
periment was performed in duplicate by using a targeting construct for the Cockayne syndrome B (CSB) locus (Table 1).
The frequency of homologous recombination at the Rb locus
was relatively low, 8.1% in wild-type cells, whereas targeting at
the CSB locus was more efficient (17.6 and 24.2%). Interestingly, in each experiment, the targeting frequency in
MmRAD522/2 ES cells was reduced to 61 to 71% of the
frequency observed in wild-type cells. This reduction is statistically significant (Cochran Q test on the odds ratio), indicating
that the MmRad52 protein does play a role in homologous
recombination.
Sensitivity of MmRAD522/2 ES cells to DNA-damaging
agents. In the yeast S. cerevisiae, genes belonging to the
RAD52 epistasis group are required for the recombinational
repair of DSBs. Mutations in these genes lead to increased
sensitivity to agents known to induce DSBs, such as ionizing
radiation and MMS (16). Therefore, the effect of these DNAdamaging agents was investigated in MmRAD522/2 ES cells.
Wild-type (IB10) ES cells and cells heterozygous and homozygous for the targeted MmRAD52 allele were exposed to different doses of X rays or MMS. The sensitivity of
MmRAD522/2 ES cells to ionizing radiation or to MMS was
similar to that of wild-type ES cells (Fig. 2).
Mitomycin C introduces interstrand cross-links in DNA, in
addition to other types of damage. In Escherichia coli and
yeast, repair of cross-links depends both on nucleotide excision repair and recombinational repair. We found that
MmRAD522/2 ES cells are not hypersensitive to mitomycin C.
UV light primarily causes adducts that are removed via nucleotide excision repair and not via recombinational repair. As
expected, the UV survival curves for MmRAD522/2 and wildtype ES cells are comparable (results not shown). These assays
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FIG. 1. Disruption of the MmRAD52 gene. (A) Schematic representation of
the MmRAD52 locus, the targeting vector, and the targeted locus. All coding
exons are indicated by numbered solid boxes, and noncoding (parts of) exons are
shown as open boxes. Relevant EcoRI (E), BsrGI (Bs), XbaI (X), BamHI (Ba),
and SacII (S) restriction sites and the positions of the probes used for Southern
blot analysis are indicated. (B) Southern blots of DNA from targeted ES cell
clones after electroporation of the cells with targeting vector TV5-3 and digestion of the genomic DNA with BsrGI (left) and subsequently with targeting
vector TV7-11 and digestion with EcoRI (right). Both blots were hybridized with
probe 1. (C) Western blot of whole-cell extracts from MmRAD521/1,
MmRAD521/2, and MmRAD522/2 ES cells, incubated with a 1:10,000 dilution
of crude anti-MmRad52 antiserum.
6426
RIJKERS ET AL.
MOL. CELL. BIOL.
TABLE 1. Homologous recombination in ES cells
Frequencya of recombination in ES cells
with targeting construct:
ES cells
CSB-pur
Rb-puromycin
1/1
MmRAD52
MmRAD522/2
% of wild type
8.1% (17/211)
4.9% (5/102)
61
Expt 1
Expt 2
17.6% (35/199)
12.5% (19/152)
24.2% (38/157)
16.2% (27/167)
71
67
a
clearly show that in contrast to S. cerevisiae, in ES cells the
MmRAD52 gene is not required for the repair of lethal DNA
damage induced by ionizing radiation, cross-links, or UV light.
Phenotype of MmRAD522/2 mice. As described above, we
generated mouse ES cells in which one MmRAD52 allele was
mutated by replacing exon 3 with a neomycin resistance cassette via gene targeting. Two heterozygous ES cell clones,
clone 6 and clone 29, were used to generate chimeric mice by
injection into C57BL/6 blastocysts. Chimeric mice that transmitted the targeted allele to their offspring were obtained with
each of the two ES cell clones. Heterozygous male and female
mice were interbred to generate two independent mouse lines,
one from each ES cell clone. Both lines produced viable and
healthy homozygous offspring in a Mendelian fashion.
MmRAD522/2 mice show no gross abnormalities, and their
viability is not affected. Since ScRad52 is essential in meiosis in
yeast and since expression of the mouse homologue is elevated
in the mouse testis, the MmRad52 protein was expected to play
a role in meiotic recombination during germ cell differentiation in the mouse (29). However, mating between homozygous
animals showed that both males and females are fertile. They
produced normal numbers of offspring with a male-to-female
ratio of 1:1. Histological examination of testes, epididymides,
and ovaries of MmRAD522/2 mice revealed no abnormalities
(results not shown). Apoptosis of male germ cells was studied
by the TUNEL assay, but no difference in the number of apoptotic cells per tubule between wild-type and MmRAD522/2
testes was observed (results not shown). Also, the weights of
all reproductive organs were normal. These results indicate
that meiosis proceeds normally in both male and female
MmRAD522/2 mice.
Development of T- and B- lymphocytes and Ig class switch.
DSBs not only occur during meiosis but are also generated
during maturation of cells of the immune system. Developing
T and B lymphocytes undergo a somatic targeted recombination process, called V(D)J rearrangement, to generate a wide
variety of antigenic specificities. The very specific types of
breaks that occur during these processes are normally repaired
via the end-to-end rejoining pathway involving the DNA-PK
protein complex (18, 41). Recent evidence has shown that
three yeast genes involved in homologous recombination,
ScRAD50, ScMRE11, and XRS2, also take part in some aspects
of end-to-end rejoining (10, 28, 55). Furthermore, the expression of mammalian RAD52-group genes, including RAD52
itself, is elevated in the thymus and spleen, which are involved
in T- and B-cell development (29). To assess a possible role for
FIG. 2. X-ray and MMS survivals of wild-type and targeted ES cells. The
survival curves of the MmRAD521/1 (IB10), MmRAD521/2 (clone 6), and
MmRAD522/2 (clone 6.7) ES cell lines are shown. The percentage of surviving,
colony-forming cells is plotted as a function of X-ray (top) or MMS (bottom)
dose. Cloning efficiencies varied from 9 to 30%, and the survival of the untreated
cells was set to 100%. The data in the upper graph represent the average of seven
independent X-ray exposure experiments; the lower graph depicts a typical MMS
survival experiment (performed twice). Experiments with an independently derived MmRAD522/2 ES cell line gave similar results.
MmRAD52 in this process, B-cell development in the bone
marrow, T-cell development in the thymus, and the splenic
T- and B-cell compartments in MmRAD522/2 mice were
analyzed by flow cytometry (Fig. 3). In bone marrow of
MmRAD522/2 mice, all the different stages of B cell development are present and, moreover, the numbers of cells in these
different development compartments are identical to those
found in wild-type mice. T-cell development as occurs in the
thymus is also undistinguishable in wild-type and MmRAD522/2
mice. Moreover, the phenotype and number of peripheral T
and B cells do not differ significantly between wild-type and
MmRAD522/2 mice. This indicates that the absence of
MmRAD52 does not influence the development of the lymphoid system.
Targeted recombination also occurs when B cells switch
from the production of immunoglobulin M (IgM) to antibodies
with a different constant region, such as IgG or IgE. Ig class
switching results from a recombination event between repetitive sequences located 59 of the Cm gene and 59 of the downstream CH gene to which switching is targeted. To examine
whether MmRAD52 interferes with this class-switching process, B cells were isolated from spleens of wild-type and
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The frequency of homologous recombination events in puromycin-resistant
ES cell clones upon electroporation with a targeting vector is shown as a percentage of correctly targeted clones relative to the total number of puromycinresistant clones analyzed (absolute numbers are given in parentheses). The
frequency of homologous recombination in MmRAD522/2 ES cells is 61% of the
frequency in wild-type cells at the Rb locus; at the CSB locus it is reduced to 71
and 67%, respectively. This reduction is statistically significant (P 5 0.015;
Cochran Q test on the odds ratio).
TARGETED INACTIVATION OF THE MOUSE RAD52 GENE
VOL. 18, 1998
6427
MmRAD522/2 mice. The cells were cultured in the presence of
anti-CD40 and interleukin-4, a culture system which is known
to give rise to high frequencies of Sm-Sε H-chain class switching (39). After 5 days, DNA from the cells was tested for the
presence of Sm-Sε switched H-chain gene loci by digestioncircularization PCR (12, 39, 60). High frequencies of Ig class
switching, but no obvious differences between wild-type and
MmRAD522/2 mice, were detected (results not shown), indicating that MmRAD52 is not necessary for the class-switching
process.
DISCUSSION
In this study, we have investigated the role of the mouse
homologue of ScRAD52, MmRAD52, in homologous recombination and the repair of X-ray-induced DNA damage. Given
the extreme phenotype in S. cerevisiae and the stimulatory
effect of both ScRad52 and HsRad52 on DNA strand exchange
in vitro (6, 32, 45, 49), the MmRad52 protein was expected to
contribute significantly to these processes. Surprisingly, however, inactivation of both alleles of the MmRAD52 gene in ES
cells did not result in increased radiation sensitivity. Moreover,
the MmRAD522/2 mice were viable and fertile and showed no
gross abnormalities.
Compared to wild-type cells, a small but significant reduction in homologous recombination of 30 to 40% was observed
in MmRAD522/2 ES cells. Interestingly, a null mutation of the
RAD52 homologue in the chicken B-cell line DT40 also has
limited effects on the frequency of homologous recombination
(61). Others have shown that monkey cells become slightly
more resistant to ionizing radiation and display an increased
level of intrachromosomal homologous recombination upon
overexpression of a human HsRAD52 cDNA (34). Similar effects were reported when ScRAD52 was overexpressed in a
human fibrosarcoma cell line (19). These findings and the
results presented in this paper suggest that recombination in
mouse ES cells can occur via both MmRAD52-dependent and
-independent pathways. The phenotypes of MmRAD52-deficient ES cells and mice are more reminiscent of those found in
Schizosaccharomyces pombe, since mutation of the RAD52 homologue in S. pombe, Sprad221, causes a less extreme phenotype than is seen in a Scrad52 mutant. The increase in radiation
sensitivity in the Sprad22 null mutant is moderate, and there is
only a twofold reduction in the efficiency of homologous integration of a linear plasmid (31).
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FIG. 3. Flow cytometric analysis of splenic lymphocytes. Flow cytometry results for splenic lymphocytes isolated from a wild-type mouse (left) and an MmRAD522/2
null mutant mouse (right) are shown. The cells were stained with the B-cell markers anti-B220 and anti-IgM antibodies (top) or with the T-cell markers anti-CD4 and
anti-CD8 antibodies (bottom).
6428
RIJKERS ET AL.
ACKNOWLEDGMENTS
This work was supported by grants from the Dutch Cancer Society
(EUR 94-858) and Euratom (F14 PCT950010). The Basel Institute for
Immunology was founded and is supported by F. Hoffmann-La Roche
& Co.
We are grateful to M. Gassmann, H. te Riele, R. Kanaar, J. Essers,
and E. Robanus-Maandag for the generous gift of cells and reagents.
We thank J. Eeken, J. Jansen, and M. Zdzienicka for comments on the
manuscript.
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To account for the mild phenotype resulting from inactivation of the MmRAD52 gene, several explanations are possible.
One is that the RAD52 gene has been duplicated in mammals
as well as in S. pombe. Duplication of repair genes in mammals
has been observed for RAD6 and RAD23 (22, 25, 40, 59).
However, hybridization of Southern blots under less stringent
conditions did not reveal the presence of closely related homologues of MmRAD52 (results not shown). Functional redundancy seems a more likely explanation for our results. The
recent identification in S. cerevisiae of a new member of the
RAD52 group, ScRAD59, provides evidence of such a redundancy. The ScRad59 protein displays weak sequence homology
to the N-terminal domain of ScRad52, whereas the C-terminal
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MmRAD52 phenotype described in this study. The defects
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ScRad52 functionally overlap. It is possible that in some organisms, including mammals and S. pombe, the redundancy
between Rad52 and Rad59 homologues is more extensive than
in S. cerevisiae and that the two genes are more nearly equal in
importance for DSB repair and recombination. These data
show that functional redundancy between RAD52 and another
gene may indeed exist, although the functions of two such
genes do not have to overlap completely. For instance, although in the Sprad22 mutant the defects in DSB repair after
X-ray treatment are mild and homologous integration is only
slightly affected, switching of the mating type is still a lethal
event (33). Another example of more specialized roles of
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and RDH54 are required for interchromosomal gene conversion, RDH54 (but not ScRAD54) is dispensable for intrachromosomal gene conversion (21, 46). Therefore, it is conceivable
that MmRAD52 is crucial in certain recombination processes
but that its role in other pathways in recombination can be
compensated for by other genes. Further analyses are required
to elucidate the exact role of MmRAD52 in DSB repair processes and recombination pathways.
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34.
TARGETED INACTIVATION OF THE MOUSE RAD52 GENE
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