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Molecular and Cellular Biology, November 1998, p. 6430-6435, Vol. 18, No. 11
Bayer-chair Department of Molecular
Immunology and Allergology,
Received 30 April 1998/Returned for modification 1 June
1998/Accepted 27 July 1998
Rad52 plays a pivotal role in double-strand break (DSB) repair and
genetic recombination in Saccharomyces cerevisiae, where mutation of this gene leads to extreme X-ray sensitivity and defective recombination. Yeast Rad51 and Rad52 interact, as do their human homologues, which stimulates Rad51-mediated DNA strand exchange in
vitro, suggesting that Rad51 and Rad52 act cooperatively. To define the
role of Rad52 in vertebrates, we generated
RAD52 The many strategies that have
evolved to deal with DNA damage attest to the vital importance of
chromosomal integrity to all organisms. Double-strand breaks (DSBs) can
lead to immediate cell death if unrepaired or to chromosomal loss or
translocation if not repaired correctly. Aside from environmentally
induced damage, DSBs of genomic DNA are generated during several
biological processes such as meiotic recombination or the development
of the vertebrate immune system. Accordingly, the enzymes and systems
of DSB repair are of great interest in biology.
The RAD52 epistasis group of genes (RAD50,
RAD51, RAD52, RAD54,
RAD55, RAD57, RAD59, MRE11,
and XRS2) has been defined by the respective
Saccharomyces cerevisiae mutants, which are hypersensitive to ionizing radiation and exhibit mitotic and meiotic
recombination defects. While phenotypic differences between
mutants distinguish between these genes genetically, it is clear that
they are constituents of a pathway for the repair of DSB damage
by homologous recombination (reviewed in references 10,
22, and 27). The high degree of
conservation of the RAD52 group of genes from yeast to
vertebrates (4, 8, 14, 23, 25) suggests a similar role for
these proteins. However, while vertebrate
RAD54 Rad52 mutants show the most pronounced phenotype among
RAD52 epistasis group mutants in S. cerevisiae. Rad52 is essential for an intermediate stage after the
formation of DSBs but before the appearance of stable recombinants
during gene conversion (26). Rad52 is also involved in
single-strand annealing (13) and other RAD51-independent forms of recombination (30),
which may explain the severe phenotype of rad52 mutants.
Recent evidence suggests that both yeast and human Rad52 interact with
the respective Rad51 protein, a structural and functional homologue of
the well-characterized Escherichia coli RecA protein
(25, 26), during recombination (31). This
interaction facilitates the Rad51 strand exchange reaction (3, 21,
28), potentially involving Rad52 binding of DNA (3, 18,
27). To define the role of Rad52 in vertebrate cells, we
generated RAD52 Plasmid constructs.
The ~11-kb genomic chicken
RAD52 locus was cloned from DT40 genomic DNA by long-range
PCR with the chicken RAD52 cDNA primers 5'-tga aag gca agg
gaa gga cag tga aag cca tg-3' and 5'-gtc tgt cca cat tta gtg ttt att
ctt gtg tt-3' (4), and the positions of the exons and
introns were determined by sequencing (Fig. 1). The Hisr or
Bsrr selection marker genes under the control of the
Cell culture, DNA transfections, and sIgM staining.
The
conditions of cell culture and DNA transfections were described
previously (29). To measure the rate of immunoglobulin (Ig)
gene conversion, cells were subcloned and the percentage of surface
IgM-positive (sIgM+) cells in 45 subclones from each
genotype was measured as described previously (5).
Measurement of Rad51 foci.
Paraformaldehyde-fixed cells were
permeabilized with 0.1% Nonidet P-40 (Sigma) and incubated with rabbit
anti-human Rad51 antiserum (33). Staining was visualized
with fluorescein isothiocyanate (FITC)-conjugated anti-rabbit IgG
(Santa Cruz), and the cells were mounted under Slow Fade solution
(Molecular Probes). All the images were taken by confocal microscopy
(MRC-1024; Bio-Rad) and processed with Adobe Photoshop version 4.0J.
Measurement of sensitivity of cells to Measurement of targeted integration frequencies.
To analyze
targeted integration events at the Generation of RAD52 Effects of Rad52 deficiency on sensitivity to DNA-damaging agents,
Rad51 focus formation, and Ig gene conversion.
The repair capacity
of cells defective in RAD52 was analyzed in a colony
survival assay with RAD54
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Homologous Recombination, but Not DNA Repair, Is
Reduced in Vertebrate Cells Deficient in RAD52
and
ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
/ mutants of the chicken B-cell line
DT40. Surprisingly, RAD52/ cells were not
hypersensitive to DNA damages induced by 
-irradiation, methyl
methanesulfonate, or cis-platinum(II)diammine
dichloride (cisplatin). Intrachromosomal recombination, measured by
immunoglobulin gene conversion, and radiation-induced Rad51 nuclear
focus formation, which is a putative intermediate step during
recombinational repair, occurred as frequently in
RAD52/ cells as in wild-type cells.
Targeted integration frequencies, however, were consistently reduced in
RAD52/ cells, showing a clear role for
Rad52 in genetic recombination. These findings reveal
striking differences between S. cerevisiae and vertebrates
in the functions of RAD51 and RAD52.
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
/ cells reflect the yeast phenotype,
namely, extreme sensitivity to -ray and defective recombination
(5, 9), RAD51 deficiency results in the death of
vertebrate cells, indicating that Rad51 is essential for cell
proliferation (16, 29, 34).
/ clones from the chicken
B-lymphocyte line DT40, which exhibits highly efficient targeted
integration following the transfection of genomic DNA constructs
(7).
MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
-actin promoter (7) were inserted between the
left and right arms derived by PCR amplification of this construct. To
make the disruption construct for the rearranged Ig
locus, Ig-neo, the Neor cassette was inserted
into the BglI site in the C region of the 13-kb
BglII-XbaI fragment from the 18-D-1 clone
(24). The RAD52 expression vector consisted of
the EcoRI-SalI fragment of chicken cDNA
(4) cloned into pApuroII (15).
-rays, MMS, and
cisplatin.
Serially diluted cells were plated in medium containing
methylcellulose and irradiated with a 137Cs source or
treated with methyl methanesulfonate (MMS) (Sigma) or
cis-platinum(II)diammine dichloride (cisplatin). Colonies
were counted 10 days after treatment. The percent survival was
determined relative to the number of colonies from untreated cells.
-Actin and
Ovalbumin loci, the disruption construct DNA for either
locus described in reference 7 was transfected into
cells and Southern blot analysis was performed following selection of
clones resistant to the appropriate antibiotic. To analyze targeted
integration events at the rearranged Ig locus,
sorter-purified sIgM+ cells were transfected with the
rearranged Ig-neo disruption construct. G418-resistant
cells were selected in bulk populations, and sIgM expression was
measured by flow cytometry (7).
RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
/ mutants.
Two
RAD52 disruption constructs, RAD52his and
RAD52bsr, were generated from an ~11-kb genomic PCR
product (4). Targeted integration of these constructs was
expected to replace the chicken RAD52 coding sequence
for amino acids 137 to 182 (which lie in the conserved N-terminal
region implicated in DNA binding [18, 19]) with the
selection markers. The disruption of the RAD52 gene in two
independently isolated clones was verified by Southern and
Northern blot analyses (Fig. 1B and C).
These RAD52/ clones were
indistinguishable from wild-type cells with respect to growth rate and
cloning efficiency (data not shown).
View larger version (26K):
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FIG. 1.
Strategy of disruption of the RAD52 gene. (A)
Schematic representation of part of the RAD52 locus, the two
gene disruption constructs, and the configuration of the targeted loci.
Solid boxes indicate the positions of exons; numbers show the 3'
nucleotide of each exon relative to the start codon (4).
Relevant EcoRI recognition sites are indicated by RI. (B)
Southern blot analysis of EcoRI-digested DNA from the
indicated genotypes with the probe shown in panel A. The positions and
sizes of the hybridizing fragments of the wild-type and targeted loci
are indicated. (C) Northern blot analysis of total RNA with the
full-length chicken RAD52 cDNA as a probe. The same filter
was rehybridized with a chicken -Actin probe
(7).
/ cells in parallel
as a control. We examined its sensitivity to ionizing radiation and to
MMS, as well as to a DNA-cross-linking agent, cisplatin. Repair of DNA
lesions induced by cisplatin is known to depend upon both nucleotide
excision repair and recombinatorial repair in yeast (12).
RAD54/ cells showed increased sensitivity to
ionizing radiation (Fig. 2A) and MMS
(Fig. 2B), as reported previously. Rad54 was also required for the
repair of cisplatin adducts (Fig. 2C), implicating the recombinatorial
repair pathway in the repair of interstrand cross-linking in vertebrate
cells. In marked contrast to rad52 mutants of S. cerevisiae, which are extremely sensitive to DNA-damaging agents,
there was no significant difference between wild-type and
RAD52/ DT40 clones in sensitivity to
ionizing radiation (Fig. 2A). Similarly, the sensitivity of
RAD52/ DT40 cells to MMS or to cisplatin was
not significantly increased over those of wild-type
(RAD52+/+) and heterozygous
(RAD52+/) cells (Fig. 2B and C). These
observations indicate that although the recombinational
repair pathway is involved in DSB repair in vertebrate
cells, it can function in the absence of Rad52.
View larger version (24K):
[in a new window]
FIG. 2.
Sensitivity of the indicated clones to DNA-damaging
agents. The fractions of colonies surviving after the indicated
treatment of cells compared to nontreated controls of the same genotype
are shown on the y axis on a logarithmic scale. (A) Ionizing
radiation; (B) MMS; (C) cisplatin. The radiation doses and the MMS and
cisplatin concentrations are displayed on the x axis on a linear scale
in each graph. Data shown are the means ± standard deviations of
at least three separate experiments.
-radiation (Fig. 3).
Since Rad51 polymerizes on DNA and promotes in vitro strand exchange
(2, 26), a Rad51 focus may reflect an intermediate structure
of recombinational repair. The formation of Rad51 foci is observed
during meiosis in S. cerevisiae and is abrogated in
rad52 mutants (10a). Furthermore, Rad51 foci are
also observed in vertebrate cells during the S phase (33),
which implies that Rad51 may be responsible for repairing spontaneous
DSBs during the cell cycle (29). Rad51 foci were induced in
DT40 cells by -irradiation in a dose-dependent manner (data not
shown), as previously reported for mammalian cells (11). We
found no significant difference between
RAD52/ and wild-type cells with respect to
the number of Rad51 foci in cycling cells and to the kinetics of Rad51
focus formation following -irradiation. This observation, along with
the capacity of RAD52/ cells to withstand
-radiation, indicates that DSB repair occurs efficiently in the
absence of Rad52.
|
)-chain locus by gene conversion, with pseudogenes serving as
donors (6). To analyze such intrachromosomal recombination, RAD52/ clones were generated from an
sIgM variant of DT40, clone 18, containing a frameshift
in its rearranged V segment in the light-chain locus. Since this
frameshift mutation can be repaired by overlapping gene conversion
events leading to reexpression of sIgM, the rate of Ig gene conversion
can be assessed by measuring the percentage of sIgM+
revertants (6). We measured the average percentage of
sIgM+ revertants of 45 subclones each from the wild-type
and the two RAD52/ clones and found no
difference between these clones (the reversion rate was 2.48 × 103 for the wild type and 2.40 × 103
and 2.89 × 103 for the two
RAD52/ clones, as calculated from the
equation in reference 17).
Reduced targeted integration frequencies in
RAD52/ cells.
We next examined
targeted integration frequencies at the Ig,
-Actin, and Ovalbumin loci, comparing
wild-type cells, two RAD52/ clones,
and two RAD52/ clones reconstituted with
chicken RAD52 cDNA. To measure targeted integration
frequencies at the Ig locus, we sorted sIgM+
cells from each of the five clones, transfected them with the Ig-neo construct (Fig. 4A),
and measured the expression of sIgM in the bulk population of
G418-resistant cells. Since integration of the Ig-neo
construct at the Ig locus results in sIgM
cells, the fraction of sIgM cells among transfectants
reflects the frequency of the targeted integration events at this
locus. Spontaneous reversion caused by Ig gene conversion was 
1%.
Representative data are shown in Fig. 4B. While transfection
efficiencies during these experiments were comparable, the averages of
targeted integration frequencies at this locus in triplicate
experiments were consistently lower (ca. threefold) in
RAD52/ clones than in the wild type. The
expression of RAD52 cDNA restored targeted integration to
wild-type levels in the two RAD52/ clones
(Table 1). Similarly, the disruption of
RAD52 caused a four- to eightfold reduction of the targeted
integration frequency at the -Actin locus when a
-Actin targeting construct was used (7). This
frequency was partially restored in a reconstituted clone
(RAD52R-1), which expressed the RAD52 cDNA
transcript at a level ca. 10-fold higher than that in wild-type DT40
cells (data not shown), confirming that Rad52 is involved in the
targeted integration of transfected genomic DNA constructs (Table
1). While RAD52/ clones showed a marked
reduction in targeted integration frequencies at the
-Actin and Ig loci, the targeted
integration frequency at the Ovalbumin locus was reduced
only to ~85% of the wild-type levels (with some variation depending
on the construct and locus in question). Taking all loci together,
there is a significant reduction in targeted integration frequency
between wild-type and RAD52/ clones
(P < 0.05, Dunnett's multicomparison test). This
reduced efficiency of targeted integration events has been also
observed in murine embryonic stem (ES) cells deficient in
RAD52 (24a).
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V1, first pseudogene; L, leader sequence;
V
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DISCUSSION |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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The above data demonstrate that Rad52 is not required for the repair of induced DSBs, Rad51 focus formation, or intragenic Ig gene conversion. Similarly, X-ray sensitivity was not increased in RAD52-deficient ES cells or mutant mice (24a). These results are perhaps surprising, given the severe phenotype associated with mutation of the RAD52 gene in S. cerevisiae. Furthermore, while the rad51, rad52, and rad54 mutants of S. cerevisiae exhibit similar defects in both DNA repair and recombination, we and other groups have revealed that RAD51-, RAD52-, and RAD54-deficient DT40 cells display quite distinct phenotypes, as do the corresponding murine ES cell mutants (5, 9, 16, 29, 34). A further example of such differences is found in Schizosaccharomyces pombe, where mutation of the RAD51 and RAD54 homologues, rhp51+ and rhp54+, leads to similar hypersensitivity to radiation and targeted integration deficiency, whereas deficiency in the RAD52 homologue, rad22+, has a less severe effect (20).
There are a number of possible explanations for the difference in phenotypes resulting from the deficiency of RAD52 homologues between S. cerevisiae and vertebrate cells, and these explanations are not mutually exclusive. There may be as yet undescribed vertebrate homologues of RAD52, which might compensate for the absence of Rad52. The recent description of S. cerevisiae RAD59, a homologue of RAD52, and its role in intrachromosomal recombination (1) suggests that other vertebrate RAD52 homologues remain to be defined. However, neither low-stringency Southern hybridization nor PCR with degenerate primers encompassing the most highly conserved N-terminal region identified any RAD52 homologue in chicken genomic DNA or cDNA, respectively. Nevertheless, a complex of Rad55 and Rad57 has been shown to act similarly to Rad52 in facilitating strand exchange by Rad51 in the presence of replication protein A in vitro (32), suggesting that any putative Rad52 homologue which is responsible for this Rad51-dependent function of Rad52 may be a functional but not necessarily a structural homologue.
In addition, the precise mechanism for recombinational repair may differ between vertebrate and yeast species. Accordingly, the relative contributions of Rad51, Rad52, and Rad54 homologues to recombinational repair may not be identical, explaining the observed weaker phenotype of the RAD52-deficient vertebrate cells compared to RAD51- and RAD54-deficient cells.
RAD52/ DT40 clones showed a significant
decrease in the frequency of targeted integration at the
Ig and -Actin loci while showing normal
sensitivity to -rays and MMS, in agreement with observations on
RAD52-deficient murine ES cells. DSB repair in DT40 cells
relies on recombinational repair, as shown by the severe effects of
RAD54 deficiency (5). While the absence of such hypersensitivity to DSBs implies a secondary role for Rad52 in recombinational repair, recombination of transfected DNA with genomic
sequences may be a more subtle method to define the involvement of
Rad52 in homologous recombination than is a survival assay. Recent in
vitro studies of strand exchange reactions have indicated that
Rad52 is required during the critical, early steps in genetic recombination (3, 21, 28). Since the DNA structure is known to play a role in determining the genetic requirements for yeast recombination in vivo (30), the in vitro reaction may
reflect the homologous recombination of transfected plasmid DNA
rather than the recombinational repair of chromosomal DNA. Rad52
may facilitate targeted integration by specifically interacting with transfected targeting construct DNA, which might explain the results obtained in the gene-targeting experiments above. Further genetic studies with knockout mouse and DT40 technology may help to clarify the
relationships in the complex mechanism(s) of recombination and
chromosomal maintenance.
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ACKNOWLEDGMENTS |
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We would like to thank T. Shibata (Riken, Wako, Japan), T. Ogawa (Institute of Genetics, Mishima, Japan), and A. Pastink (University of Leiden, Leiden, The Netherlands) for critically reading the manuscript; Y. Kubota (Caltech, Pasadena, Calif.) for statistical analysis; and M. Hashishin, Y. Sato, O. Koga, and M. Hirao for their excellent technical assistance.
C.M. is the recipient of a JSPS Postdoctoral Fellowship. The Bayer-chair of the Department of Molecular Immunology and Allergology is supported by Bayer Yakuhin, Kyoto, Japan. This work was supported in part by a Grant-in-Aid for Scientific Research on Priority Areas from the Ministry of Education, Science and Culture of Japan and by a grant from The Mochida Memorial Foundation for Medical and Pharmaceutical Research. The Basel Institute for Immunology was founded and is supported by F. Hoffmann La-Roche Ltd., Basel, Switzerland.
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FOOTNOTES |
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* Corresponding author. Mailing address: Bayer-chair Department of Molecular Immunology and Allergology, Faculty of Medicine, Kyoto University, Konoe Yoshida, Sakyo-ku, Kyoto 606-8501, Japan. Phone: 81-75-771-8159. Fax: 81-75-771-8184. E-mail: stakeda{at}mfour.med.kyoto-u.ac.jp.
Present address: Department of Cellular Immunology,
Heinrich-Pette-Institute, 20251 Hamburg, Germany.
Present address: Department of Radiation and Cellular Oncology,
University of Chicago, Chicago, IL 60637.
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Abstract
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Materials & Methods
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Discussion
References
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Molecular and Cellular Biology, November 1998, p. 6430-6435, Vol. 18, No. 11
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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284: 31945-31952
[Abstract] [Full Text] -
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