Repression of mutagenesis by Rad51D-mediated
Nagasawa1, Salustra S. Urbin, Joel S. Bedford1and, Larry H. Thompson*
Biosciences Directorate, Lawrence Livermore National Laboratory, Livermore, CA 94551, USA and1Department of
Environmental and Radiological Health Sciences Colorado State University, Fort Collins, CO 80523, USA
Received December 20, 2005; Revised and Accepted February 14, 2006
Homologous recombinational repair (HRR) restores
chromatid breaks arising during DNA replication
and prevents chromosomal rearrangements that
can occur from the misrepair of such breaks. In
vertebrates, five Rad51 paralogs are identified that
contribute in a nonessential but critical manner to
HRR proficiency. We constructed and characterized
a knockout of the paralog Rad51D in widely studied
CHO cells. The rad51d mutant (clone 51D1) displays
sensitivity to a diverse spectrum of induced DNA
damage including g-rays, ultraviolet (UV)-C radiation,
and methyl methanesulfonate (MMS), indicating the
chromatid breaks/gaps and isochromatid breaks are
elevated 3- to 12-fold, but the chromosome number
distribution remains unchanged. Most importantly,
51D1 cells exhibit a 12-fold-increased rate of hprt
mutation, as well as 4- to 10-fold increased rates of
gene amplification at the dhfr and CAD loci, respect-
ively. Xrcc3 irs1SF cells from the same parental CHO
line show similarly elevated mutagenesis at these
three loci. Collectively, these results confirm the a
priori expectation that HRR acts in an error-free
(chromosomal aberrations, loss of gene function
and increased gene expression), all of which are
associated with carcinogenesis.
A major goal in cancer biology is to understand the mechan-
isms of origin of genomic alterations that promote the
progressive conversion of normal cells to a fully malignant
phenotype. Double-strand break (DSB) repair pathways play a
pivotal role in carcinogenesis, as chromosomal rearrange-
ments are a fundamental feature of cancer cells. In model
systems, mutations in DSB repair pathways consistently
manifest phenotypes of spontaneous chromosomal instability
as elevated aneuploidy, chromosome breaks and exchanges
and micronuclei. However, there is little information on the
contributions oftheDSBrepairpathwaystoothercrucial types
of spontaneous genetic alterations that are integral to carci-
nogenesis, such as gene mutation or amplification.
The DNA repair pathways of nonhomologous end joining
(NHEJ) and homologous recombinational repair (HRR) are
responsible for eliminating DSBs arising from endogenous
processes or DNA damage caused by exogenous agents. In
mammalian cells HRR is critical for restarting broken
replication forks that encounter single-strand breaks or
other lesions (1,2) (www.landesbioscience.com), and for the
error-free repair of DSBs occurring in chromosomal regions
that have already replicated during S phase. HRR is essentially
inactive during G1 phase since DSB-mediated recombina-
tion between homologous chromosomes occurs at a very
low frequency (10?5to 10?6) (3). HRR activity is responsible
for the classical S-phase resistance of cells to ionizing radi-
ation (IR) and may decline in G2phase (4,5).
In vertebrate cells, HRR is mediated by the Rad51 strand
transferase acting with other proteins that include the five
Rad51 paralogs (XRCC2-3, Rad51B-C-D), as reviewed in
(6,7). Although cycling cells die rapidly without Rad51 (8),
the ancillary function(s) provided by the Rad51 paralogs are
not essential, as mutants can grow, but with impaired viability.
Mutant cell lines defective in XRCC2, XRCC3 or RAD51C
were produced by random mutagenesis in Chinese hamster
cells, isolated on the basis of IR sensitivity (9,10), and used
to clone the complementing human cDNAs (11–14). These
mutants consistently show high levels of chromosomal
aberrations, extreme sensitivity to crosslinking agents, and
IR-induced Rad51 focus formation and defective HRR
They also showdefective
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Nucleic Acids Research, 2006, Vol. 34, No. 5
measured by the repair of an enzymatically induced DSB in a
direct repeat substrate (6). In general, the rodent Rad51 para-
log mutants are phenotypically similar to BRCA2-defective
cells as reviewed previously (6), but it is noteworthy that only
BRCA2 is essential for cell viability (15). Although the ham-
ster cellRad51 paralog mutants have provedvaluablein study-
ing HRR, none is isogenic with its parental cell line because of
induced mutagenesis, and they are often not fully complemen-
ted by transfected human cDNAs or genes. In chicken DT40
cells, gene targeting has produced mutants for all five paralogs
(16,17). Although they share some common phenotypic traits
and resemble the hamster mutants in many respects, among
them they have differential sensitivity to the crosslinker
cisplatin and the DSB-induced agent camptothecin (18).
Gene knockouts of Xrcc2, Rad51b and Rad51d in mice
cause embryonic lethality, usually early in development
(19–21). In the Xrcc2 mouse knockout study (22), early
growth arrest of MEFs from 13.5-day-old mutant embryos
in culture was observed. However, it was possible to obtain
immortalized MEF cultures at a very low frequency. Both
primary and immortalized MEFs displayed substantial gain
and loss of chromosomes in addition to elevated chromosomal
aberrations (22). Using the rad51d knockout mouse, MEF
cell lines could be established in a Trp53-deficient back-
ground, and these cells exhibited chromosomal instability,
aneuploidy and centrosome fragmentation, but no reduction
in spontaneous sister-chromatid exchange (SCE) (23).
In this study we describe a new isogenic rad51d mutant of
CHO cells and characterize its phenotype, with emphasis on
spontaneous genetic instability. As expected, we find greatly
enhanced spontaneous chromosomal breakage and exchange
although, paradoxically, SCE is unchanged. Most importantly,
we demonstrate increased spontaneous rates of mutagenesis in
the form of gene amplification of the CAD and dhfr loci, as
well as a greatly increased mutation rate at the hprt locus. We
confirm these findings with another Rad51 paralog CHO
mutant, xrcc3 irs1SF. These studies provide the first deter-
mination of the quantitative contribution of Rad51 paralogs
in preventing these two classes of gene-specific alterations that
are intrinsically relevant to carcinogenesis.
MATERIALS AND METHODS
Cell culture and cell cycle analysis
CHO AA8 cells (24) were grown in monolayer or suspension
culture in aMEM supplemented with 10% fetal bovine serum,
100 mg/ml streptomycin and 100 U/ml penicillin. Cells were
counted and analyzed on a Coulter?Multisizer II. The plating
efficiency of AA8 and other repair-proficient cell lines was
?90%, and that of 51D1 was ?70%; the doubling times for
AA8 and 51D1 were ?13 and ?16 h, respectively.
To determine the cell cycle distribution of each cell line
5 · 105cells were treated with 10 mg/ml BrdUrd for 20 min
at 37?, fixed with 70% ethanol and stained with fluorescein
isothiocyanate (FITC)-conjugated anti-BrdUrd antibody (BD
of cells in each phase and DNA content, respectively.
Fluorescence measurements of each sample were made on a
FACscan (Becton Dickinson) and the data analyzed using Cell
Mutagen sensitivity was determined by colony formation in
10 cm dishes. When most colonies were clearly visible by eye,
disheswere rinsed with phosphate-bufferedsaline(PBS),fixed
with 95% ethanol and stained with Gram Crystal Violet
(Becton Dickinson). Exposure to genotoxic agents was as
follows: UV radiation, as described (24);137Cs g-irradiation,
at 5 · 105cells in 15 ml tubes kept on ice; methyl methanes-
ulfonate (MMS) and mitomycin C (MMC), at 1 · 106cells in
10 ml suspension cultures were exposed to drug at 37?C for 60
min, chilled on ice, centrifuged, resuspended in fresh medium.
Mitotic cells were collected by a shake-off procedure,
obviating the need for colcemid collection, and centrifuged
at 200 · g for 3 min. The cell pellet was gently broken up,
resuspended in 10 ml of 37?C 75 mM KCl hypotonic buffer,
and incubated for 7 min in a 37?C water bath. Two ml of fresh
3:1 methanol:acetic acid (Carnoy’s) fixative was added
directly to the cell suspension in hypotonic buffer and gently
mixed. The suspensions were centrifuged at 200 · g for
4 min, the supernatant was removed and the cell pellet was
gently broken up and fixed dropwise with 4 ml of fresh
fixative. This procedure was repeated two more times, and
cell suspensions were dropped on to cold, wet slides,
air-dried and desiccated for 24 h at 37?C. The next day, slides
were stained in a 10% Giemsa solution (Gurr), dried and
mounted with CytoSeal? 60 mounting medium (Microm
International) and a coverslip. Non-polyploid metaphase
chromosome spreads of good quality were examined under
a 100· objective and 2· optivar using a Nikon Microphot
aberration frequencies (mainly chromatid-type) were scored.
Chromatid gaps were defined as fully achromatic lesions less
than the width of a chromatid arm, chromatid breaks being
separated at a width greater than the chromatid arm or
displaced from the main chromatid axis (25). Sister chromatid
previously (26) by measuring 50 cells for each cell line in
each of two experiments.
Rad51D targeting vector construction
puro.LARA were derived from a universal backbone targeting
vector designated pTnT.neo (details available on request).
pTnT-neo.LARA was constructed by first inserting a
285 bp PCR fragment containing RAD51D exon 4 between
XhoI and AflII of pTnT-neo. A 2.8 kb HincII/HindIII blunted-
end fragment of the left arm containing the sequences of
Rad51D intron 3 region was ligated into pTnT-neo at the
BstEII site to create a 9.1 kb pTnT-neo.LA. The right arm
containing a 2.7 kb KpnI/XmnI blunted-end fragment from
regions of exons 5 and 6 and introns 4–6 was inserted into
pTnT-neo.LA at the filled-in AscI site to create the 11.8 kb
pTnT-neo.LARA construct used to target the first RAD51D
allele. The left arm of the gene-targeting vector containing the
puromycin gene was created by first ligating a 4.2 kb BamHI/
PacI fragment of pTnT-puro and a 5.0 kb BamHI/PacI frag-
ment from pTnT-neo.LA to produce a 9.2 kb pTnT-puro.LA.
A 335 bp AscI/XhoI fragment containing exon 4 derived from
Nucleic Acids Research, 2006, Vol. 34, No. 51359
pTnT-neo.LARA was cloned into the AscI/XhoI sites of
pTnT-puro.LA, and the 2.7 kb AscI/ClaI right arm fragment
was inserted into the modified pTnT-puro.LA at the AscI/ClaI
restriction sites containing exon 4 to create the 12.1 kb
pTnT-puro.LARA, which was used to disrupt the second
For gene targeting, 3 · 107cells were washed and resus-
pended in 1 ml cold electroporation buffer [20 mM HEPES
(pH 7), 137 mM NaCl, 5 mM KCl, 0.7 mM Na2HPO4, 6 mM
glucose], mixed with 10 mg linearized (pTNT.neo.LARA
or pTNT.puro.LARA) DNA, electroporated at 260 V/1600
mF, incubated for 5 min on ice and plated in T150
flasks for 24 h to allow for selective marker expression
Targeting RAD51D alleles
Cells transfected with pTNT.neo.LARA were plated into
10 cm dishes at ?2 · 106cell/dish in 20 ml medium contain-
ing 1.7 mg/ml G418 (Gibco Invitrogen) and incubated for
5 days at 5% CO2and 37?C, after which the medium was
replaced with fresh medium (supplemented with 10% dialyzed
arabinofuranosyl-5-iodouracil (FIAU); cells were incubated
an additional 5 days. Each dish contained a pool of
?150 drug-resistant colonies, which were harvested for freez-
ing and DNA isolation (QIAamp?DNA Blood Mini Kit,
Qiagen Inc.). The frequency of G418 resistant colonies
averaged2 · 10?4, and the
?6.3-fold. A single pool of clones containing a targeting
event was processed through two successive rounds of PCR
screening, first in 30 sub-pools of 15 clones each, and then as
single clones. Transfectant pools were screened by PCR in
three steps. First, we tested for HR of the left arm of the
targeting vector by PCR amplification from neo to upstream
Rad51D sequence. Second, pools were tested by PCR across
the right arm from neo to downstream Rad51D sequence, and
finally across the entire targeted (extended) gene. The
experiment yielded at least three independent clones (one
electroporation yielded three positive dishes). One positive
clone was then used to target the second RAD51D allele by
10.5 mg/ml puromycin for 2 days. Selection media was chan-
ged to G418/FIAU medium at 1.7 mg/ml and 0.1 mM, respect-
ively (10% dialyzed serum) and grown for 5 days. Selection in
G418 ensured elimination of clones that retargeted the allele
already targeted in the first round of targeting. Puromycin
(10.5 mg/ml) was then added back to the dishes and incubated
an additional 4 to 5 days to ensure all killing of cells with-
out puromycin integration. Each dish contained a pool of
?150 drug-resistant colonies. The frequency of puro-
resistant colonies averaged ?5 · 10?4, and the FIAU enrich-
ment was ?6.5-fold. Gene-targeted cells were identified and
cloned as detailed above. Three clones independently targeted
for the second allele were identified.
Two positive clones were treated with Cre recombinase
plasmid, pBS185. We identified single clones that underwent
Cre-mediated recombination between the LoxP sites flanking
and selecting in
the targeted 51D exon 4 and selectable marker sequences. The
identification was based on sensitivity to both selectable
markers, as well as MMC.
A gene-complemented clone of 51D1, called 51D1.3, was
created by transfecting 51D1 cells with a bacterial artificial
RAD51D gene, followed by continuous selection in 15 nM
MMC. Single colonies were picked, and Rad51D expression
was verified by western blot.
Nuclear extracts were prepared from CHO cells using the
NE-PER kit (Pierce Biotechnology) and separated on a
12% Bis–Tris gel (NuPage system, Invitrogen) after normal-
ization for loading by Bradford analysis. After blotting to
PVDF membrane, the blot was blocked overnight in PBS-T
containing 5% milk at 4?C. Anti-Rad51D 5A8/4 (Novus
Biologicals) was used at 1:300 for 2 h at room temperature
in PBS-T/5% milk and washed with PBS-T before incubation
with secondary antibody, anti-mouse-horseradish peroxidase
(HRP) (Santa Cruz Biotechnology Inc.) in PBS-T/5%milk.
Final development was achieved using a chemiluminescent
HRP substrate (BioRad). Antibody against Lamin A/C
(H-110) (Santa Cruz Biotechnology Inc.) was used as a
loading control, as described above at 1:300 with an anti-
rabbit-HRP secondary (Santa Cruz Biotechnology Inc.).
A total of 10 ml of cell suspension at 1 · 105cell/ml was
treated with 5 mM MMC or 75 mg/ml MMS for 1 h, washed
once with medium, and resuspended in 10 ml fresh medium.
Cells were incubated for 4 h and then centrifuged on to glass
slides at 2000 r.p.m. for 5 min using a Cytospin?4 cytocentri-
fuge (Thermo Shandon). Cells were fixed in 2% paraformal-
dehyde for 15 min, permeabilized in cold 0.2% Triton
X-100 for 5 min, and blocked in 1% BSA for 1 h. The slides
were incubated with anti-Rad51 antibody (clone H-92, Santa
Cruz Biotechnology Inc.) at 4?C overnight (1:1000 dilution in
1% BSA), and Alexa Fluor?488 goat anti-rabbit secondary
antibody (A-11008; Molecular Probes) at room temperature
for1h.Glass slideswere mountedusingVectashieldmounting
medium with DAPI (H-1200; Vector Laboratories). Fluo-
rescence images were captured on Quips PathVysion using
an Axiophot II fluorescence microscope and Rad51 foci were
Mutation and gene amplification rates
Hprt mutation rate and gene (dhfr and CAD) amplification
rates were determined by fluctuation analysis (27). Replica
cultures (12–24 per experiment) were seeded with 100 cells
and grown in suspension to 1–2 · 106cells/replica, plated and
incubated under 6S-Gua selection for hprt mutant recovery
(24). To recover cells having amplified dhfr or CAD genes,
selection was done in 300 nM methotrexate (28) or in 360 mM
PALA [N-(phosphonacetyl)-L-aspartate] and 1 mM dipyri-
dimole (29), respectively. Hprt, dhfr and CAD mutation
rates were calculated using the Poisson P0term (27) or the
method of the mean (30).
1360 Nucleic Acids Research, 2006, Vol. 34, No. 5
Targeted conditional disruption of RAD51D
The strategy for inactivating RAD51D was to delete exon 4
(amino acids 88–115), which contains the GKT Walker A-box
for ATPase activity. This deletion also changes the reading
frame and results in a highly truncated polypeptide
(S88!R...114X). In order to disrupt the two alleles of
RAD51D [as determined by fluorescence in situ hybridization
(FISH) analysis;data notshown],alleleswere targeted one ata
time using two different selectable markers (Figure 1A).
Targeting vectors were designed to maintain functional
RAD51D alleles such that LoxP sites flank both exon 4 and
the associated selectable marker. After transfection with the
targeting vector containing the neo gene and selecting G418
resistant clones, pools of clones were screened by PCR
analysis, using primers specific for DNA sequence in neo
and genomic DNA sequence outside of the homologous
arms in the vector, to determine the presence of correctly
targeted cells. Positive pools were reduced to pure clones
as detailed in Materials and Methods, and such a clone was
then transfected with the vector containing the puro selectable
marker.Transfectants were selected in puromycinand G418 to
insure the initial allele was not re-targeted. Pools of clones
were screened by PCR analysis in the same manner as in the
first-allele targeting. Two clones, from independent pools and
having both Rad51D alleles targeted, were isolated and
designated 51D1Lox and 51D2Lox. Simultaneous deletion
of exon 4 in both alleles was accomplished by transfection
with Cre recombinase, resulting in mutant clones 51D1 and
51D2, respectively. Disruption was verified by the inability of
Cre-treated cells to grow in the presence of puromycin, G418,
and MMC (Figure 1B). Western analysis was performed to
verify absence of the Rad51D protein in the knockout cells.
Figure 1C shows the absence of the 39 kDa band in the 51D1
cells. The level of Rad51D appears higher in the AA8 parental
cells than in the 51D1Lox and the 51D1.3 gene-complemented
cells. RAD51D expression may be reduced in 51D1Lox cells
because of the embedded neo and puro gene promoters, which
may retard transcription. The 51D1.3 cells may possess only
one copy of the gene, whose expression could also be
influenced by its ectopic location.
Exponentially growing 51D1 cells show clear abnormalities
in their cell cycle distribution (Figure 1D). There is a much
with the AA8 and 51D1.3 control lines. Moreover, there is a
measurable increase in the proportion of tetraploid cells (5%)
in 51D1 cultures. Each of these features is qualitatively similar
Figure 1. Targetingvectordesignandconfirmationoftargeting.(A)Configurationoftargetingvectors.ThetargetingvectorscontainedaHSV-TKgenefornegative
selection and, forpositive selection, a purogene forone allele and a neo gene forthe other allele.Step 1 resultsin a targeted recombination event in which exon 4 is
replaced by a functional exon 4 flanked by neo/puro and LoxP sites. After each allele has been targeted, without affecting RAD51D function, treatment with
After Cre transfection and phenotypic expression, single-cell clones were picked, distributed into 24-well trays, grown for 8 days in media containing, geneticin,
parental, mutant and gene-complemented mutant cells. (D) DNA profiles of mutant and control cell lines.
Nucleic Acids Research, 2006, Vol. 34, No. 51361
to what was seen in rad51d trp53 knockout mouse embryonic
Increased sensitivity of rad51d cells to killing by diverse
DNA damaging agents
We used colony formation assays to determine survival of
rad51d cells after exposure to four commonly used and
distinctly different DNA-damaging agents (Figure 2). The
rad51d 51D1 cells show exquisite sensitivity (?80-fold) to
the interstrand crosslinking agent MMC relative to par-
ental AA8 and 51D1Lox, and the gene-complemented clone
51D1.3 (Figure 2A). (Fold sensitivity is measured as the dose-
reduction factor at 37% cell survival.) With g-rays, both the
51D1 and 51D2 mutant clones show a ?1.5-fold sensitivity
(Figure 2B), similar to that of the xrcc3 irs1SF mutant. 51D1
cells show substantial sensitivity (?5-fold) to the alkyla-
ting agent MMS (Figure 2C), as well as ?2-fold sensitivity
to UV-C (Figure 2D). For each agent the mutant cells are fully
complemented when they express the hamster RAD51D gene
(clone 51D1.3). These results show that the impairment of
HRR through loss of Rad51D compromises the ability of
CHO cells to deal with very diverse types of DNA damages.
Reduced Rad51 foci in rad51d cells after exposure to IR
Lack of Rad51 nuclear focus formation after exposure to
DNA-damaging agents is generally associated with HR
deficiency in Rad51 paralog mutants of rodent and chicken
cells[reviewed in (6)], including mouse rad51d knockout cells
(23). As expected, 51D1 cells exposed to 8 Gy g-rays or
5 mM MMS were grossly defective in Rad51 focus formation
(Figure 3A). Whereas the control cultures treated with g-rays
or MMS showed 62–85% of cells with >5 foci per cell,
51D1 showed only 7 and 1% of cells with foci, respectively
(Figure 3B). In untreated cultures the 51D1 cells showed a less
severe focus defect, i.e. an average of 2 foci per cell versus
3 foci per cell in the two control cell lines (data not shown).
Figure 2. Sensitivityofrad51dknockoutcellstoMMC,IR,MMSandUV.TheAA8parentalcelllinewasusedtocreatetwoconditionalknockoutclones,51D1Lox
and 51D2Lox e.g. (B), which have undergone independent gene targeting in the second allele of RAD51D. The rad51d 51D1 cells were stably transfected with the
(A) MMC survival curves; note log scale for dose. (B) g-ray survival curves. (C) MMS survival curves. (D) UV-C survival curves.
1362 Nucleic Acids Research, 2006, Vol. 34, No. 5
Increased spontaneous chromosomal aberrations in
Chromosomal instability is a hallmark of HR-deficient cells.
To determine the role of Rad51D in maintaining chromo-
somal integrity, we measured spontaneous chromosomal
aberrations in a large population of cells. Relative to the
Rad51D-proficient cell lines tested, the 51D1 cells showed
a significant increase in the levels multiple types of aberrations
(Table 1): chromatid breaks (5- to 12-fold), chromatid gaps
(?3-fold), and isochromatid breaks (5- to 6-fold). Also seen in
the 51D1 cells was a low level of chromatid exchanges, as
illustrated in Figure 3C, which were not detected in the
Rad51D-proficient cells. Though dicentric chromosomes
were no more prevalent in the 51D1 cells than in the parental
51D1Lox cells, ringed chromosomes were only detected in the
51D1 cells. Aneuploidy (chromosome gain or loss) has been
associated with CHO and MEF cells deficient in the Rad51
paralogs XRCC2 and XRCC3 (22,31). We wished to
determine if the Rad51D deficiency caused a similar problem
with maintenance of chromosome number. CHO AA8 cells
have a modal chromosome number of 21 (32), which was
maintained in both the 51DLox and 51D1 lines (Table 2).
The percentage of cells having a gain or loss of one or two
chromosomes from the modal value also remained cons-
tant. Furthermore, the Rad51D-complemented 51D1.3 cells
maintained the same distribution of chromosome numbers.
These findings suggest that aneuploidy is not associated
with the rad51d mutation in CHO cells.
Normal spontaneous SCE in rad51d cells
As a classical, cytological manifestation of crossing over
between sister chromatids, SCE could be expected to be
reduced in 51D1 mutant cells. The average value from two
experiments was 0.30 ± 0.1 SCE per chromosome in each
of the AA8, 51D1 and 51D1.3 cell lines. Thus, some-
what surprisingly, there was no detectable reduction in the
An increased mutation rate at the hprt locus is a measure of
genomic instability at the single-gene level. Using fluctuation
analysis to measure mutation rates (27), we found that rad51d
cells have a greatly increased rate (?12-fold) of hprt mutation
calculated by both the P0method and the method of the mean
(27,30) (Figure 4). Importantly, the gene-corrected 51D1.3
Figure 3. Defective Rad51 focus formation and spontaneous chromosomal aberrations in rad51d mutant cells. (A) Immunofluorescence images of parental and
scoring 100 cells per sample. (C) Examples of spontaneous aberrations. IB, isochromatid break; G, gap; AE, asymmetrical exchange; SE, symmetrical exchange,
which derives from pairing between homologs. The most common aberrations, single-chromatid breaks, are not seen in this cell.
Nucleic Acids Research, 2006, Vol. 34, No. 5 1363
cells and the pre-Cre-treated 51D1Lox cells had mutation
rates like that of the parental AA8 cells. Hypermutability
was also seen with the xrcc3 irs1SF cells, which had an
even higher hprt mutation rate (?20-fold increased over
AA8) than the 51D1 cells. 1SFwt8 cells, which express
human XRCC3 cDNA, were partially corrected for hprt
mutagenesis (Figure 4), consistent with the incomplete
complementation previously measured for cell survival
after g-irradiation and MMC treatment (33). The aprt locus
in AA8 cells is heterozygous due to a point mutation at one of
the two alleles (24,34), and aprt is known to have ?10-fold
higher mutation rate than hprt because of a high rate of
deletion (35). There was no significant increase in mutation
rate at the aprt locus in either rad51d or xrcc3 cells. The aprt
rate for AA8 was (10 ± 3) · 10?6and for 51D1 was
(13 ± 4) · 10?6, based on the method of the mean. An
increase in mutability at the aprt locus in the 51D1 cells
could be difficult to detect, as the spontaneous rate is so
high in AA8 cells that a Rad51D-dependant increase compar-
able to that at hprt would be less than a doubling in the
Cell line Cell
Fraction of cells with chromosome number
0.09 ± 0.04 0.88 ± 0.05 0.03 ± 0.01 0.003 ± 0.005
0.06 ± 0.01 0.91 ± 0.04 0.02 ± 0.03 0.009 ± 0.008
0.06 ± 0.03 0.90 ± 0.02 0.03 ± 0.04 0.006 ± 0.010
Figure 4. Mutationratesinrad51d,xrcc3,andcontrolcelllines.(A)Methodofthemeancalculations.(B)P0calculationmethod.Experimentsweredonethreetosix
times for the Rad51D series and twice for irs1SF and 1SFwt8. Each experiment had 12 or 18 replicate dishes. Error bars are SEM values.
Table 1. Spontaneous chromosomal aberrations in rad51d mutant and control cell linesa
Cell lineNo. of cellsBreaksGaps Isochromatid
51D1 vs. 51D1Lox
51D1 vs. 51D1.3
0.07 ± 0.04
0.03 ± 0.03
0.36 ± 0.09
0.7 ± 0.1
0.8 ± 0.1
2.2 ± 0.2
0.1 ± 0.05
0.15 ± 0.07
0.7 ± 0.1
*Indicates P < 0.05 and ** for P < 0.01 using the student t-test.
aValues are given as mean number of aberrations per cell ± SEM. Each value is based on the data in three experiments.
bP ¼ 0.11.
cP ¼ 0.09.
1364 Nucleic Acids Research, 2006, Vol. 34, No. 5
Increased spontaneous amplification rates
in rad51d cells
Gene amplification is another form of mutagenesis associated
with tumor cells, in which megabase regions of DNA are
replicated in excess and maintained chromosomally or as
extrachromosomal elements. The role of HRR in preventing
such events is not known, but CHO cells deficient in the DSB
repair pathway NHEJ, because of mutant DNA-PKcs, have
increased rates of amplification (29). We determined that the
rate of gene amplification was increased 3- to 10-fold in
HRR-deficient 51D1 and irs1SF cells at both the dhfr and
CAD loci (Table 3). The gene-corrected mutant cell lines
showed rates of amplification similar to those of the parental
cells. To see if this amplification phenotype could be seen in
another paralog mutant, we tested the V79-derived xrcc2 cell
line, irs1. It also displayed an increased amplification rate
compared to the corresponding XRCC2-complemented cells
(GT619) (13), consistent with a general role for the paralogs in
preventing gene amplification.
In this study, we created in CHO AA8 cells a knockout
mutant of RAD51D (clone 51D1) that is isogenic with respect
to AA8 and 51D1Lox parental lines, and to the BAC
gene-complemented control (clone 51D1.3). To further ensure
an isogenic relationship, the 51D1 mutant was complemented
with the hamster RAD51D gene, rather than a human homolog
as has often been done historically. Although the level of
Rad51D expression in 51D1.3 cells appears to be lower
than in AA8 cells (Figure 1C), the level is adequate for full
complementation of all aspects of the mutant phenotype. 51D1
is the first isogenic mutant in DSB repair to be constructed in
Comparison of the phenotype of 51D1 cells with other
Rad51 paralog mutants
The rad51d CHO cells resemble rad51d knockout mouse
MEFs in showing very high sensitivity to killing by MMC
and lesser sensitivity to IR, UV-C and MMS (23). Irs1SF
xrcc3 CHO cells have also been shown to be very sensitive to
DNA-replication inhibitors (camptothecin and hydroxyurea)
that result in broken replication forks (36,37). Thus, HRR
allows cells to cope with a broad range of genotoxic agents,
all of which result in one-sided DSBs when replication forks
stall and then break (2). Such DSBs will also arise when
replication forks encounter single-strand breaks produced
either directly by the DNA-damaging agent (e.g. IR) or as
an intermediate during repair (e.g. MMS and MMC). Another
theoretical possibility is a role for HRR in bypassing dama-
ged bases in an error-free manner through a process of fork
regression/reversal followed by restart of the replication fork
(i.e. ‘chickenfoot’ intermediate) (38).
The profile of sensitivity of the rad51d CHO cells to various
DNA-damaging agents is also similar to that seen previously
for hamster cells deficient in other Rad51 paralogs: XRCC2
(irs1), XRCC3 (irs1SF) and RAD51C (irs3) cells (11–
13,39,40). The 80-fold sensitivity of 51D1 cells to MMC is
dramatically higher than the ?3-fold sensitivity of the DT40
rad51d mutant to MMC (16). Similar differences have been
seen between systems for other Rad51 paralog mutants,
emphasizing that there are inherent differences between
these model systems. The similarities among these Rad51
paralog mutants within each system suggest that the paralogs
have a common role in HRR.
Elevated gene amplification rates associated with HRR
Gene amplification, a form of mutagenesis in which large
regions of genomic DNA are multiply replicated and main-
tained in the genome, results in tumor cells having increased
copies of oncogenes and multi-drug resistance phenotypes
[reviewed in (41)]. DNA damage appears to play an important
role in promoting gene amplification, as it is enhanced when
cells are exposed to agents that break DNA (i.e. g-rays and
hydrogen peroxide) (42). Defects in DNA repair systems have
previously been associated with increased rates of gene amp-
lification, including NHEJ deficiency in CHO cells (29) and
mismatchrepair deficienciesinhuman tumorcells(43,44).We
have also found increased gene amplification rates in a fancg
CHO knockout mutant defective in the Fanconi anemia chro-
mosome stability pathway (J.M. Hinz and L.H. Thompson,
unpublished data). As shown in Table 3, we have made the
novel observation that HRR also plays an important role in
preventing gene amplification as evidenced by the highly
elevated rates in rad51d cells, and confirmed in the xrcc3
irs1SF and xrcc2 irs1 mutants. However, the highest reported
rates of CAD amplification occur in dna-pkcs cells, in which
the elevation was 20- to 150-fold (29). From these collective
findings it appears that multiple DNA repair pathways, includ-
ing HRR, contribute to the prevention of gene amplification.
Normal rate of SCE in rad51d cells
Sister chromatid exchange, which is thought to be a cytolo-
gical manifestation of HRR, occurs only a few times during
each S phase. SCE is greatly increased by a broad range of
genotoxic agents including inhibitors of DNA replication
(45,46). Evidence that SCE is caused at least in part by
HRR was presented in chicken cells (47). However, models
have also been proposed in which fork breakage and rejoining
Cell line Methotrexate
(units · 10?6)
(units · 10?6)
bEach experiment was done two or three times with 20 replicate cultures.
cEach experiment was done twice with 20 replicates.
dEach experiment was done once.
Nucleic Acids Research, 2006, Vol. 34, No. 51365
by NHEJ could be responsible (45,48). SCE can presumably
occur when broken replication forks are restarted by HRR (2).
A priori, one might expect that the rad51d deletion would
partially suppress SCE, but we found that 51D1 cells had
no change compared with control cell lines. Although the
rad51d mutant and other Rad51 paralog mutants in chicken
DT40 cells have 2- to 3-fold reduced rates of SCE (16), mouse
rad51d trp53 knockout MEFs also show no reduction
compared with the trp53 control cells (23).
These findings of normal SCE rates in mammalian rad51d
mutants have several possible explanations. First, Rad51D
may have no role in the putative HRR exchange event
associated with restarting a broken replication fork through
the processing of a Holliday junction (HJ) intermediate (2).
This interpretation was put forth to explain the mouse rad51d
MEF data (23). However, given the very high levels of
spontaneous chromatid breaks in the mouse and hamster
rad51d cells, which most likely derive from unrepaired broken
replication forks, this explanation seems unlikely. Second,
SCE in wild-type cells might arise primarily from NHEJ by
a process in which both parental strands are broken and forks
are restored by rejoining parental strands with daughter
strands. However, the normal frequency of SCE in xrcc5/
ku80 CHO mutants argues against this possibility (49).
Third, the restart of broken forks by HRR may normally
occur primarily through the ‘crossover’ mode of HJ resolution,
which would not produce SCE as cytologically identifiable
events (2). The visible SCE could arise in a different manner,
e.g. ‘non-crossover’ mode of HR resolution, perhaps
independently of Rad51D. In any event, other studies show
that the loss of Rad51 paralogs in mammalian cells has only a
modest or no influence on spontaneous SCE. Neither xrcc2
irs1 nor xrcc3 irs1SF shows a significant reduction in SCE
(10,50), and xrcc2 knockout mouse cells (both primary and
immortalized) show only a 30% reduction (22). Two rad51c
V79 mutants have a slightly reduced rate of spontaneous SCE
(11,12). Thus, mammalian Rad51 paralogs differ from the
chicken homologs in having a lesser quantitative contribution
to the rate of SCE. However, since the rad51d mutant MEF
cells have a clear deficiency in MMC-induced SCEs (23), the
mechanistic details of induced SCEs must differ from spon-
Role of Rad51D in chromosome stability
We observed substantially elevated chromosomal aberrations
in rad51d cells, particularly chromatid and chromosome
breaks, which presumably arise from broken replication
forks that remain unrepaired. An essential role for Rad51D
in telomere maintenance also has been reported for
telomerase-deficient cells. Rad51D was shown to localize at
the sites of telomeres in HeLa cells, and rad51d trp53 MEFs
have a high level of telomere end-to-end fusions as well as
chromatid breaks and other chromosomal abnormalities pos-
sibly associated with telomere dysfunction (23,51). The asso-
ciation of Rad51D with telomeric sequences appeared to be
specific compared with other Rad51 paralogs. In addition,
Rad51D-deficient human cells exhibited telomeric DNA
repeat shortening (51). CHO and other immortalized Chinese
hamster cells lack cytologically visible telomeres (52), but
they express telomerase activity (53) and exhibit interstitial
telomeric bands that are subject to amplification (54). The
absence of a requirement for Rad51D in telomere maintenance
in CHO cells may explain why our rad51d mutant cells grow
relatively robustly compared with the rad51d trp53 mouse
cells. Importantly, 51D1 cells show only a mild growth
retardation, having a doubling time of ?16 h compared
with ?13 h for 51D1Lox and AA8 cells.
High mutability of Rad51 paralog mutants
Genomic alterations in nucleotide sequence that are undetect-
able by cytogenetics are a crucial aspect of cancer progression,
as reviewed by (41). Mutation rate measurements at the hprt
gene have been widely used in human, Chinese hamster, and
other mammalian cells for quantifying mutagenesis in a locus
that is responsive to both point mutations and deletions
(24,55–57). Due to the functionally hemizygous nature of
the hprt locus [and physical hemizygosity in CHO cells
(32)], point mutations, insertions and gene-size deletions
are detectable, but large-scale multigenic deletions and inter-
chromosomal rearrangements are not recovered (58). High
levels of spontaneous hprt mutagenesis are often associated
with defects in mismatch repair due to increased tolerance for
mis-incorporated nucleotides (59,60), or with overexpression
of translesion polymerases, such as DINB1, Pol k and Pol b
(61–63), as such polymerases synthesize DNA in an error-
In the rad51d cells constructed in this study, and in the
commonly used CHO model cell line for HRR-deficiency
(xrcc3 irs1SF), we find highly elevated rates of mutagenesis,
showing a clear role for these Rad51 paralogs in suppressing
spontaneous mutagenesis. HRR-defective brca2 V-C8 cells
were reported to have ?4-fold elevated hprt mutation rate
compared with the non-isogenic parental hamster V79 line,
and the mutant spectrum contained an increased proportion of
deletions (64). These results, along with our data, highlight the
importance of HRR in preventing loss-of-function mutations
that are presumed to arise during DNA replication when bro-
ken forks are inaccurately repaired by NHEJ.
In conclusion, the tumorigenic progression of somatic cells
toward malignancy depends predominantly on two kinds of
altered gene expression: loss of tumor suppressor gene func-
tion and gain of inappropriate gene expression (e.g. oncogenes
and multi-drug resistance). In this study, we determined the
contribution of HRR to two classes of spontaneous gene-level
mutagenesis: (i) loss of hprt gene function measured as 6-
thioguanine resistance, and (ii) dhfr and CAD gene amplifica-
tion, which confer drug resistance via increased gene dosage
(overexpression). The rad51d cells have >10-fold higher hprt
mutagenesis and 4- to 10-fold elevated gene amplification
rates. For each of the three marker genes, we confirmed the
instability phenotype in non-isogenic xrcc3 cells from the
same parental CHO line. These genetic instabilities measured
at the single-gene level should be viewed in concert with the
high, classically HRR-associated cytological measurements of
chromosomal breakage and exchange, which are elevated by a
similar magnitude in rad51d cells, but only represent a minor
facet of the complex genomic alterations required for tumor
1366 Nucleic Acids Research, 2006, Vol. 34, No. 5
The authors thank Lynn Carr and Tricia Allen, teachers from
Burroughs High School, Ridgecrest, CA, for their assistance
with fluctuation analyses. The authors also thank Angela Hinz
for her technical expertise. Deserving recognition is the Drug
Synthesis and Chemistry Branch, Division of Cancer
Treatment, National Cancer Institute, for providing PALA.
This work was performed under the auspices of the U.S.
Department of Energy by the University of California,
Lawrence Livermore National Laboratory under Contract
No. W-7405-Eng-48. The DOE Low-Dose Program and
NCI/NIH grant CA89405 funded this work. Funding to pay
the Open Access publication charges for this article was
provided by NIH grant 1 R01 CA112566-01A1.
Conflict of interest statement. None declared.
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