MOLECULAR AND CELLULAR BIOLOGY, Nov. 2004, p. 9305–9316
Copyright © 2004, American Society for Microbiology. All Rights Reserved.
Vol. 24, No. 21
Genetic Steps of Mammalian Homologous Repair with Distinct
Jeremy M. Stark,1Andrew J. Pierce,1† Jin Oh,1‡ Albert Pastink,2and Maria Jasin1*
Molecular Biology Program, Memorial Sloan-Kettering Cancer Center, New York, New York,1and
Department of Toxicogenetics, Leiden University Medical Center, Leiden, The Netherlands2
Received 22 July 2004/Returned for modification 3 August 2004/Accepted 6 August 2004
Repair of chromosomal breaks is essential for cellular viability, but misrepair generates mutations and gross
chromosomal rearrangements. We investigated the interrelationship between two homologous-repair path-
ways, i.e., mutagenic single-strand annealing (SSA) and precise homology-directed repair (HDR). For this, we
analyzed the efficiency of repair in mammalian cells in which double-strand break (DSB) repair components
were disrupted. We observed an inverse relationship between HDR and SSA when RAD51 or BRCA2 was
impaired, i.e., HDR was reduced but SSA was increased. In particular, expression of an ATP-binding mutant
of RAD51 led to a >90-fold shift to mutagenic SSA repair. Additionally, we found that expression of an ATP
hydrolysis mutant of RAD51 resulted in more extensive gene conversion, which increases genetic loss during
HDR. Disruption of two other DSB repair components affected both SSA and HDR, but in opposite directions:
SSA and HDR were reduced by mutation of Brca1, which, like Brca2, predisposes to breast cancer, whereas SSA
and HDR were increased by Ku70 mutation, which affects nonhomologous end joining. Disruption of the
BRCA1-associated protein BARD1 had effects similar to those of mutation of BRCA1. Thus, BRCA1/BARD1
has a role in homologous repair before the branch point of HDR and SSA. Interestingly, we found that Ku70
mutation partially suppresses the homologous-repair defects of BARD1 disruption. We also examined the role
of RAD52 in homologous repair. In contrast to yeast, Rad52?/?mouse cells had no detectable HDR defect,
although SSA was decreased. These results imply that the proper genetic interplay of repair factors is essential
to limit the mutagenic potential of DSB repair.
An increased frequency of mutations and gross chromo-
somal rearrangements is widely observed in a variety of tumor
types and aged cells from a number of organisms (3, 9). To
understand the etiology of these genetic alterations, it is critical
to delineate the factors and pathways that influence the muta-
genic potential of DNA damage repair. A critical type of DNA
damage is a chromosomal double-strand break (DSB), which can
be formed by reactive oxygen species, topoisomerase failure, ra-
diation treatment, and DNA replication. In mammalian cells,
such damage can be repaired through multiple pathways: non-
homologous end joining (NHEJ), which can result in deletions
and insertions at the DSB site, or pathways that utilize homology
for repair, which can also be variably mutagenic (2, 17, 26, 34).
Pathways that utilize sequence homology for repair are broad-
ly characterized into two types based on whether homologous
associations arise from strand exchange or strand-annealing
activities. Homologous recombination, also called homolo-
gy-directed repair (HDR), is initiated by a strand exchange
protein, the prototype being RecA or its eukaryotic equivalent,
RAD51 (51). Strand exchange, which involves a single strand
invading a DNA duplex, results in a gene conversion, since the
duplex templates repair. The mutagenic potential of HDR,
which is generally considered to be quite low compared to
other DSB repair pathways, is related to the choice of tem-
plate, as well as to the extent or outcome of gene conversion.
For example, HDR involving the identical sequence on a sister
chromatid should be nonmutagenic. Because the sister chro-
matid is the preferred template for HDR in mammalian cells
(20), HDR is presumed to be predominantly a nonmutagenic,
precise type of repair. However, HDR is also well known to
have the potential to result in genetic loss when the template is
the allele on the homologous chromosome, given the heterozy-
gosity between maternal and paternal chromosomes. Although
apparently rare on a per allele basis, genetic loss from these
repair events can arise from extensive gene conversion or
crossing over (40, 50). Since this loss of heterozygosity de-
creases genetic variability and can uncover recessive genetic
mutations, it is considered to be mutagenic.
The other DSB repair pathway involving sequence homol-
ogy is single-strand annealing (SSA). SSA arises from the an-
nealing of complementary single strands formed after resec-
tion at a DSB. Thus, when sequence repeats are present near
a DSB, they can undergo SSA, resulting in a deletion of se-
quences between the repeats. In contrast to HDR, therefore,
SSA is always mutagenic. Because ?50% of the mammalian
genome consists of repeat sequences (15), SSA is potentially
an important pathway of mutagenesis. Consistent with this
notion, mutagenic deletions associated with cancer and human
genetic diseases often exhibit homology at the breakpoint junc-
tion, which is suggestive of SSA. For instance, both somatic
and germ line rearrangements and deletions involving homol-
ogous Alu sequences are associated with human disease (8).
Although the structure-specific endonuclease ERCC1/XPF,
* Corresponding author. Mailing address: Molecular Biology Pro-
gram, Memorial Sloan-Kettering Cancer Center, 1275 York Ave., New
York, NY 10021. Phone: (212) 639-7438. Fax: (212) 717-3317. E-mail:
† Present address: Department of Microbiology, Immunology, and
Molecular Genetics, University of Kentucky College of Medicine, Lex-
‡ Present address: Department of Life Science, College of Natural
Sciences, Hangyang University, Seoul 133-791, Korea.
the homologue of yeast Rad10/Rad1, has been implicated in
strand-processing steps during SSA (1, 43), the protein(s) in-
volved in the annealing step of SSA has not been as well
studied in mammalian cells. The RAD52 protein is implicated
in this step because Rad52 has a key role in SSA in Saccharo-
myces cerevisiae (52) and human RAD52 has strand-annealing
activity in vitro (46, 52). In yeast, Rad52 also has a critical role
in the HDR pathway (52); its role in mammalian homologous
repair is uncertain, however, because Rad52 mutation in the
mouse leads to only a small decrease in gene targeting (42), a
pathway that utilizes other homologous-repair components.
HDR and SSA are expected to share mechanistic interme-
diates, such as resected single strands, and thus some protein
factors, but other intermediates and factors are expected to be
specific for each pathway, suggesting that the two homologous-
repair pathways may be competitive. Although a weak com-
petitive interaction of these pathways has been suggested (11,
55), a comparative analysis using mutants from each repair
pathway has not been performed. Given the strikingly different
mutagenic outcomes of these homologous-repair pathways, we
investigated the efficiency of SSA and HDR in multiple genetic
backgrounds in mammalian cells. We present evidence that
RAD51 function is critical to limit the mutagenic potential of
homologous repair; in the most extreme example, we found
that disruption of RAD51 can lead to a ?90-fold shift in path-
way usage toward SSA. Additionally, we found that expression
of an allele of RAD51 that is defective for ATP hydrolysis
results in more extensive gene conversion, which increases the
mutagenic potential of HDR. Furthermore, we present evi-
dence that two additional protein complexes involved in DSB
repair, i.e., BRCA1/BARD1 and KU70/KU80, have opposite
effects on the homologous-repair pathways. Interestingly, Ku70
mutation partially suppresses the effect of BRCA1/BARD1
disruption, suggesting early and late roles for this complex.
Finally, we provide evidence that mammalian RAD52 pro-
motes SSA but not HDR, highlighting a striking difference
between yeast and mammalian cells in the reliance on partic-
ular repair factors. Our results clearly demonstrate that the
genetic interplay of DSB repair factors is essential to limit the
mutagenic potential of homologous repair in mammalian cells.
MATERIALS AND METHODS
Plasmids and cell lines. Green fluorescent protein gene (GFP) fragments for
SA-GFP were derived from pEGFP-N1 (Clontech); hprtSAGFP was generated
similarly to hprtDRGFP (36). After linearization with KpnI/SacI, the hprt vectors
were electroporated into embryonic stem (ES) cells suspended in 650 ?l of
phosphate-buffered saline in a 0.4-cm-diameter cuvette by pulsing at 800 V and
3 ?F. Puromycin was added 24 h later at 1 ?g/ml for all cells except the Brca1?/?
and Brca2L1/L2cells, for which the concentration was 2 ?g/ml. After 6 days of
puromycin selection, 6-thioguanine was added at 10 ?g/ml for all lines except
Brca1?/?and Brca2L1/L2. Colonies were isolated and analyzed by Southern
blotting with a GFP probe (36, 37). Mouse J1 (wild-type), Ku70?/?, Brca1?/?,
and Brca2L1/L2(also known as Brca2lex1/lex2) ES cell lines and their DR-GFP
derivatives were previously described (27, 31, 32, 36, 47), as were the Ercc1?/?
and Rad52?/?ES cells (33, 42).
For stable RAD51 expression in E14 ES cells containing H-DR-8mu inte-
grated at the hprt locus (12), the P59-CAGGS-RAD51K133R and P59-CAGGS-
RAD51WT cassettes (49) were targeted to the Pim1 locus. Two independently
targeted clones were used in the recombination experiments. The generation of
pCAGGS expression vectors for human RAD51-WT, RAD51-K133A, RAD51-
K133R, KU70, BRC3, BARD1-WT, and BARD1-hB202 were previously de-
scribed (36, 49, 58); for ERCC1 and RAD52, vectors were generated by PCR of
cDNAs, followed by sequence confirmation.
Repair assays. To measure the repair of an I-SceI-generated DSB, 50 ?g of
the I-SceI expression vector pCBASce (40) was mixed with 5 ? 106ES cells
suspended in 650 ?l of phosphate-buffered saline in a 0.4-cm-diameter cuvette,
followed by pulsing the cells at 250 V and 950 ?F, except for the ERCC1- and
RAD52-deficient cell lines, which were pulsed at either 250 or 280 V. The
pCAGGS expression vectors for other factors were added as follows: 30 ?g for
RAD51, 50 ?g for BARD1, 45 ?g for BRC3, and 15 ?g for KU70, ERCC1, or
RAD52. GFP-positive cells were quantitated by flow cytometric analysis 2 days
after electroporation on a Becton Dickinson FACScan. To calculate the differ-
ence in homologous repair relative to wild-type cells (see Fig. 2 and 4d), the
percentage of GFP?cells from each individual transfection was divided by the
mean value for wild-type cells transfected only with the I-SceI expression vector.
The calculation for the KU-deficient cells (see Fig. 5b) is exactly analogous. To
calculate the difference in homologous repair for the ERCC1- and RAD52-defi-
cient cell lines, the percentage of GFP?cells from individual transfections that
included the complementing vector was divided by the percentage of GFP?cells
from the parallel transfection with the I-SceI expression vector alone, since trans-
fection conditions varied between experiments. From this calculation, the relative
value from the transfection of I-SceI alone in the ERCC1- and RAD52-deficient cell
lines is always equal to 1, so that there is no error bar for these values. For
H-DR-8mu, recombination frequencies were determined by dividing the number
of G418-resistant colonies by the number of cells that survived electroporation.
Homologous-repair frequencies are the mean of at least three independent trans-
fections, and error bars represent the standard deviation from the mean. Statistical
analysis of homologous-repair frequencies was performed using the unpaired t test.
PCR analysis. For H-DR-8mu cell lines, PCR analysis was described previ-
ously (12). Statistical analysis of the distribution of gene conversion tract lengths
was performed using the unpaired t test. To determine the percent I-SceI site loss
following SA-GFP repair, genomic DNA was isolated 6 days after transfection.
The primer sequences for PCR were as follows: SAGFP3A, 5?-GCCCCCTGC
TGTCCATTCCTTATT, and SAGFP3B, 5?-ATCGCGCTTCTCGTTGGGGTC
TTT. Amplification of PCR products and determination of the fraction of I-SceI-
cleaved product were performed as previously described for DR-GFP (36).
Reporters for DSB repair by HDR and SSA. To investigate
the genetic interrelationship of pathways of homologous DSB
repair, we used chromosomal reporter substrates that allow the
introduction of a DSB, i.e., DR-GFP, which assays HDR (37),
and a novel reporter, SA-GFP, which assays SSA. In both
reporters, the DSB is generated by the rare-cutting endonu-
clease I-SceI, whose 18-bp recognition sequence has been in-
tegrated within the green fluorescent protein gene (GFP) in
such a way that it disrupts the gene.
The SA-GFP reporter consists of the GFP gene fragments
5?GFP and SceGFP3?, which have 266 bp of homology (Fig.
1a). Repair of the I-SceI-generated DSB in SceGFP3? by SSA
results in a functional GFP gene when a DNA strand from
SceGFP3? is annealed to the complementary strand of 5?GFP,
followed by appropriate DNA-processing steps. As a result,
SSA between the homologous sequences in the GFP gene
fragments produces a 2.7-kb deletion in the chromosome. The
SA-GFP reporter can also be repaired by HDR, but this repair
does not restore a functional GFP gene. We measured the
efficiency of HDR separately using the previously described
DR-GFP reporter (37). In this reporter, the I-SceI site is in-
tegrated into a full-length GFP gene (SceGFP). Repair of the
I-SceI-generated DSB by HDR results in a functional GFP
gene when repair is directed by the downstream internal GFP
fragment, iGFP (Fig. 1b). HDR does not alter the structure of
the reporter; the only change is the conversion of the I-SceI
site to a BcgI site. Presumably, if SceGFP on the sister chro-
matid is uncleaved, it also provides a template for repair, but it
is not possible to score these types of sister repair events. With
both reporters, the presence of a functional GFP gene was
9306STARK ET AL.MOL. CELL. BIOL.
scored in individual cells by green fluorescence using flow
cytometric analysis (Fig. 1c). Furthermore, the relevant repair
product was confirmed for both reporters by Southern blot
analysis of genomic DNA from enriched populations of green
fluorescent cells, as shown previously for DR-GFP (32, 37) and
here for SA-GFP (Fig. 1a).
To compare the efficiencies of HDR and SSA in mammalian
cells with these reporters, we integrated DR-GFP and SA-GFP
FIG. 1. Reporters for measuring homologous DSB repair by SSA and HDR. (a) hprtSAGFP reporter. The structure of the reporter is shown
before and after I-SceI cleavage and SSA. Note that repair by SSA results in a 2.7-kb deletion in the chromosome. Southern blot analysis is shown
from an untransfected cell line with the SA-GFP reporter and from the same line after I-SceI expression and flow sorting to enrich for a pool of
GFP?cells. Large black triangles depict the 3? end of the AFP cassette. (b) hprtDRGFP reporter. The structure of the reporter is shown before
and after I-SceI cleavage and HDR. Note that repair by HDR converts the I-SceI site but otherwise maintains the structure of the reporter, as
confirmed previously by Southern blot analysis (37). (c) Flow cytometric analysis of wild-type ES cells containing the hprtDRGFP and hprtSAGFP
reporters targeted to the hprt locus, either untransfected or transfected with the I-SceI expression plasmid. Green fluorescence (FL1) is plotted
on the y axis, with orange fluorescence (FL2) on the x axis.
VOL. 24, 2004STEPS OF HOMOLOGOUS REPAIR WITH MUTAGENIC CONSEQUENCES9307
at the hprt locus in the mouse J1 ES cell line. The reporters
were integrated separately to generate an SA-GFP-containing
cell line and a DR-GFP-containing cell line. In the absence
of I-SceI expression, GFP?cells were rare or undetectable
among cells carrying either reporter (?0.01%) (Fig. 1c).
Transfection of the I-SceI expression vector resulted in 1.9%
GFP?cells for the DR-GFP cell line and 1.4% GFP?cells for
the SA-GFP cell line (Fig. 1c), indicating that SSA, as mea-
sured with this reporter, is nearly as efficient as HDR in wild-
type ES cells. It should be noted that the efficiency of SA-GFP
repair resulting in a functional GFP gene is consistent with a
mechanism involving SSA; HDR involving crossing over, which
would have given an identical product, is estimated to be at
least 30-fold less frequent in mammalian cells (20, 40, 50).
RAD51 promotes HDR but suppresses SSA. We next com-
pared the relative efficiencies of HDR and SSA in multiple
genetic backgrounds, using mutant cell lines and cells express-
which forms nucleoprotein filaments on DNA, because its strand
exchange activity is central to HDR. Because RAD51 has been
shown to be essential for mammalian cell viability (25, 54), we
investigated its function using putative dominant-negative forms.
We focused on two ATPase mutants: RAD51-K133A, which is
defective for ATP binding and hence is inactive for strand ex-
change in vitro, and RAD51-K133R, which is defective for ATP
hydrolysis but competent for ATP binding and hence retains
significant biological activity (28). We previously demonstrated
that stable expression of RAD51-K133R in otherwise wild-type
mammalian cells reduces HDR (49) so that RAD51-K133R
acts as a dominant-negative peptide during HDR. The mecha-
nism of interference is not certain; however, the mutant RAD51
peptides may form mixed nucleoprotein filaments with the
endogenous wild-type RAD51 to partially disrupt its function.
To test the effects of these RAD51 alleles on both pathways
of homologous repair, RAD51 expression vectors were tran-
siently cotransfected with the I-SceI expression vector into the
ES cell lines containing the integrated reporters. Under these
conditions, RAD51-K133R expression reduced HDR eight-
fold, while additional expression of wild-type RAD51 (RAD51-
WT) had little or no effect (Fig. 2), similar to previous results
with cells constitutively expressing exogenous RAD51 (49).
With RAD51-K133A, we found that the efficiency of HDR was
reduced even more than with RAD51-K133R, i.e., 22-fold,
which would be consistent with its more severely impaired
biochemical activity. The magnitude of the recombination de-
fect in this dominant-negative context suggests that complete
loss of RAD51 would abrogate most or all HDR.
In contrast to the inhibition of HDR, both RAD51-K133R
and RAD51-K133A enhanced the efficiency of SSA (1.9- and
4.2-fold, respectively [Fig. 2]). Expression of wild-type RAD51
also resulted in a slight (1.6-fold) increase in the efficiency of
SSA, which may indicate a mild dominant-negative effect with
elevated RAD51 (21, 41). Expression of yeast Rad51 in mam-
malian cells has been shown to reduce HDR, but SSA was not
obviously increased (22); it is not clear if this is due to different
experimental approaches or different allelic effects. Consider-
ing the relative efficiencies of HDR and SSA, expression of the
ATPase mutants of RAD51 led to an overall shift of 16-fold
(RAD51-K133R) and 93-fold (RAD51-K133A) toward muta-
genic homologous repair. These results suggest that the ATP-
dependent functions of RAD51 are important to promote
HDR and inhibit SSA during DSB repair.
Disruption of RAD51 function shifts HDR toward more
extensive gene conversion. Although HDR is usually consid-
ered to be a precise type of repair, some outcomes of HDR,
such as extensive gene conversion, have the potential to lead to
genetic loss. We considered the possibility that normal RAD51
function could be important to limit the extent of gene con-
version during HDR. In this regard, RAD51-K133R has been
shown in vitro to promote the formation of strand invasion
intermediates that are more stable than those formed by wild-
type RAD51 (45); in vivo these hyperstable recombination
intermediates may result in longer gene conversion tracts
during HDR. Alternatively, the reduced level of HDR with
RAD51-K133R may indicate impaired initiation of strand ex-
change, which could result in elevated strand resection prior to
HDR, thereby extending the gene conversion tracts.
We examined the effect of RAD51-K133R on the outcome
of recombination using the reporter H-DR-8mu (12) (Fig. 3a).
In this reporter, which is stably integrated at the hprt locus, the
I-SceI site has been introduced as a disrupting mutation into
the neomycin resistance gene (S2neo). HDR of the I-SceI-
induced DSB directed by the downstream internal neo fragment
pneo-8mu results in the replacement of the I-SceI site with an
NcoI site, which is the wild-type neo sequence at this position,
giving rise to a functional neo?gene. In addition, pneo-8mu also
contains single-base-pair silent mutations, which generate restric-
tion sites at various distances from the DSB (Fig. 3a). Incorpo-
ration of these restriction sites into the S2neo gene during re-
combination provides markers for the extent of gene conversion.
We constitutively expressed either RAD51-K133R or, as a
control, RAD51-WT in otherwise wild-type ES cells containing
the H-DR-8mu reporter. By Western blot analysis, RAD51-
K133R is estimated to be expressed at a level similar to that
of the endogenous RAD51 (data not shown) (49). We used
FIG. 2. RAD51-K133R and RAD51-K133A expression reduces
HDR but increases SSA. Wild-type (wt) ES cells with either the DR-
GFP or SA-GFP reporter were cotransfected with the I-SceI and
RAD51 expression vectors, as indicated. The efficiency of homologous
repair is indicated relative to transfection with the I-SceI expression
vector alone, which is set to 1. The asterisks indicate a statistically
significant difference from transfection with the I-SceI expression vec-
tor alone, with P ? 0.0001, except for SA-GFP repair with RAD51-
WT, with P ? 0.02. The error bars indicate standard deviations.
9308STARK ET AL.MOL. CELL. BIOL.
FIG. 3. Expression of RAD51-K133R increases gene conversion of markers distant from the DSB during HDR. (a) Structure of the H-DR-8mu
reporter and summary of gene conversion tracts in neo?recombinants. The pneo-8mu fragment contains the wild-type NcoI site at the site of the
DSB, along with 1-bp silent mutations, which create the following restriction site polymorphisms: A, ApaI; L, ApaL1; P, PstI; B, BamHI; X, XbaI;
Nr, NruI; and Pm, PmlI. HDR of the I-SceI-generated DSB in S2neo, using the pneo-8mu gene as the template for repair, results in restoration
of a wild-type neo?gene, so that all recombinants have an NcoI site incorporated. HDR may be associated with incorporation of the other
restriction sites. The filled bars represent the incorporation of all restriction site markers up to and including the indicated site for various
recombinants derived from the parental cell line or after expression of RAD51-WT or RAD51-K133R. (b) Summary of gene conversion
frequencies of each restriction site marker for the recombinants shown in panel a. The conversion frequency for each restriction site is graphed
as a function of distance from the DSB.
this constitutive-expression approach rather than transient
transfection so that we could be assured that each recom-
binant analyzed was derived from a cell in which RAD51-
K133R was expressed. We did not attempt a similar approach
with RAD51-K133A, since efforts to derive cell lines with sta-
ble expression of RAD51-K133A were unsuccessful.
The RAD51-expressing H-DR-8mu cell lines were trans-
fected with the I-SceI expression vector, and neo?colonies
were selected. As observed previously, the overall efficiency of
recombination is much lower with the H-DR-8mu reporter
than with the DR-GFP reporter due to the sequence heterol-
ogy between the S2neo and pneo-8mu sequences (12). Expres-
sion of RAD51-K133R resulted in a further reduction in HDR
of the H-DR-8mu reporter (1.6 ? 10?4compared to 5.2 ?
10?4for the parental cells). Thus, HDR between heterologous
sequences is also reduced by impaired RAD51 function.
We next examined gene conversion in the residual HDR
events by PCR amplification of the neo?genes from individual
recombinants and subsequent restriction digestion analysis of
the PCR products (Fig. 3a). From this analysis, we found that
expression of RAD51-K133R resulted in a shift toward con-
version of markers more distant from the DSB (Fig. 3) (P ?
0.0012). A mean gene conversion tract length of 145 bp was
found in the neo?recombinants from RAD51-K133R-express-
ing cells compared to only 60 and 59 bp in the recombinants
from RAD51-WT-expressing cells and the parental cells, re-
spectively (Fig. 3). Considering individual markers, the ApaLI
restriction site marker, for example, which is 394 bp from the
DSB, was incorporated in 23.7% of the neo?recombinants
from RAD51-K133R-expressing cells compared to 7 and 6.2%
of recombinants from RAD51-WT-expressing cells and the
parental cells, respectively (Fig. 3b). Thus, as well as suppress-
ing SSA, ATP hydrolysis by RAD51 limits the extent of gene
conversion during HDR. Our results indicate, therefore, that
proper RAD51 function is essential to limit the mutagenic
potential of homologous repair on multiple levels.
BRCA2 promotes HDR and suppresses SSA, whereas BRCA1
promotes both pathways. We next examined the generality of
the RAD51 suppression of SSA with respect to other HDR
factors. Other factors with key roles in HDR are the breast can-
cer susceptibility genes Brca1 and Brca2, both of which have
been shown to promote HDR and colocalize with RAD51 at
sites of DNA damage (18). BRCA2 appears to have a direct
role in modulating RAD51 activity through direct interaction;
the role of BRCA1, however, is unclear, since a direct BRCA1-
RAD51 interaction has not been found. As with RAD51, com-
plete loss of BRCA1 and BRCA2 can lead to cellular lethality
(18). Therefore, to test the roles of these factors in SSA, we
integrated the SA-GFP reporter into mouse ES cells with hy-
pomorphic alleles of Brca1 and Brca2 (Fig. 4a) so that the
presence of an intact copy of the reporter in several indepen-
dent clones was confirmed by Southern blotting (data not
shown). The hypomorphic alleles in Brca1?/?cells contain a
deletion in exon 11, which encodes approximately half of the
protein, so that an exon 10-to-12 splice product is formed (47).
The hypomorphic alleles in Brca2L1/L2cells contain deletions
of the terminal coding exon(s), so that one allele encodes a
C-terminal truncation of the BRCA2 protein lacking one of
the RAD51 binding motifs whereas the other appears to be
essentially a null allele (10, 27).
We compared the efficiency of SSA in these mutant cells to
that of wild-type cells by transfection of the I-SceI expression
vector and subsequent quantification of the percentage of GFP?
cells (Fig. 4b). We found that Brca2L1/L2cells exhibited an
increased efficiency of SSA (1.9-fold increase; P ? 0.002), like
the RAD51-disrupted cells. In contrast, Brca1?/?cells surpris-
ingly exhibited a decreased efficiency of SSA (1.8-fold de-
crease; P ? 0.004). It should be noted that there is some pre-
cedent for these results (30, 55); however, the previous lack of
a direct comparison of the Brca1 and Brca2 mutants, using
similar repair substrates, has until now precluded a definitive
conclusion about a distinct role for these proteins in DSB re-
pair. Under the same transfection conditions, HDR, as mea-
sured with DR-GFP reporter, was reduced in both the Brca1?/?
and Brca2L1/L2cells (5.3- and 6-fold decreases, respectively), con-
sistent with previous results (31, 32). Thus, while both hypomor-
phic Brca1 and Brca2 mutations impair HDR, they have opposite
affects on SSA.
Since the SA-GFP reporter in each of the examined Brca1
and Brca2 mutant cell lines was randomly integrated rather
than hprt targeted as in the parental cell line, we wanted to be
certain that the decreased SSA in the Brca1 mutant was not
due to decreased I-SceI endonuclease accessibilities at differ-
ent loci. To examine this, we directly assayed loss of the I-SceI
site by PCR amplification of the SceGFP3? gene and subse-
quent cleavage of the PCR product by I-SceI (Fig. 4c). In this
approach, DSB repair products arising from the other repair
pathways, i.e., HDR and NHEJ, were specifically quantitated.
The SSA product was not amplified, because the upstream
primer-binding site is lost during SSA. While this assay is not
a direct quantification of I-SceI endonuclease cleavage effi-
ciency, it allows a determination of whether a change in the
efficiency of SSA is specific or associated with a general change
in overall repair by other pathways. Using this assay, we mea-
sured the percent I-SceI site loss for each of the transfections
described in Fig. 4b. Wild-type and Brca1?/?cells had similar
percentages of site loss (6 and 7%, respectively), suggesting
that the SSA defect in these cells is not attributable to a
decreased accessibility of the I-SceI site for cleavage; it also
further suggests that decreased homologous repair is at least
partially compensated for by increased NHEJ (30). By con-
trast, site loss is reproducibly decreased in Brca2L1/L2cells
(3%). The decrease in site loss in Brca2L1/L2cells is consistent
with the associated shift of HDR toward SSA repair (Fig. 4b).
The Brca1 and Brca2 alleles used in the above analysis main-
tain large portions of the BRCA1 and BRCA2 proteins. To be
certain that the directions of the SSA effects we observed were
not allele specific, we tested the effects of dominant-negative
peptides on BRCA1 and BRCA2 function (Fig. 4a). To inves-
tigate BRCA1, we turned to its heterodimeric partner, BARD1.
BRCA1 and BARD1 interact via their N-terminal RING do-
mains, and expression of an N-terminal fragment of BARD1
(BARD1-hB202) has been shown to decrease HDR (58). We
examined BRCA2 function using the BRC3 peptide, which can
disrupt RAD51 function in vitro (7) and has been shown to
inhibit HDR in vivo (49).
To compare the effects on both HDR and SSA, expression
vectors for BARD-hB202 and BRC3 were transiently cotrans-
fected with the I-SceI expression vector in otherwise wild-type
cells containing the DR-GFP or SA-GFP reporter (Fig. 4d).
9310STARK ET AL.MOL. CELL. BIOL.
FIG. 4. BRCA1/BARD1 promotes both HDR and SSA, whereas BRCA2, like RAD51, promotes HDR and suppresses SSA. (a) Schematic of
wild-type and mutant proteins used in this analysis. Note that although the Brca1 mutant is indicated as Brca1?/?for simplicity, an exon 11-deleted
peptide is expressed in this mutant (58). For BRCA2, RAD51 binding regions at the BRC repeats and C terminus are indicated. Brca2L1/L2has
the C-terminal RAD51 binding domain in exon 27 deleted. BRC3 is tagged with a nuclear localization signal (NLS). BARD1-hB202 is the
N-terminal 202 amino acids of human BARD1, which interacts with BRCA1 (58). (b) Homologous repair in Brca1?/?and Brca2L1/L2cell lines.
The efficiency of homologous repair after I-SceI expression is indicated for the wild-type (wt) and mutant cell lines. SA-GFP repair frequencies
are the mean (? standard deviation) of 11 and 6 independent transfections for the Brca1?/?and Brca2L1/L2cell lines, respectively. The asterisks
indicate a statistically significant difference from the wild type, with P ? 0.004, except for DR-GFP repair in Brca2L1/L2, with P ? 0.008. (c) Analysis
of I-SceI site loss in the SA-GFP reporter arising from non-SSA repair. The genomic region surrounding the I-SceI site in SceGFP3? was PCR
amplified using the primers depicted as arrows. The PCR product from untransfected wild-type cells is efficiently cleaved by I-SceI, whereas after
I-SceI expression and HDR or NHEJ, a portion of the PCR product is not cleaved by I-SceI. (Note that the SSA product is not amplified because
the upstream primer is lost during SSA repair.) After I-SceI expression, Brca1?/?cells have a portion of noncleaved PCR product similar to that
of wild-type cells, whereas Brca2L1/L2cells reproducibly show a smaller amount of noncleaved product, consistent with the shift toward SSA. (d)
Expression of peptides predicted to interfere with BRCA1 and BRCA2 function mimics the effects of Brca1 and Brca2 mutation on homologous
repair. Wild-type ES cells with either the DR-GFP or SA-GFP reporter were cotransfected with the I-SceI and BARD1-WT, BARD1-hB202, or
BRC3 expression vectors, as indicated. The efficiency of homologous repair is indicated relative to transfection with the I-SceI expression vector
alone, which is set to 1. The asterisks indicate a statistically significant difference from transfection with the I-SceI expression vector alone, with
P ? 0.002, except for DR-GFP repair with BRC3, with P ? 0.01.
VOL. 24, 2004 STEPS OF HOMOLOGOUS REPAIR WITH MUTAGENIC CONSEQUENCES9311
Expression of BARD1-hB202 decreased the efficiencies of
both HDR and SSA (2.4- and 1.9-fold decreases, respectively),
similar to the Brca1?/?cell line, whereas the control full-
length BARD1 had no effect. In contrast, expression of the
BRC3 peptide from BRCA2 reduced HDR but also in-
creased SSA (2-fold decrease and 3.6-fold increase, respec-
tively), similar to the Brca2L1/L2cell line. Together with
those for the mutant cell lines, these results indicate that while
BRCA1/BARD1 promotes both HDR and SSA, BRCA2 pro-
motes HDR and suppresses SSA. These results suggest that the
BRCA1/BARD1 complex functions during a step common to
both homologous-repair pathways, whereas BRCA2 functions
downstream with RAD51 to affect the choice between HDR
FIG. 5. Ku70 mutation results in elevated HDR and SSA and suppresses the effect of BARD1-hB202 on homologous repair. (a) Homologous
repair in Ku70?/?ES cells. The efficiencies of homologous repair after I-SceI expression are indicated for the wild-type (wt) and Ku70?/?cell lines,
as well as the Ku70?/?cell line in which the I-SceI and KU70 expression vectors were cotransfected. The asterisks indicate a statistically significant
difference from the wild type, with P ? 0.0001. The error bars indicate standard deviations. (b) Influence of BARD1-hB202 on homologous repair
in Ku70?/?cells. Ku70?/?cells with either the DR-GFP or SA-GFP reporter were transfected with the I-SceI expression vector alone or together
with the expression vector for KU70, BARD1-hB202, or RAD51-K133R. The efficiency of homologous repair is indicated relative to transfection
with the I-SceI expression vector alone, which is set to 1. The asterisks indicate a statistically significant difference from the Ku70?/?cells
transfected with the I-SceI expression vector alone, with P ? 0.004.
9312 STARK ET AL.MOL. CELL. BIOL.
Ku70 mutation increases both HDR and SSA and partially
suppresses the repair defects of BRCA1/BARD1 disruption.
Previously, NHEJ mutants were reported to exhibit increased
HDR (4, 36), with a KU mutant showing the largest increase in
HDR among the tested mutants (36). The KU70/KU80 het-
erodimer binds DNA ends to promote their repair by NHEJ
(19, 57), and this end-binding activity has been postulated to
compete with HDR factors for access to DNA ends (36, 56).
We were interested in determining if KU, like BRCA1/
BARD1, influences steps common to both pathways of homol-
ogous repair or if its effect is limited to HDR.
To test the role of KU70 in SSA, we targeted the SA-GFP
reporter to the hprt locus in Ku70?/?cells and compared the
results with those for a previously described Ku70?/?cell line
containing the DR-GFP reporter, also targeted to the hprt
locus (36). Using each of these cell lines, the efficiency of
homologous repair in cells transfected with I-SceI alone was
compared to that in cells additionally transfected with a KU70
expression vector that allows partial complementation of the
KU deficiency (36) (Fig. 5). We found that the Ku70?/?cells
exhibited elevated levels of both SSA and HDR (eight- and
sevenfold increases, respectively), each of which was partially
reduced by cotransfection of the KU70 expression vector.
These results suggest that KU70 acts during a step common to
both homologous-repair pathways to inhibit HDR and SSA.
The above-mentioned finding that KU70 and BRCA1/
BARD1 influence both HDR and SSA, albeit in opposite di-
rections, suggested that these factors may both act during an
early step of homologous repair. To investigate this further, we
tested whether there is a genetic association between these
factors by comparing the effect of BARD1-hB202 on homolo-
gous repair in Ku70?/?cells. In particular, we wanted to de-
termine if the loss of KU would limit the effect of this peptide
on either or both pathways. For comparison, we also tested the
effect of RAD51-K133R in Ku70?/?cells, as this is expected to
act at a later step. Transfection of the BARD1-hB202 expres-
sion vector into Ku70?/?cells resulted in a 1.7-fold decrease in
HDR repair (Fig. 5b), a somewhat smaller effect than that seen
in wild-type cells (2.4-fold) (Fig. 4d). However, transfection of
the BARD1-hB202 expression vector into Ku70?/?cells had
no effect on SSA, although it reduced SSA repair in wild-type
cells (1.9-fold). As expected, transfection of the RAD51-
K133R expression vector into Ku70?/?cells resulted in de-
creased HDR and increased SSA, similar to its effect on wild-
type cells (14- and 1.9-fold, respectively). Thus, disruption of
KU function partially suppresses the homologous-repair de-
fects caused by BARD1-hB202 but does not suppress the ho-
mologous-repair defects caused by RAD51-K133R. These re-
sults are consistent with the notion that BRCA1/BARD1 and
KU70 both act at an early step in homologous repair, whereas
RAD51 acts at a step distinct from that of KU70. However, the
finding that the BARD1-hB202 inhibition of HDR is not com-
pletely abolished by Ku70 mutation, indicates that BRCA1/
BARD1 may also play an additional later role in HDR.
RAD52 and ERCC1 promote SSA but are not essential for
HDR. RAD52 is essential for multiple aspects of homologous
repair in yeast, including SSA and HDR (34, 52), whereas
mouse cells deficient in RAD52 have been shown to exhibit
only a mild gene-targeting defect (42). However, the role of
mammalian RAD52 in HDR and SSA has not been examined.
ERCC1 is involved in nucleotide excision repair, but it has also
been shown to be important for gene targeting (33), as has its
yeast ortholog (34), and to promote SSA in mammalian cells
(1, 43). Recently, human RAD52 has been shown to form a
complex with ERCC1 and its associated factor XPF (29).
To investigate the roles of RAD52 and ERCC1 in homolo-
gous repair in mammalian cells, we integrated the DR-GFP
and SA-GFP reporters into Rad52?/?and Ercc1?/?ES cells
using the hprt targeting vectors. For the Rad52?/?cells, hprt-
targeted clones were readily obtained; however, for the
Ercc1?/?cells targeting was severely reduced, although a few
targeted clones were obtained (data not shown). In the
Ercc1?/?clones in which the reporter was randomly inte-
grated, the presence of a single intact copy of each reporter
was verified by Southern blotting (data not shown). The effi-
ciency of homologous repair in cells transfected with I-SceI
alone was compared to that in cells additionally transfected
with the complementing expression vector (Fig. 6). We find
this analysis more informative than a comparison to our wild-
type line, since the Ercc1?/?and Rad52?/?cells, which are
derived from a different ES cell background, appear to have a
higher transfection efficiency (data not shown). In these exper-
iments, the complemented Rad52?/?and Ercc1?/?cells had
no higher efficiency of HDR than uncomplemented cells; how-
ever, SSA was increased (two- and fivefold, respectively) (Fig.
6). These results indicate that RAD52 and ERCC1 specifically
promote homologous repair by SSA.
Repair of chromosomal DSBs is essential to maintain
genomic integrity, yet different repair pathways are variably
FIG. 6. RAD52 and ERCC1 promote SSA but are not essential for
HDR. The efficiency of homologous repair is indicated for the
Ercc1?/?and Rad52?/?ES cells transfected with the I-SceI expression
vector alone or together with the ERCC1 or RAD52 complementing
expression vector, respectively. The efficiency of homologous repair in
individual transfections is calculated relative to transfection with the
I-SceI expression vector alone for each mutant, which is set to 1 (see
Materials and Methods). SA-GFP repair frequencies are the means (?
standard deviations) of seven and six independent transfections for the
Rad52?/?and Ercc1?/?cell lines, respectively. The asterisks indicate a
statistically significant difference from the mutant transfected with the
I-SceI expression vector alone, with P ? 0.008.
VOL. 24, 2004STEPS OF HOMOLOGOUS REPAIR WITH MUTAGENIC CONSEQUENCES9313
mutagenic. In this report, we have examined the interrelation-
ship of the homologous-repair pathways in mammalian cells.
HDR is primarily a precise type of repair and thus much less
prone than SSA to causing gross chromosomal rearrangements
(39) or chromosomal deletions, as assayed in this study. How-
ever, HDR can lead to genetic loss under some circumstances,
such as when conversion is extensive. Thus, the proper balance
and regulation of DSB repair pathways would be predicted to
be critical for the maintenance of genomic integrity. In support
of this, HDR mutants exhibit chromosome aberrations and
higher rates of mutagenesis (48, 53), apparently as a result of
misrepair of spontaneously arising DNA lesions, including
DSBs. As yet, however, a comparative analysis of distinct
classes of DSB repair mutants has not been reported to pro-
vide an understanding of the interrelationship of the homolo-
gous-repair pathways in mammalian cells.
Our analysis has determined that some factors affect the
competitive choice between HDR and SSA (RAD51 and
BRCA2), whereas others factors promote or suppress both
homologous-repair pathways (BRCA1/BARD1 and KU, re-
spectively), and we have identified an activity that limits ge-
netic loss from HDR (ATP hydrolysis by RAD51) (see below)
(Fig. 7). Failure to specifically utilize HDR as a result of
impaired RAD51 function—either directly by RAD51 muta-
tion or indirectly by BRCA2 disruption—leads to an increased
reliance on SSA. The RAD51 ATP hydrolysis mutant not only
shifts homologous pathway use to SSA but also fails to limit the
extent of gene conversion in the remaining HDR events, in-
creasing the potential for genetic loss.
The finding that the proper functioning of RAD51 is essen-
tial to promote precise homologous repair indicates that con-
trol of strand exchange may be a deciding factor in suppressing
mutagenic outcomes of DSB repair. BRCA2 is a particularly
intriguing factor for such control, since it can interact directly
with RAD51 at multiple motifs, as well as with single-stranded
DNA, and thus may modulate the association of RAD51 with
damaged DNA (7, 44, 59). Interestingly, we found a shift from
HDR to SSA, whether BRCA2 is mutated by the loss of only
the C-terminal RAD51 binding motif so that 94% of the pro-
tein coding region is left intact, including the eight BRC re-
peats (23, 55), or whether a single 70-amino-acid BRC repeat,
which can disrupt RAD51 nucleoprotein filament formation in
vitro, is overexpressed. Another Brca2 mutant, as yet unchar-
acterized, also shows increased deletional homologous-recom-
bination events which may reflect SSA (23, 55). Thus, a shift
from HDR to SSA appears to be a generalized outcome when
RAD51 or BRCA2 function is disrupted. Consistent with this,
loss of RAD54, a protein that promotes RAD51-mediated
strand exchange activity in vitro (51), also results in a shift from
HDR to SSA (11), although the magnitude of the shift is much
smaller than that seen with RAD51 or BRCA2. Thus, this shift
in homologous pathway usage appears to be a common out-
come when factors that affect steps after the branch point of
HDR and SSA are disrupted. Although key distinctions exist
for some yeast and mammalian DSB repair factors, a similar
shift to SSA has been noted in yeast for some HDR mutants
(16). The shift to SSA may result from hyperresection of DSBs
when RAD51 activity is impaired: whereas HDR requires min-
imal resection, SSA requires ?2 kb of resection in our sub-
strate. Since repetitive elements are at various distances from
each other, the extent of resection is expected to be an impor-
tant factor in the efficiency of SSA in the mammalian genome.
In addition to the shift to SSA, we found that genetic loss
arising from more extensive gene conversion tracts also oc-
curred when ATP hydrolysis by RAD51 was disrupted. Muta-
tion of the RAD51 paralog XRCC3 also results in more ex-
tensive gene conversion in the residual recombinants that are
obtained with this mutant, although the tracts are often dis-
continuous (5). As we find no evidence for discontinuous tracts
with RAD51-K133R expression, RAD51 and XRCC3 appear
FIG. 7. Genetic steps of homologous repair with distinct mutagenic consequences. Shown are different pathways of DSB repair and the roles
of individual factors in pathway choice. Whereas homologous repair by HDR with limited gene conversion (blue) is a precise type of repair, SSA,
NHEJ, and HDR with extensive gene conversion (red), as well as crossing over (not shown), are much more prone to being mutagenic. The arrows
do not necessarily reflect a temporal order of events. Homologous-repair results were obtained using DR-GFP and SA-GFP, except for RAD54
and XRCC3 (see the text).
9314 STARK ET AL.MOL. CELL. BIOL.
to have different roles in the extent of conversion. Perhaps
XRCC3 is essential to promote the stability of recombination
intermediates and thus maintain the continuity of DNA syn-
thesis during gene conversion, whereas ATP hydrolysis by
RAD51 is important for processing recombination intermedi-
ates to limit such DNA synthesis. Alternatively, ATP hydrolysis
by RAD51 may result in greater protection of DNA ends,
limiting the amount of single- or double-strand resection that
In contrast to RAD51 and BRCA2, we found that BRCA1/
BARD1 or KU70/KU80 affected both homologous-repair path-
ways in the same direction; defects in BRCA1/BARD1 de-
creased the efficiencies of both homologous-repair pathways,
while loss of KU increased the efficiencies of both pathways.
Thus, these factors appear to affect a step(s) of homologous
repair that is common to both HDR and SSA, although with
opposite outcomes. Furthermore, loss of KU70 partially sup-
presses the homologous-repair defects of BARD1 disruption,
which indicates that BRCA1/BARD1 and KU70 may both be
acting at an early step during homologous repair. The initial
strand resection step to generate single-stranded DNA for
strand exchange or annealing is one such step. Ku70-deficient
yeast cells exhibit elevated rates of strand resection, indicating
that Ku70 is important in limiting resection in yeast (24). Thus,
while KU70 could limit strand resection, BRCA1/BARD1
could act to promote strand resection. An early role for the
BRCA1/BARD1 complex in homologous repair is consistent
with its rapid localization to DSBs (35). The exonuclease(s)
that promotes resection has not been identified; however,
BRCA1/BARD1 could affect nuclease activity or access of a
nuclease to DNA ends via one of the identified BRCA1/
BARD1 biochemical activities, e.g., DNA binding, ubiquitina-
tion, or phosphopeptide binding (6, 18). Unlike SSA, the in-
hibitory effect of BARD1 disruption on HDR is not totally
abrogated by Ku mutation; thus, BRCA1/BARD1 could still
have a later role in HDR, possibly through its interaction with
HDR-specific factors like RAD51 or BRCA2 (18).
We also determined that mammalian RAD52 promotes
SSA, although loss of RAD52 does not overtly affect HDR.
That RAD52 does not play a significant role in promoting
HDR in mammalian cells is surprising, given its critical role in
HDR in yeast (52), its postulated role as a “gatekeeper” for
HDR in mammalian cells through its biochemical activity of
binding to DNA ends (56), and the fact that human RAD52
stimulates homologous pairing by human RAD51 in vitro (51).
The nonessentiality of RAD52 in mammalian cells suggests
this function is assumed in vivo by some other factor. BRCA2,
which also binds single-stranded DNA and RAD51 and stim-
ulates RAD51 activity (7, 44, 59), is an excellent candidate.
However, a role for RAD52 in HDR may still be uncovered in
other, more complex genetic contexts, as has been observed in
chicken cells by combined mutation with the RAD51 paralog
In summary, the multiple DSB repair pathways have a com-
plex interrelationship which affects whether repair occurs faith-
fully. Understanding these interrelationships at the molecular
level in mammalian cells is essential, given that many DSB
repair mutants have markedly different phenotypes, including
development defects, aging phenotypes, and tumor suscepti-
bility (38), and in some cases even have tumors of the same
tissue type yet with markedly different characteristics, i.e., as in
humans for BRCA1- and BRCA2-associated tumors (14).
We thank members of the Jasin laboratory, especially Beth Elliott,
Ulrica Westermark, and Mary Ellen Moynahan, for materials and
discussions and Laura Niedernhofer and Roland Kanaar (The Neth-
erlands) for the Ercc1?/?ES cell line.
This work was supported by NIH grants GM54668 and CA94060
and a P20 grant from the MSKCC Cancer and Aging Program.
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