Mutation in Rpa1 results in defective DNA double-strand

break repair, chromosomal instability and cancer in mice

Yuxun Wang
1
, Christopher D Putnam
2
, Michael F Kane
2
, Weijia Zhang
3
, Lisa Edelmann
4
, Robert Russell
5
,
Danaise V Carrio n
3
, Lynda Chin
6
, Raju Kucherlapati
3
, Richard D Kolodner
2
& Winfried Edelmann
1
Most cancers have multiple chromosomal rearrangements;
the molecular mechanisms that generate them remain largely
unknown. Mice carrying a heterozygous missense change in
one of the DNA-binding domains of Rpa1 develop lymphoid
tumors, and their homozygous littermates succumb to early
embryonic lethality. Array comparative genomic hybridization
of the tumors identied large-scale chromosomal changes as
well as segmental gains and losses. The Rpa1 mutation resulted
in defects in DNA double-strand break repair and precipitated
chromosomal breaks as well as aneuploidy in primary
heterozygous mutant mouse embryonic broblasts. The
equivalent mutation in yeast is hypomorphic and semi-
dominant and enhanced the formation of gross chromosomal
rearrangements in multiple genetic backgrounds. These
results indicate that Rpa1 functions in DNA metabolism are
essential for the maintenance of chromosomal stability and
tumor suppression.
Replication protein A (Rpa) is a heterotrimeric single-stranded DNA
binding complex consisting of subunits Rpa1, Rpa2 and Rpa3. Rpa1,
the largest subunit, is conserved in eukaryotes and has essential roles
in DNA replication, recombination and repair
1
. In yeast, the L221P
missense change in one of three DNA binding domains led to gross
chromosomal rearrangements (GCRs) that resembled those frequently
found in human cancers, suggesting that RFA1 (Rpa1 in multicellular
eukaryotes) has a crucial role in maintaining genomic stability
2,3
. This
hypomorphic mutation also causes sensitivity to DNA-damaging
agents, including those that induce double-strand breaks (DSBs);
homologous recombination defects in meiosis associated with exten-
sive resection of the 5 ends of meiosis-specic DSBs much like those
seen in rad51, rad52, rad55 and rad57 mutants
4
; and checkpoint
defects but no substantial defect in DNA replication
2,3,58
. To study
the effect of Rpa1 mutations in mammals, we generated a mouse line
carrying the corresponding mutation Rpa1 689T-C (resulting in the
amino acid substitution L230P) using a knock-in gene-targeting
approach (Fig. 1a,b). We conrmed expression in the tissues of F
1
offspring by RT-PCR analysis and direct sequencing (Fig. 1c,d). The
mutant Rpa1 L230P protein was present in heterozygous mutant cells
at levels equivalent to those of Rpa1 in wild-type cells; this is
consistent with modeling studies indicating that the L230P mutation
disrupts one of the three Rpa1 DNA-interacting domains but does not
interfere with domain folding or stability (Fig. 1e,f and Supplemen-
tary Fig. 1 online).
Matings between Rpa1
689C/+
males and females produced no homo-
zygous mutant Rpa1
689C/689C
mice, indicating that the 689T-C
mutation caused embryonic lethality. Genotyping of mutant embryos
showed that homozygous Rpa1
689C/689C
embryos were pre-
sent at embryonic day (E) 3.5 but not at later developmental
stages (Table 1). The E3.5 embryos consisted of only a few cells that
were unable to proliferate in the in vitro culture, in contrast to inner
cell mass cells of wild-type and heterozygous mutant E3.5 embryos
(Fig. 2a,b). This observation indicates that homozygosity with
respect to the Rpa1 689T-C missense mutation is lethal and is
consistent with the idea that Rpa1 has an essential role in replicating
cells. In contrast, the corresponding mutation rfa1 662T-C (resulting
in the amino acid substitution L221P, also called rfa1-t48) is not lethal
in yeast
2,3,8
. This difference might be caused by the homologous
recombination defect, possibly in conjunction with the checkpoint
defect caused by the mutation. In mice, homologous recombination
defects like those caused by a Rad51 mutation also cause embryonic
lethality between E3.5 and E7.5, whereas such defects do not cause
lethality in yeast
911
.
To study the effect of the Rpa1 689T-C mutation in heterozygous
mutant mice, we monitored the status of mutant mice during a 2-y
period. Rpa1
689C/+
mice developed normally but had signicantly
lower survival than their wild-type littermates (P o 0.01, log-rank
test; Fig. 2c). As early as 4 months of age, Rpa1
689C/+
mice began to
die, and by 12 months, 420% of them had died, whereas all wild-type
mice were still alive. To determine the cause of death, we killed
moribund Rpa1
689C/+
mice between the ages of 4 and 14 months and
Published online 19 June 2005; doi:10.1038/ng1587
1
Department of Cell Biology, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
2
Ludwig Institute for Cancer Research, Cancer Center and Departments
of Medicine and Cellular and Molecular Medicine, University of California San Diego, La Jolla, California 92093, USA.
3
Harvard Medical School-Partners Healthcare
Center for Genetics and Genomics, Harvard Medical School, Boston, Massachusetts 02115, USA.
4
Department of Human Genetics, Mount Sinai School of Medicine,
New York University, New York, New York 10029, USA.
5
Department of Pathology, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
6
Department
of Medical Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts 02115, USA. Correspondence should be addressed to R.D.K. (rkolodner@ucsd.edu) or
W.E. (edelmann@aecom.yu.edu).
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subjected them to detailed histopathological analysis. All Rpa1
689C/+
mice had marked lymphoid hyperplasia as well as altered hemato-
poiesis in the bone marrow. Notably, most of the mice (9 of 14)
developed lymphomas, and several of the lymphomas were highly
disseminated, inltrating multiple tissues (Fig. 2d and Table 2). Only
one of the wild-type mice died at 14 months of age, of unknown
causes but with no detectable tumors. The difference in survival
between the two cohorts continued to increase, and by 2 years of
age, only 40% of heterozygous mutant mice were still alive, compared
with 470% of wild-type mice.
To identify the genetic events associated with tumorigenesis in
Rpa1
689C/+
mice, we analyzed the genomic DNA of Rpa1
689C/+
tumors
for loss of heterozygosity at the Rpa1 locus after laser-capture micro-
dissection. None of the six tumors analyzed showed loss of either the
mutated Rpa1
689C
allele or the wild-type allele (Supplementary Fig. 2
online). The retention of the wild-type Rpa1 allele is consistent with
the essential role of Rpa1 in DNA metabolism and suggests that the
mutated Rpa1
689C
allele has a dominant effect. Genetic analysis of the
corresponding rfa1 662T-C mutation in yeast indicates that rfa1
662C
is a partially dominant allele.
Mutations in RFA1 in yeast, including the missense mutation
662T-C (also called rfa1-t48), cause increased accumulation
of GCRs reminiscent of genome rearrangements seen in human
cancers
2,3
. We therefore analyzed tumors in Rpa1
689C/+
mice for
chromosomal abnormalities by array comparative genomic hybridiza-
tion (CGH) and found that the genomes in all tumors analyzed
(n 9) contained multiple alterations (Supplementary Table 1
online). Notably, most tumors showed gains of the entire chromo-
somes 6 and 15 (Fig. 3a,b). We also identied segmental gains and
losses of regions on other chromosomes (Fig. 3b). None of the losses
involved regions known to contain tumor-suppressor genes, but the
gains on chromosomes 6, 15 and 16 involved regions containing
Figure 1 Generation of Rpa1
689C
mutant mice.
(a) Gene-targeting scheme. The 689T-C point
mutation (red asterisks; resulting in the amino
acid substitution L230P) in exon 8 of Rpa1, as
well as a silent mutation resulting in a XhoI site
(at codon 233), was introduced by homologous
recombination (dotted line) into embryonic stem
cells. The Rpa1 wild-type locus, the pRpa1
689C
gene targeting vector and the modied Rpa1
allele are shown. After germline transmission of
the modied allele, the PGKneo cassette was
deleted by Cre-loxPmediated recombination
by mating of F
1
male offspring to Zp3-Cre
transgenic female mice. (b) Southern-blot
analysis of wild-type (+/+) and heterozygous
Rpa1
689Cneo/+
(+/m
neo
) F
1
offspring showing
germline transmission of the modied Rpa1
allele. (c) RT-PCR analysis of total RNA in wild-
type (+/+) and heterozygous Rpa1
689C/+
(+/m)
mutant mice. Top, diagnostic scheme. The wild-
type allele contains a HindIII restriction site, and
digestion with HindIII generates fragments of
235 and 121 bp. The mutated allele has lost the
HindIII restriction site and contains a newly
introduced XhoI site. Digestion with XhoI
generates fragments of 224 and 132 bp. Bottom,
the presence of the 689T-C mutation was
veried by restriction digestion of RT-PCR
products in wild-type (lanes 1 and 3) and
Rpa1
689C/+
cells (lanes 2 and 4) with HindIII
(lanes 3 and 4) and XhoI (lanes 1 and 2),
yielding diagnostic fragments of 235 and 121 bp
with HindIII in wild-type cells and of 224 and
132 bp with XhoI in Rpa1
689C/+
cells. M, 100-bp
DNA markers. (d) Sequence analysis of the
RT-PCR product in Rpa1
689C/+
mice conrmed
the presence of the 689T-C mutation (red
asterisk). (e) Comparative western-blot analysis of
wild-type (+/+) and heterozygous Rpa1
689C/+
(+/m) MEF cells indicates stable expression of the Rpa1 L230P protein. (f) Human Leu221 (equivalent to
mouse Leu230) is in a b-ribbon that generates a hydrophobic patch at the DNA-binding interface and is illustrated from the complex of oligonucleotide
binding (OB) domains 1 and 2 with single-stranded DNA (ssDNA). Prolines cannot adopt an extended conformation, and the L221P mutation would be
expected to disrupt the DNA-binding interface but not the OB1 domain (Supplementary Fig. 1 online).
E 6 E 7 E 8 E 9 E 13
aag
K
gga
G L F
HincII HincII
E 6 E 7 E 8 E 9 E 13
HincII KpnI
*
PGKneo
Kpn I
E 6 E 7 E 8
HincII
*
E 9 E 13
HincII
E 6 E 7 E 8 E 9 E 13
HincII
*
HincII
PGKneo
G418
Cre
Genomic Rpa1 locus
pRpa1
689C
targeting vector
Modified Rpa1 allele
Rpa1
689C
allele
+
/
+
+
/
m
n
e
o
+
/
+
+
/
m
n
e
o
7.6 kb
5.6 kb
M 1 2 3 4
356 bp
224 bp
132 bp
356 bp
235 bp
121 bp
HindIII
XhoI
121 bp 235 bp
224 bp 132 bp
Rpa1
+
Rpa1
m
*
+/m
+/+
+
/
+
+
/
m
+
/
m
+
/
m
+
/
+
+
/
+
30 g 15 g 7.5 g
Rpa1
OB1 OB2
-actin
a
b
c d
e f
Phe
238 210
212
216
221
Leu
Leu221
Arg
Arg
Trp
ssDNA
ctt ttc
S
tct
Table 1 Viability analysis of mouse embryos
Genotype
Developmental stage +/+ +/m m/m ND Unfertilized eggs Total
E3.5 8 15 7 12 12 54
E7.5 10 21 0 0 NA 31
E9.5E14.5 6 10 0 0 NA 16
Live birth 31 62 0 0 NA 93
Wild-type (+/+) and heterozygous Rpa1
689C/+
(+/m) mice were born at a 1:2 ratio, but no
homozygous Rpa1
689C/689C
(m/m) mice were detected at birth. Rpa1
689C/689C
embryos
were present at E3.5 but not at later developmental stages. NA, not applicable; ND,
not determined.
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oncogenes. Although their involvement in lymphomagenesis in
Rpa1
689C/+
mice remains to be established, two of these oncogenes,
Myc on chromosome 15 and Raf1 on chromosome 6, are frequently
involved in human and mouse neoplasia
1214
.
The Rpa1 689T-C mutation predisposed the mice to increased
tumorigenesis, with tumors bearing widespread chromosomal rear-
rangements, but whether this mutation caused genomic instability and
predisposed Rpa1
689C/+
mice to increased cancer susceptibility, or
whether the chromosomal aberrations in Rpa1
689C/+
tumors were
secondary events during tumor progression, was not known. To
address this issue, we analyzed the karyotypes of primary mouse
embryonic broblast (MEF) strains derived from wild-type and
Rpa1
689C/+
mice. For each genotype, we
examined several hundred metaphase spreads
from three independent MEF lines for chro-
mosome abnormalities. The frequency of
abnormal karyotypes was signicantly greater
in Rpa1
689C/+
MEFs than in wild-type MEFs
(P o 0.01; Fig. 3c,d and Supplementary
Table 2 online). In addition to an increase
in aberrant metaphases containing aneuploid
karyotypes (Fig. 3c and Supplementary
Table 2 online), we also detected chromoso-
mal breaks in Rpa1
689C/+
mutant cells that
were not found in wild-type cells (Fig. 3d).
DNA DSBs in mammalian cells are potent
inducers of chromosomal instability
15
. The
Rpa1 689T-C mutation may result in
increased DSBs by causing defects in DSB
repair or by causing checkpoint defects that
result in failure to process replication errors
correctly and thus lead to mutagenic repair of
spontaneous DNA damage and to GCRs
similar to those observed in Rpa1
689C/+
mutant MEFs
28,16
. To investigate this possi-
bility, we studied DSB repair after exposure
to the replication inhibitor aphidicolin. We
observed substantial levels of phosphorylated
H2AX (g-H2AX), a marker for DSBs in
mammalian cells, in Rpa1
689C/+
MEFs after
treatment with aphidicolin (Fig. 3e). In con-
trast, there was little induction of g-H2AX in
wild-type cells. Moreover, high levels of
g-H2AX remained even 2 h after withdrawal
of aphidicolin from the culture medium
(Fig. 3e), indicating that DSB repair was impaired in Rpa1
689C/+
MEFs.
To gain mechanistic insight into the development of lymphomas in
Rpa1
689C/+
mice, we carried out parallel experiments in yeast. We
introduced the equivalent rfa1
662C
allele at the chromosomal locus or
expressed the wild-type RFA1 or rfa1
662C
alleles under control of the
native RFA1 promoter on either low-copy ARS CEN or high-copy
2-micron plasmids in different yeast strains (Table 3). Western-blot
analysis showed that expression of Rpa1 from an ARS CEN plasmid
did not alter the levels of Rpa1 or Rpa2, whereas expression of Rpa1
from a 2-micron plasmid resulted in no more than a twofold increase
of both Rpa1 and Rpa2 (data not shown; Rpa3 was not analyzed).
Expression of the two different plasmid rfa1
662C
alleles in a wild-type
M 1 2 3 4 5 6 7 8 9
+/+
m/m
+/m
MLN (200) Liver (400) Kidney (100)
Brain (630) Spinal cord (100) Spleen (200)
Rpa1
+
(277 bp)
Rpa1
m
(360 bp)
0 2 4 6 8 10 12 14 16 18 20 22 24
0
20
40
60
80
100
+/+
+/m
Months
S
u
r
v
i
v
a
l

(
%
)
a
d c
b
+1 +3 +4 Time (d)
Figure 2 Phenotypes of Rpa1 mutant mice. (a) Homozygous mutant Rpa1
689C/689C
(m/m) embryos had
severe growth retardation, and blastocyst outgrowth experiments showed that, in contrast to wild-type
(+/+) and heterozygous mutant (+/m) embryos, homozygous embryos could not proliferate and further
degenerated in the in vitro culture. (b) Representative PCR genotyping analysis of wild-type (lanes 2,
6 and 8), heterozygous (lanes 1, 4, 5 and 9) and homozygous (lanes 3 and 7) mutant embryos at 4 d
in culture. M, 100-bp DNA markers. (c) Kaplan-Meier survival curve. The survival of wild-type (+/+;
n 62) and heterozygous Rpa1
689C/+
(+/m; n 54) mice was monitored for a period of 24 months.
Rpa1
689C/+
mice had a signicantly lower survival rate than did their wild-type littermates (P o 0.01,
log-rank test). (d) Lymphoma development in Rpa1
689C/+
mice. Representative histopathological
sections of disseminated lymphomas inltrating into multiple tissues showing pronounced enlargement
with the lymphomas replacing the architecture in mesenteric lymph node (MLN), liver and spleen as
well as inltration of lymphomas into kidney, spinal cord and brain.
Table 2 Histopathological analysis of lymphomas in Rpa1
689C/+
mice
Mouse Sex Age (months) Genotype Histological type Anatomic site
1 F 9 +/m Lymphoma Bone marrow, thymus, liver, spleen, lymph nodes
2 F 8.5 +/m Lymphoma Spleen
3 F 4 +/m Disseminated lymphoma Lymph nodes, thymus, liver, kidney, brain
4 F 6 +/m Disseminated lymphoma Brain, spleen, thymus, kidney, liver, lung, lymph nodes
5 F 8 +/m Disseminated lymphoma Lymph nodes, liver
6 F 11 +/m Disseminated lymphoma Spleen, liver, kidney, lymph nodes, thymus, skeletal muscle,
brain, spinal cord, stomach, bone marrow
7 M 10 +/m Lymphoma Lymph nodes
8 F 11 +/m Lymphoma Spleen, lymph nodes
9 M 14 +/m Lymphoma Gastric lymph nodes, mesenteric lymph nodes, spleen, bone marrow
+/m, Rpa1
689C/+
.
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strain resulted in increased sensitivity to DNA-damaging agents
including ultraviolet light, hydroxyurea, methylmethanesulfonate
(high-copy plasmid only) and HO endonuclease-induced DSBs
(high-copy plasmid only), as well as increased rates of accumulating
GCRs, though not to the extent caused by chromosomal rfa1
662C
(Table 3 and Supplementary Tables 35 and Supplementary Fig. 3
online). When the plasmid rfa1
662C
alleles were expressed in strains
carrying mutations that inactivate homologous recombination (mre11
or rad52), suppression of de novo telomere addition (pif1-m2) or
different checkpoints (mre11, rad24, mec1 or rad53), the GCR rate
increased by vefold to more than 200-fold. The increased GCR rates
were not as great as those seen in double mutants carrying the
chromosomal rfa1
662C
allele (Table 3). In addition, the chromosomal
rfa1
662C
allele showed synthetic lethality when combined with mre11D
and rad27D mutations, whereas the plasmid rfa1
662C
alleles did not
17
(Table 3 and data not shown). These results support the view that the
rfa1
662C
allele is both hypomorphic and semidominant when
expressed from both low-copy and high-copy plasmids in the presence
of a single copy of the wild-type RFA1 gene. The pattern of genetic
interactions that we observed supports the hypothesis that the plasmid
rfa1
662C
mutation causes partial defects in both homologous recom-
bination
4
and checkpoints
5
(Supplementary Tables 35 online) and
Chromosome number
1
0
0.5
0.5
1
1
0
0.5
0.5
1
1
0
0.5
0.5
1
1
0
0.5
0.5
1
T2
T5
Y1T
T1
1 2 3 4 5
6 7 8 9 10
11 12 13 14 15
16 17 18 19 X
L
o
g
2

r
a
t
i
o
-H2AX
-actin
a b
c d
e
Aphidicolin 0 1 2 4 +2 0 1 2 4 +2 Time (h)
+/m +/+
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 X
Figure 3 Chromosome analyses of tumors and MEFs in Rpa1
689C/+
mice.
(a) Representative array CGH results showing gains (green) and losses (red) of
chromosomes in individual tumors. (b) Ideograms highlighting gains (green) and losses
(red) of chromosomal regions. (c) Example of an aneuploid metaphase in Rpa1
689C/+
MEFs. (d) An example of chromosome breaks in Rpa1
689C/+
MEFs. (e) Aphidicolin
treatment results in increased induction of g-H2AX in Rpa1
689C/+
MEFs. Cells were
cultured in the presence or absence of 5 mM aphidicolin, and protein was isolated 0, 1,
2 and 4 h after aphidicolin treatment. Protein was also prepared from cells 2 h (+2) after
aphidicolin withdrawal after the cells had been treated with aphidicolin for 4 h.
Table 3 Expression of the rfa1
662C
allele in yeast shows partial dominance for GCR formation
GCR rate (10
10
) (plasmid allele/chromosome allele)
Genotype Plasmid type Vector/RFA1 RFA1/RFA1 rfa1
662C
/RFA1 Vector/rfa1
662C
Wild-type Low o50 o0.80 16 27,000
High o2 o3.1 75 39,000
mre11D Low 1,900 600 3,300 SL
High 2,800 920 9,100 SL
rad52D High 56 o43 10,000 38,600
rad57D High o41 67 1,200 33,000
pif1-m2 High o31 22 6,200 ND
rad24D High 6.4 o45 820 85,000
mec1D sml1D Low 110 o30 330 ND
High o9.6 9.8 1,700 57,000
rad53D sml1D High o16 o20 3,000 120,000
GCR rates were determined for the indicated strains (1028 cultures from at least two and an average of three independent isolates) containing the wild-type RFA1 allele or the
rfa1
662C
allele on either lowcopy number (ARS CEN, pRS415) or highcopy number (2-micron, pRS425) LEU2 plasmids or vector only. Plasmid allele is listed rst; chromosome
allele second. ND, not determined; SL, synthetic lethal mutation combinations. Extra copies of wild-type RFA1 seem to partially suppress the GCR rate of the mre11D mutant. All 11
rearrangements recovered from the wild-type strain containing a highcopy number plasmid with a rfa1
662C
allele were de novo telomere additions. All 19 mre11D spores from the
cross between the mre11D and rfa1
662C
mutants were RFA1, indicating that mre11D and rfa1
662C
are synthetically lethal (P 0.00013; w
2
test).
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hence shows genetic interactions with checkpoint defects
7
and homo-
logous recombination defects, respectively
1820
. The cell-cycle check-
point and homologous recombination defects observed in yeast might
underlie the chromosomal gains and rearrangements in the observed
lymphomas, as such defects cause both genome rearrangements and
mis-segregation of chromosomes
18,2022
. But the specicity of both the
induced cancers and the associated altered chromosomes could also
occur because the DNA damage promoted by the Rpa1
689C/+
back-
ground provides a selection for activation or inactivation of a specic
set of oncogenes or tumor-suppressor genes, respectively.
Human RPA1 maps to chromosome 17p13.3 (ref. 23), and loss of
this chromosomal region has repeatedly been implicated in various
malignancies including colorectal cancers, breast cancers, lymphomas
and leukemias
2429
. In addition, comparisons of CGH data suggest
that human tumors with loss of 17p also have more total genomic
aberrations (SKY/M-FISH and CGH database). Imbalances at a
nearby locus, 17p13.1, have been reported in several types of cancers.
TP53 maps to 17p13.1, and its loss is believed to be responsible for the
associated cancer phenotypes
25
. Notably, allelic changes occur at a
substantially higher frequency at 17p13.3 than at 17p13.1 in human
cancers
27
. Although the relevance of 17p13.3 loss for tumorigenesis is
not known, defects in other proteins associated with DNA metabolism
underlie several human genetic disorders associated with cancer
susceptibility
23
. Our results indicate that Rpa1 is essential for cell
survival and for maintaining chromosomal stability and tumor
suppression in mammalian cells. These ndings may also have
implications for tumor formation in humans.
METHODS
Generation of Rpa1
689C
mutant mice and preparation of MEFs. We intro-
duced the missense mutation 689T-C (resulting in the amino acid substitu-
tion L230P) in exon 8 of Rpa1 into the mouse genome by gene targeting. We
introduced the targeting vector into WW6 embryonic stem cells by electro-
poration, isolated G418-resistant colonies and screened them by PCR (primer
sequences available on request). We identied ve positive clones and con-
rmed the correct targeting event by digestion of genomic DNA with HincII
and Southern-blot analysis. We injected three correctly targeted embryonic
stem cell lines into C57BL/6J blastocysts to generate male chimeras that
transmitted the Rpa1
689C
allele through the germ line. We then mated F
1
males carrying the mutated allele to Zp3Cre transgenic females (C57BL/6J) to
eliminate the resistance cassette by loxP-mediated recombination. We inter-
crossed male and female mice carrying the modied allele to generate Rpa1
+/+
,
Rpa1
689C/+
and Rpa1
689C/689C
mutant mice. To produce Rpa1
+/+
and Rpa1
689C/+
MEF lines, we intercrossed Rpa1
689C/+
mice, isolated E13.5 embryos and
prepared MEF lines from embryos by standard procedures. We cultured MEFs
in Dulbeccos modied Eagle medium with 10% fetal calf serum and genotyped
them by PCR.
RT-PCR. We isolated total RNA from the liver, thymus, spleen, muscle,
intestine and testis of Rpa1
+/+
and Rpa1
689C/+
mice (RNeasy Mini kit, Qiagen)
and carried out RT-PCR (primer sequences available on request; Titan One
Tube RT-PCR System, Roche Diagnostics Corporation). We digested the
amplied 356-bp cDNA fragment with either HindIII to detect the wild-type
transcript or XhoI to detect the mutant transcript. To conrm expression of the
Rpa1 689T-C mutation, we sequenced the cDNA fragment.
Western-blot analysis. We isolated protein from MEFs using Trizol reagent
(Invitrogen) and quantied it (BCA Protein Assay Reagent, Pierce). We created
serial dilutions (30, 15 and 7.5 mg) of protein of each cell line, separated
samples by 10% SDS-PAGE and transferred them onto PVDF membranes
(Immobilon P, Millipore). We then incubated the membranes with antibodies
directed against Rpa1 (Ab-1, Oncogene Sciences) and b-actin (ACTN05,
NEOMARKERS, Inc.) as a loading control. For the DSB repair assay, we used
the antibody to H2AX phosphorylated at Ser139 (g-H2AX; Upstate Biotech-
nology) at a 1:1,000 dilution in TBST containing 3% nonfat dried milk.
Viability analysis and blastocyst outgrowth of Rpa1
689C
mutant mice. We
isolated mouse embryos at stages E14.5, E9.5, E7.5 and E3.5 and genotyped
them by PCR. We isolated Rpa1
+/+
, Rpa1
689C/+
and Rpa1
689C/689C
blastocysts,
cultured them in vitro on gelatinized tissue culture plates and monitored their
growth and morphology for 4 d.
Histopathology. We killed mice when they became moribund and subjected
them to systematic histopathological analysis. We processed tissues in accor-
dance with standard procedures and evaluated tumor and normal tissue
parafn sections by staining with hematoxylin and eosin.
Chromosome analysis. We prepared metaphase spreads from three indepen-
dent lines of wild-type and Rpa1
689C/+
MEFs using standard protocols. We
stained the spreads with Giemsa (BDH Laboratory Supplies) and examined
them for chromosomal abnormalities.
Array CGH analysis. The methods used for array CGH analysis are described
in Supplementary Methods online.
Yeast procedures. We generated the high-copy plasmids containing the RFA1
(pRDK1139) and rfa1
662C
(pRDK1140) alleles by subcloning the HindIII-SalI
fragment from the low-copy plasmids pKU1 and pKU1-t48 (ref. 8) into
pRS425 (ref. 30). Yeast mutants were isogenic to the wild-type strain
RDKY3615 (MATa, ura3-52, his3D200, trp1D63, leu2D1, hom3-10, ade2D1,
ade8, yel069::URA3)
21
. We generated double mutants by crossing an isogenic
MATa rfa1
662C
strain with mutants of interest and identied mutations by
sequencing PCR products amplied from genomic DNA preparations and by
plating onto selective media. We used media and methods for determining
GCR rates as previously described
3
, except that we grew liquid cultures in SC-
Leu medium for 2 d at 30 1C to reach saturation before plating them on GCR
selection medium. We determined Rfa1 and Rfa2 levels by western blotting
using antibodies provided by S. Brill (Rutgers University).
URL. The SKY/M-FISH and CGH database is available at http://www.
ncbi.nlm.nih.gov/sky/skyweb.cgi/.
Accession numbers. GenBank: mouse Rpa1, NM_026653; yeast RFA1,
M60262. PDB: single-stranded DNA-binding domain of human RPA1 bound
to single-stranded DNA, 1JMC.
Note: Supplementary information is available on the Nature Genetics website.
ACKNOWLEDGMENTS
We thank H. Hou, Jr. and B. Jin for technical assistance and S. Brill for providing
antibodies. This work was supported by grants from the US National Institutes of
Health (to R.K., R.D.K. and W.E.).
COMPETING INTERESTS STATEMENT
The authors declare that they have no competing nancial interests.
Received 24 August 2004; accepted 16 May 2005
Published online at http://www.nature.com/naturegenetics/
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