Targeted deletion of mouse Rad1 leads to
deficient cellular DNA damage responses
Chunbo Zhang1,2,4*, Yuheng Liu1,3*, Zhishang Hu1, Lili An1, Yikun He2, Haiying Hang1✉
1National Laboratory of Biomacromolecules, and the Center for Computational and Systems Biology, Institute of Biophysics,
Chinese Academy of Sciences, Beijing 100101, China
2College of Life Science, Capital Normal University, Beijing 100037, China
3Graduate School of the Chinese Academy of Sciences, Beijing 100049, China
4Current address: School of Pharmacy, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, Hong Kong, China
✉ Correspondence: email@example.com
Received April 23, 2011Accepted May 5, 2011
The Rad1 gene is evolutionarily conserved from yeast to
human. The fission yeast Schizosaccharomyces pombe
Rad1 ortholog promotes cell survival against DNA
damage and is required for G2/M checkpoint activation.
In this study, mouse embryonic stem (ES) cells with a
targeted deletion of Mrad1, the mouse ortholog of this
gene, were created to evaluate its function in mammalian
cells. Mrad1−/−ES cells were highly sensitive to ultravio-
let-light (UV light), hydroxyurea (HU) and gamma rays,
and were defective in G2/M as well as S/M checkpoints.
These data indicate that Mrad1 is required for repairing
DNA lesions induced by UV-light, HU and gamma rays,
and for mediating G2/M and S/M checkpoint controls. We
further demonstrated that Mrad1 plays an important role
in homologous recombination repair (HRR) in ES cells,
but a minor HRR role in differentiated mouse cells.
ing, DNA repair, homologous recombination repair
Rad1, DNA damage, checkpoint signal-
Cells face endogenous and exogenous assaults that damage
genomic DNA. But eukaryotic cells have conserved surveil-
lance mechanisms, which could detect the DNA lesions and
send the signals to the DNA repair system and the cell cycle
control machinery, to coordinate DNA repair and minimize
negative effects of these lesions. The cell cycle delay induced
via the checkpoint mechanism is thought to provide extra time
for DNA damage repair, and to prevent cell cycle progression
into critical phases that could lead to lethality (Hartwell and
Rad9, Rad1 and Hus1 are a group of genes conserved
from yeast to human that play key roles in the cell cycle
signaling networks. Their protein products form a ring-shaped
heterotrimer, named the 9-1-1 complex (Doré et al., 2009;
Sohn and Cho, 2009; Xu et al., 2009). It is believed that this
complex is important for the functions of DNA repair as well as
the activation of cell cycle checkpoints (Shiomi et al., 2002;
Bermudez et al., 2003; Ellison and Stillman, 2003). Interest-
ingly, human Rad1 (i.e., RAD1) also exists as monomer
besides forming the 9-1-1 complex in cells, and the function of
this form of the protein is unknown (Burtelow et al., 2001). In
fission yeast Schizosaccharomyces pombe, disruption
mutants of the three genes resulted in similar phenotypes,
including viability, sensitivity to UV-light, the replication
inhibitor hydroxyurea (HU), as well as gamma rays, and
defective S/M and G2/M checkpoint control (al-Khodairy and
Carr, 1992; Enoch et al., 1992; Lieberman et al., 1992; Murray
et al., 1991; Rowley et al., 1992). Disruption of the budding
yeast Saccharomyces cerevisiae counterparts, Mec3
(schus1), Rad17 (scrad1) and Ddc1 (scrad9), also caused
similar phenotypes in the corresponding mutants, including
hypersensitivity to UV light, HU and gamma rays, and G2/M
checkpoint defect, but not a disruption of the S/M checkpoint
defect (Longhese et al., 1997; Lydall and Weinert, 1997).
Mouse cells with a disruption of Mrad9 or Mhus1, the mouse
homologues of rad9 or hus1, were successfully created, and
also exhibited significantly higher sensitivity to UV light, HU
and gamma rays than the wild-type cells (Weiss et al., 2000;
*These authors contributed equally to the work.
410© Higher Education Press and Springer-Verlag Berlin Heidelberg 2011
Protein Cell 2011, 2(5): 410–422
Protein & Cell
Hopkins et al., 2004). The cell cycle checkpoint functions of
Mrad9 and Mhus1 were reported to be different but the
comparison was based on the data using two different cell
types (Weiss et al., 2000; Hopkins et al., 2004; Wang et al.,
2004). The Mhus1−/−cells are mouse embryonic fibroblasts
(MEF), while the Mrad9−/−cells are mouse embryonic stem
cells (ES). Mhus1−/−MEFs were defective in the UV light-
induced intra-S phase checkpoint, but functioned normally
with respect to the G2/M checkpoint (Weiss et al., 2003). In
contrast, Mrad9−/−ES cells were not markedly defective in the
UV light-induced intra-S phase checkpoint, but failed to
maintain G2/M checkpoint control following the exposure to
gamma rays (Weiss et al., 2000; Hopkins et al., 2004).
Results from human RAD1 knockdown using siRNA
suggested that the gene is an important element for cell
growth and is required for the recovery of DNA synthesis
following HU treatment (Bao et al., 2004). The same study
showed that reduced RAD1 protein level caused a defect in
the intra-S phase checkpoint but did not affect the G2/M
checkpoint. However, rad1-disrupted yeast cells failed to
arrest in response to ionizing radiation exposure (al-Khodairy
and Carr, 1992; Enoch et al., 1992; Rowley et al., 1992; Lydall
and Weinert, 1997).
Although targeted deletion of Mrad9 and Mhus1 in mouse
cells and mice have been reported (Weiss et al., 2000;
Hopkins et al., 2004; Levitt et al., 2005; Levitt et al., 2007; Hu
et al., 2008; Yazinski et al., 2009; An et al., 2010), equivalent
studies for Mrad1 have not been published. Such investiga-
tion is important to reveal the gene functions that are not
detectable when RAD1 protein is only partially expressed in
siRNA knockdown cells (Bao et al., 2004) or heterozygous
cells (Han et al., 2010). In the present study, we constructed
mouse ES cells with a targeted deletion of Mrad1 gene and
investigated Mrad1 function in these cells. Our results
showed that Mrad1 homozygously deleted ES cells were
viable, but were defective in G2/M checkpoint maintenance as
well as the HU-induced S/M checkpoint, and were highly
sensitive to UV light, HU and gamma rays. Interestingly, the
differentiation of Mrad1−/−ES cells modulated the capability of
double-strand breaks (DSB) repair.
Construction of mouse ES cells with homozygous
disruptions of Mrad1
Mrad1+/−ES cells were obtained as previously described
(Han et al., 2010). The neo gene product can destroy
antibiotic G418, and the Mrad1+/−ES cells contained one
allele of disrupted genomic Mrad1 bearing a copy of neo
gene. We hypothesize that increasing G418 concentration in
the medium might force the amplification of the copy number
of neo and even replace the remaining wild type genomic
Mrad1 with the neo-bearing disrupted genomic Mrad1. To
obtain Mrad1−/−clones, the Mrad1+/−ES cells were incubated
with 3.2–4.4mg/mL G418 instead of the original 300μg/mL
G418 for 20 days, and from 96 survivors, six colonies bearing
Mrad1 homozygous deletion were identified by Southern
blotting (Fig. 1A). These results were confirmed using
Northern blotting (Fig. 1B) and RT-PCR (Fig. 1C).
Mrad1 deletion retards cell proliferation and alters cell
cycle phase distribution
RAD1 knockdown by siRNA reduced the proliferation rate of
human cells (Bao et al., 2004). Consistent with this result,
Mrad1−/−ES cells grew significantly slower than the wild type
control population (Fig. 2A), and formed much smaller
colonies (Fig. 2B). We examined the cell cycle phase
distributions of Mrad1−/−and Mrad1+/+cells with flow
cytometry, and found that significantly more Mrad1−/−cells
accumulated in the G2/M phase than the wild-type cells
(Fig. 2C), suggesting the mutant cells proceeded through
G2/M at a significantly slower pace. Bromodeoxyuridine
(BrdU) incorporation analysis showed that S phase progres-
sion rate was reduced by homozygous deletion of Mrad1 (Fig.
2D). All the aforementioned changes in the cell cycle caused
by Mrad1 deletion were reversed by ectopically expressing
blot of Mrad1 in mouse ES cells. Genomic DNA from wild-type
and targeted ES clones were digested with HindIII and
hybridized with probes corresponding to flanking sequences.
Bands indicate wild-type and deleted Mrad1 alleles. (B)
Northern blot of Mrad1 RNA in mouse ES cells. The β-actin
gene was used as a control to demonstrate equivalent sample
sequence of genomic Mrad1 gene. (C) RT-PCR to assess
Mrad1 RNA levels. Total RNA was isolated from Mrad1+/+and
Mrad1−/−ES cells, the latter ectopically expressing Mrad1.
Gapdh RNA levels were used as an internal control. Primer
pairs and other experimental details are described in MATERI-
ALS AND METHODS.
Targeted deletion of mouse Mrad1. (A) Southern
© Higher Education Press and Springer-Verlag Berlin Heidelberg 2011411
Roles of mouse Rad1 in cells
Protein & Cell
Protein & Cell
Proliferation of Mrad1+/+and Mrad1−/−ES. The average results were derived from three independent experiments. (B) Cells were
grown on Petri dishes at 37°C with 5% CO2for 10 days and then stained to visualize colony formation. Mrad1−/−ES cells (left) formed
much smaller colonies than Mrad1+/+ES cells (right). (C) Asynchronously dividing ES cells were fixed and stained with PI. Cell cycle
distribution was analyzed by flow cytometry. Deletion of Mrad1 resulted in an aberrant accumulation of cells in the G2/M phase,
suggesting a slower progression through this phase of cell cycle. The percentage of each cell population in G1, S and G2/M phases is
showninthe graphsas indicated. (D)S-phaseDNA replicationwas assayedby simultaneousmeasurement of DNA contentand BrdU
incorporation. Deletion of Mrad1 resulted in a slowdown of S phase DNA synthesis. The number inside each graph is the geometric
mean of BrdU incorporation per 10min. All the above experiments had been repeated at least three times. Only one set of
representative data was presented here.
Deletion of Mrad1 in mouse ES cells retards cell proliferation and changes cell cycle phase distribution. (A)
412© Higher Education Press and Springer-Verlag Berlin Heidelberg 2011
Chunbo Zhang et al.
Mrad1 (Fig. 2C and 2D; data not shown), and thus these
alterations were due to the lack of Mrad1 function.
The increased accumulation of Mrad1−/−cells in the G2/M
phase might result from activation of the G2/M checkpoint by
DNA lesions, and therefore, we monitored the DNA breaks in
wild type and Mrad1−/−ES cells using an alkaline comet
assay for all types of DNA lesions and a histone γ-H2AX
assay for DSBs. The comet tail moment in Mrad1−/−ES cells
was significantly higher than that in Mrad1+/+cells (Fig. 3A
and 3B), indicating the presence of more DNA lesions in the
mutant. These results were confirmed by the histone γ-H2AX
assay, in which more foci (Fig. 3C and 3D) as well as a higher
level of histone γ-H2AX were detected in the mutant
population (Fig. 3E), reflecting enhanced DNA DSBs in
Failure of Mrad1−/−ES cells to maintain ionizing
radiation-induced G2/M checkpoint control
DNA damage-induced arrest in G2phase is one of the most
prominent cell cycle checkpoints in eukaryotic cells. Fission
yeast S. pombe rad1 is required for this cell cycle arrest in
for DNA lesionsusing a CometAssaykit as describedin MATERIALSAND METHODS. (B)Thetail momentof Mrad1−/−EScells was
measured and the mean±SD is depicted. The tail moment of the mutant cells was significantly larger than wild-type control
(p<0.002). (C) Spontaneous DNA double-strand breaks were detected by γ-H2AX labeling. (D) Quantitative assessments were
made by counting foci in at least 100 cells of each phenotype, and the percentage of foci containing cells is shown. (E) Whole cell
lysates from Mrad1+/+and Mrad1−/−ES cells were subjected to western blotting using anti-γ-H2AX antibody, with tubulin served as a
Deletion of Mrad1 leads to increased frequency of DNA lesions. (A) Mrad1−/−and Mrad1+/+ES cells were analyzed
© Higher Education Press and Springer-Verlag Berlin Heidelberg 2011413
Roles of mouse Rad1 in cells
Protein & Cell
response to ionizing radiation exposure (Freire et al., 1998;
Udell et al., 1998). Therefore, we examined whether the role
of Mrad1 in the G2/M checkpoint is evolutionarily conserved.
Mrad1+/+and Mrad1−/−cells, as well as the Mrad1−/−cells
ectopically expressing Mrad1 were irradiated with 10Gy of
gamma rays, harvested at 4, 6, 8, 10 and 12h after exposure,
and then processed for flow cytometric analysis to assess cell
cycle phase distribution. Only data from untreated, 6 and 10h
time points are presented here because the rest of data
essentially indicated the same trends. The percentage of cell
populations in each phase of the cell cycle is shown in graphic
(Fig. 4) and tabular formats (Table 1). Subpopulations of both
Mrad1+/+and Mrad1−/−ES cells increased in the G2/M phase
and decreased in the G1and S phases post irradiation. This
pattern, lacking G1arrest but exhibiting radiation-inducible G2
arrest, is a typical response of wild-type ES cells to gamma
rays (Aladjem et al., 1998). This result indicated that Mrad1 is
not indispensable to activate the G2/M checkpoint. However,
in contrast to the wild-type cells, Mrad1-deficient cells
accumulated in the G1phase (arrows in Fig. 4). To assess
whether the small G1subpopulation of cells came from the
G2/M phase post irradiation, colcemid, which disrupts the
mitotic spindle and traps cells in mitosis, was added to the
cells. The results showed that incubation of the cells with
colcemid eliminated the small G1subpopulation accumula-
tion, and therefore the cells progressed from the G2/M phase
(Fig. 4), suggesting the important role of Mrad1 in maintaining
the DNA damage-induced G2/M checkpoint control. This
conclusion was confirmed by the fact that the G2/M
checkpoint defect was rescued by the ectopic expression of
Protein & Cell
arrest induced by ionizing radiation exposure. Mrad1+/+,
Mrad1−/−ES cells, and Mrad1−/−ES cells ectopically expres-
sing Mrad1 were mock-treated or treated with 10Gy of gamma
rays in the absence or presence of colcemid, and then
analyzed by flow cytometry. G1, S and G2/M regions of the
profiles are delineated on top of graph for the calculations of
cell distribution in each phase as indicated in Table 1.
Mrad1 deletion leads to a deficiency in G2
Percentage of cells in different phases of the cell cycle at indicated times post-irradiation with 10 Gy of gamma rays
Post-irradiation time (h)Percentage of population in all cycle phase (%)
6 1.5645.14 52.95
6+ colcemid 0.6443.18 56.05
10+ colcemid0.38 23.0176.26
0 23.58 44.9431.36
6 1.15 46.8650.40
6+ colcemid 0.5942.9055.25
10+ colcemid 0.4430.35 66.40
414© Higher Education Press and Springer-Verlag Berlin Heidelberg 2011
Chunbo Zhang et al.
Mrad1 disruption in ES cells alters S/M checkpoint
S. pombe rad1 is required to block cells with incomplete DNA
replication from moving into the M phase of cell cycle (S/M
checkpoint), while the S. cerevisiae ortholog, Rad17
(scRad1), is dispensable for the checkpoint. To determine
whether the S/M checkpoint in mouse ES cells is Mrad1-
dependent, we examined the level of phospho-histone-H3 (γ-
H3) throughout the cell cycle. Histone-H3 is specifically
phosphorylated during mitosis. After treatment with HU for
different times, ES cells were labeled with anti-γ-H3 antibody,
stained with propidium iodide (PI) for DNA content, and then
analyzed by flow cytometry. Incubation with HU reduced the
number of γ-H3 positive Mrad1+/+cells with 2N DNA content
because most cells were blocked in S phase and thus fewer
moved into the M phase. Few Mrad1+/+cells with less than 2N
DNA content were γ-H3 positive after HU treatment, indicat-
ing that the wild type cells have a normal S/M checkpoint
(Fig. 5 and Table 2). In contrast, the number of γ-H3 positive
Mrad1−/−cells with less than 2N DNA content dramatically
increased after HU treatment. Mrad1-deficient ES cells
ectopically expressing Mrad1 showed the same pattern as
wild-type cells. Therefore, Mrad1 is essential for the S/M
Mrad1 is not essential for the intra-S phase checkpoint
induced by UV light
The intra-S phase cell cycle checkpoint monitors DNA
replication and delays DNA synthesis in the presence of
DNA damage. We demonstrated that Mrad1-null cells are
highly sensitive to UV light (see below). Therefore, we
determined whether the UV-induced intra-S phase checkpoint
of the mutant cells was aberrant. Mrad1+/+and Mrad1−/−cells
were treated with UV light, and then pulse-labeled with
10 μmol/L BrdU at designated times post treatment to detect
DNA replication by flow cytometry. The incorporation rates of
BrdU into DNA in both cell populations dramatically reduced
at 40, 90 and 180min after irradiation, and the kinetics were
similar (Fig. 6). Thus, these findings indicate that deletion of
Mrad1 does not affect the intra-S phase checkpoint control
after exposure to UV light.
Mrad1-deleted ES cells are hypersensitive to UV light,
HU and gamma rays
Previous research showed that Rad1 associates with Hus1
and Rad9 in a 9-1-1 heterotrimer to respond to DNA damage
(Hang and Lieberman, 2000; Rauen et al., 2000; Lindsey-
Boltz et al., 2001; Roos-Mattjus et al., 2002; Parrilla-Castellar
et al., 2004). Both Mhus1−/−MEF cells and Mrad9−/−mouse
ES cells are highly sensitive to genotoxins, including UV light,
HU and gamma rays (Weiss et al., 2000; Hopkins et al., 2004;
Wang et al., 2004; Wang et al., 2006). S. pombe rad1::ura4+
control defect. Mrad1+/+, Mrad1−/−ES cells, and Mrad1−/−ES
cells ectopically expressing Mrad1 were treated or mock-
treated with 1 mmol/L HU for various times. Cells were
collected and labeled with antibodies against the mitotic
marker phosphor-histone H3, stained with PI, and analyzed
by flow cytometry. Staining intensity for PI (x-axis) is plotted
versus staining intensity of phosphor-histone H3 (y-axis). Cells
in the boxed region correspond to the prematurely condensed
chromosome mitotic fraction. The percentage of boxed cells in
the graphs is listed in Table 2.
Mrad1 deletion leads to an S/M checkpoint
somes (phosphor-histone H3 labeled cells with less than 2N DNA)
treated for indicated times with 1mmol/L HU
Percentage of cells with premature condensed chromo-
Percentage of γ-H3 positive
with less than 2N DNA (%)
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Roles of mouse Rad1 in cells
Protein & Cell
cells are also extremely sensitive to these DNA damaging
agents (Freire et al., 1998; Udell et al., 1998). Therefore, we
examined whether Rad1 in mouse ES cells plays an
important role in promoting resistance to these genotoxins.
As shown in Fig. 7, Mrad1−/−ES cells were extremely
sensitive to UV light, HU and gamma rays compared to the
wild type control population. To confirm the sensitivities are
due to a defect in Mrad1, resistance was examined in the
mutant cells ectopically expressing the wild-type gene. As
indicated in Fig. 7, expression of wild-type Mrad1 compen-
sated the resistance to UV light, HU and gamma rays in
Mrad1−/−ES cells, thus indicating that Mrad1 gene mediates
the resistance to these agents.
Deletion of Mrad1 does not affect expression of other cell
cycle checkpoint genes
p21, p53, Hus1 and Rad9 are important cell cycle checkpoint
genes, and the expression levels of p21, p53, Hus1 and Rad9
were examined by northern blotting to gain a mechanistic
insight into the potential influence of Mrad1 deletion on the
regulation of these genes. The results indicated that homo-
zygous deletion of Mrad1 did not affect expression of these
cell cycle checkpoint genes (Fig. 8). Mrad1−/−cells bearing
the Mrad1 cDNA also displayed similar expression levels of
these cell cycle checkpoint RNAs, except for the increased
expression of Mhus1, and the deletion of Mrad1 did not affect
Mhus1 RNA level. Therefore, Mrad1 deletion did not cause a
dramatic shift in RNA levels corresponding to this group of cell
cycle checkpoint genes, suggesting that the deletion caused
Protein & Cell
delay in DNA synthesis in response to UV light exposure.
Mrad1+/+, Mrad1−/−ES cells, and Mrad1−/−ES cells ectopically
expressing Mrad1 were treated or mock-treated with 20J/m2
UV, labeled with BrdU at the indicated times post exposure,
stained with FITC-conjugated anti-BrdU antibody and PI, and
then analyzed by flow cytometry. Staining intensity for PI (x-
axis) versus staining intensity for BrdU (y-axis) is indicated.
Geometric means of the FITC fluorescence in BrdU-positive
cells, which reflects the BrdU uptake rate by the S phase
subpopulation of cells, are shown in each sample.
Mrad1-deficient ES cells demonstrate a normal
ity to DNA-damaging agents. Mrad1+/+, Mrad1−/−ES cells,
and Mrad1−/−ES cells ectopically expressing Mrad1 were
treated as described in MATERIALS AND METHODS, and
colony formation was used to assess their sensitivity to
hydroxyurea (A), UV (B), and gamma rays (C). Points in all
the graphs represented the average of three independent
experiments, with bars indicating standard deviation.
Mrad1-deficient cells have increased sensitiv-
416 © Higher Education Press and Springer-Verlag Berlin Heidelberg 2011
Chunbo Zhang et al.
defects of cell checkpoints and altered cell cycle distribution
not through regulating the expression of p21, p53, Hus1 or
Differentiated Mrad1-deleted ES cells have more efficient
It has been reported that ES cells have more efficient DNA
repair than differentiated ES cells in response to various
DNA-damage agents (Maynard et al., 2008; Tichy and
Stambrook, 2008). However, in the cell survival assay, we
found that Mrad1−/−ES cells were hypersensitive to IR, but
retinoic acid (RA)-induced differentiated Mrad1−/−ES cells
had nearly identical sensitivity as the wild type cells (Fig. 9A).
Meanwhile, undifferentiated and differentiated Mrad1+/+ES
cells displayed similar resistance to the same doses of
irradiation (Fig. 9B). Leukemia inhibitory factor (LIF) is
routinely added to ES cell medium to prevent ES cells from
differentiation. Here we obtained similar results when
Mrad1−/−ES cells were cultured in RA-containing medium
as well as LIF-free medium (Fig. 9C), confirming that mouse
ES cell differentiation compensated for DNA repair defects
caused by Mrad1 deletion. Treatment by gamma rays causes
DSBs, which are repaired by two major pathways, non-
homologous end joining (NHEJ) and homologous recombina-
tion (HR). Using an established in vivo HR assay (Pierce et
al., 2001), we found that the loss of Mrad1 caused significant
reduction in HR repair capacity, but differentiation could
largely compensate it in Mrad1−/−ES cells (Fig. 9D and 9E).
In fission yeast S. pombe, rad1 is a key component that
mediates multiple cellular responses to DNA damage,
including a role in cell cycle checkpoint (Murray et al., 1991;
al-Khodairy and Carr, 1992; Enoch et al., 1992; Lieberman
et al., 1992; Rowley et al., 1992; Parker et al., 1998).
However, the function of this gene in mammals is not clear.
In this report, we examined the activities of Mrad1, the mouse
ortholog of S. pombeRad1, by creating and characterizing the
mouse ES cells with deletion of Mrad1. We demonstrated that
Mrad1-deficient ES cells were highly sensitive to UV light, HU
and gamma rays (Fig. 7), defective in S/M and G2/M cell cycle
checkpoint controls (Fig. 4–6), and prone to accumulate DNA
lesions under normal growth conditions (Fig. 3). These data
indicate that Mrad1 plays essential roles in the resistance to
UV light, HU and gamma rays, as well as in the S/M and G2/M
As shown by previous reports (Burtelow et al., 2001; Roos-
Mattjus et al., 2002), as well as 9-1-1 complex crystal
structure (Doré et al., 2009; Sohn and Cho, 2009; Xu et al.,
2009), Rad1 along with Rad9 and Hus1 in a trimeric
checkpoint complex were believed to have similar functions.
Indeed as we showed above, many phenotypes such as the
hypersensitivity to HU, UV light and gamma rays are similar
among Mrad1-deletion, Mrad9-deletion and Mhus1-deletion
mouse cells (Fig. 7) (Weiss et al., 2000, 2003; Hopkins et al.,
2004). In addition, mouse ES cells with Mrad1-deletion and
Mrad9-deletion are similarly deficient in G2/M and S/M
Mrad1+/+, Mrad1−/−ES cells, and Mrad1−/−ES cells ectopically expressing Mrad1 was subjected to Northern blotting hybridization
with indicated32P-labeled cDNA probes. Gapdh served as a loading control. (B) Quantitative analysis of RNA levels corresponding
to the checkpoint control genes. The ratio of radioactive intensity of indicated gene over Gapdh levels in (A) was quantified with
AlphaEaseFCTMsoftware (AlphaImager 2200, Alpha Innotech Corp., San Leandro, CA).
Northern blotting analyses of cell cycle checkpoint genes in mouse ES cells. (A) Total RNA prepared from
© Higher Education Press and Springer-Verlag Berlin Heidelberg 2011417
Roles of mouse Rad1 in cells
Protein & Cell
checkpoint maintenance, but intact in intra-S phase check-
point, which is in contrast to Mhus1 embryonic fibroblasts
(EF) (Weiss et al., 2000, 2003; Hopkins et al., 2004). Taken
together, Rad1, Rad9 and Hus1 are likely to work in the 9-1-1
complexfor the resistanceto HU, UV light and gammarays as
well as for the maintenance of S/M and G2/M checkpoints in
mouse ES cells. The phenotype differences in intra-S phase,
S/M and G2/M checkpoints between Mrad1 or Mrad9-deleted
mouse ES cells and Mhus1-deleted mouse EF cells are
probably due to the various differentiation states, suggesting
the different functions of these genes in cell cycle checkpoints
in ES and EF cells.
Consistent with the above hypothesis, we found in this
study that the differentiated Mrad1−/−cells induced by RA and
LIF-free media had similar resistance to gamma rays as
undifferentiated or differentiated Mrad1+/+cells (Fig. 9).
Interestingly, the resistance to HU or UV light was similar
between undifferentiated and differentiated Mrad1−/−cells
(our unpublished data). These results together suggest that
differentiation has various influence on different DNA repair
pathways. It is still unknown whether differentiation of
Mrad9−/−cells can also enhance their resistance to gamma
rays. This experiment is critical to clarify whether the
differentiation-associated resistance change is 9-1-1 complex
dependent or only Rad1-dependent. Indeed, there are
significant amounts of individual Rad1 molecules in human
cells (Burtelow et al., 2001; our unpublished data).
ES cells were reported to have higher DNA repair abilities
than differentiated cells (Maynard et al., 2008; Tichy and
Stambrook, 2008). Our results are inconsistent with these
reports. It is possible that repair factors work differently at
various stages of differentiation, and the comparison between
ES and differentiated cells only at certain stages probably
does not reflect all the DNA repair situations of mammalian
cells during differentiation. In addition, various DNA repair
pathways are probably differently influenced by cell differ-
entiation as shown in this study while only the resistance to
gamma rays, but not to HU or UV light, was altered by the
differentiation of mouse ES cells. As already shown by many
studies, differentiation is largely regulated and reflected by
chromatin status and many chromatin remodeling factors play
important roles in DNA repair pathways. DNA repair at
different stages of differentiation attracts more researches
and will generate further insights into DNA repair mechan-
HR repair was a major component that was altered in DSB
Protein & Cell
resistance was observed in differentiated Mrad1−/−ES cells (A), but not Mrad1+/+ES cells (B). Other differentiation-inducing method
indicated the same results (C). Points in all the graphs represented the average of three independent experiments with bars
indicating standard deviation. Meanwhile, flow cytometric analysis demonstrated attenuated HR in Mrad1−/−ES cells, but partial
compensation in differentiated Mrad1−/−ES cells. ES cells containing a chromosomal DR-GFP reporter were cotransfected with the
expression vectors for the I-SceI endonuclease. In vivo cleavage of DR-GFP reporter at the I-SceI site of SceGFP gene and repair
by the downstream iGFP repeat directed HR resulted in GFP-positive cells (D). Summary of the percentage of HR deficient cells
fromeachcelllinesis presented.Bars representtheaverageofthreeindependently isolatedhprtDRGFPsubclonesforeachcellline
(E). Error bars are±S.D. (n = 3).
Differentiated Mrad1−/−ES cells have increased DSBs repair capability. After treating with gamma rays, enhanced
418© Higher Education Press and Springer-Verlag Berlin Heidelberg 2011
Chunbo Zhang et al.
repair from mouse ES Mrad1−/−cells to RA-induced
differentiated mouse Mrad1−/−cells (Fig. 9D and 9E), but it
only accounted for half of the altered DSB. It is likely that
NHEJ also changed during the differentiation. If this is true,
the differentiation would modulate the common part(s) of both
repair pathways, and the chromatin status during DSB
repairing process might be modulated
A study of human HCT116 cells with RAD1 siRNA
demonstrated no effect of the corresponding reduced protein
levels on the G2/M checkpoint, but impaired intra-S phase
checkpoint control was observed (Bao et al., 2004). Our study
using Mrad1-deficient ES cells revealed the opposite results:
a defective G2/M and an intact intra-S phase checkpoint
(Fig. 4 and 6). The difference in cell types might contribute to
the different checkpoint responses. As for the lack of a role of
human RAD1 in G2/M checkpoint as shown by knockdown
strategy, a possibility also exists that a low level of RAD1 is
sufficient to support G2/M checkpoint function.
MATERIALS AND METHODS
Growth of ES cells, gene targeting, and generation of
Mrad1+/−ES cells were prepared as previously described (Han et al.,
2010). To generate Mrad1−/−ES cells, Mrad1+/−ES cells were grown
in a medium containing 3.2–4.4mg/mL G418. For the construction of
Mrad1−/−ES cells that ectopically express wild-type gene, the cells
were transfected with pZeoSV2-Mrad1, grown in the presence of
identify Mrad1 transcription.
The Mrad1 expression vector was made by PCR from mouse
cDNA with the primers: 5′-ATTCGGCCGACTCGAGTCAAGACT-
CAGGAACTTCTTCATCAG-3′ and 5′-GTCCATAAGCTTGCCGC-
CACCATGCCTCTCCTAACCCAGTACAATG-3′. The product was
cut with XhoI/HindIII and subcloned into pZeoSV2 (Invitrogen).
Retinoic acid (RA)-induced differentiated ES cells were prepared
using normal Mrad1+/+and Mrad1−/−ES cells cultured in 8μmol/L RA
for 5 days.
Southern blotting and PCR assays to assess genotypes
Genomic DNA was isolated from ES cells and mouse tails using
published methods (Weiss et al., 2000). For southern blotting,
DNA was digested with HindIII, separated on a 0.7% agarose gel,
then transferred to a nylon membrane, and hybridized to a32P-
labeled probe, which was generated by PCR using primers: 5'-
GTGGCCTAGGTGGTTGCGTATCTGAAC-3' and 5'-GTCGGCTCC-
GAGAAGAAGGATGCTCC-3' with mouse genomic DNA as template.
To genotype ES cells and mice by PCR, the reaction was
performed using genomic DNA templates and the following primer
pairs: 5'-GTCTCAGGTTTTCACACATCTTCC-3' and 5'-GCTTA-
TATTCTAGAAACCTTCCTGTATG-3'. After denaturation at 94°C for
5min, 35 cycles of amplification (94°C for 13s, 59°C for 30s, at
72°C for 3min 10s) were followed, with a final extension at 72°C
Northern blotting and RT-PCR
Total RNA was isolated from ES cells using RNeasy Mini kit
(QIAGEN) as described by the manufacturer. For Northern blotting,
10μg RNA was fractionated in a 1.2% (w/v) formaldehyde-agarose
gel and then transferred to a Hybond-N membrane. Templates for
probes were made by PCR using the following primers: Mhus1, 5'-
ATGAAGTTTCGCGCCAAGAT-3' and 5'-AGTCTGGGATG-
GAGGGTTCT-3'; Mrad9, 5'-ACTATTGAGGATTCCTTGCTGGATG-
3' and 5'-ACAGTGAACGAAACTTCTTGGGTG-3'; Mrad1, 5'-
GGAGTTTCCTGCATTTCCAAAAG-3' and 5'-GTCCATAAGCTT-
CCTCTCCTAACCCAGTACAATGAAGAG-3'; neo, 5'-CTACGCGTC-
GACATTGAACAAGATGGATTGCACGC-3' and 5'-AGGAATTCAGA-
CATGATAAGATACATTGATGAG-3'; p21, 5'-ATGTCCAATC-
CTGGTGATGTCCG-3' and 5'-CAGGCTGGTCTGCCTCCGTTTTC-
3'. Then, the membrane was hybridized with the probes, which were
made using [α-32P]-dCTP and the Prime-a-gene labeling system
(Amersham). The labeled membrane was washed and used to
expose X-ray film.
For RT-PCR, 2μg total RNA was reverse-transcribed to cDNA
using the SuperScript First-Strand Synthesis System for RT-PCR
(Invitrogen). PCR amplification was carried out using the following
primer pairs: Mrad1 ORF, 5'-TCCATAAGCTTCCTCTCCTAACCCAG-
TACAATGAAGAG-3' and 5'-ACTGCCATAACTCGAGTCAAGACT-
CAGGAACTTCTTCATCAGG-3'; Mrad1 upstream, 5'-ATGCCTCT-
CCTAACCCAGTACAATG-3' and 5'-TTCTTCCTGAATGA-
CAAATTCCTG-3'; Gapdh, 5'- GCAAAGTGGAGATTGTTGCC-3'
Cell lysate for western blotting was prepared in 1 × SDS-sample
buffer, with the final concentration of 104cells/μL. 3μL lysates were
resolved on a 10% SDS-PAGE gel, and proteins were transferred to a
polyvinylidene difluoride membrane. The membrane was probed
consecutively with primary and peroxidase-conjugated secondary
antibodies, and the signal was detected using the SuperSignal West
Pico Chemiluminescence Substrate system (Prod #34077, Pierce).
Primary and secondary antibodies used in this study were mouse
anti-phospho-H2AX (Upstate), mouse anti-tubulin (Sigma), mouse
anti-p21 (Santa Cruz), rabbit anti-p53 (Santa Cruz), chicken anti-
RAD9, anti-HUS1, peroxidase-conjugated anti-chicken IgY (A9046,
Sigma), peroxidase-conjugated anti-mouse IgG (A9044, Sigma), and
peroxidase-conjugated anti-rabbit IgG (A9169, Sigma). The anti-
RAD9 and anti-HUS1 antibodies were isolated from the eggs of
chickens immunized with full-length human RAD9 and HUS1
Cell survival assays
ES cells were plated in duplicate or triplicate and grown for 16h
before treatment. To test hydroxyurea (HU) sensitivity, the drug was
added to the medium to achieve the designated final concentrations.
After 24h incubation, cells were washed twice with phosphate-
buffered saline (PBS), a fresh medium without HU was added back,
and the cells were incubated for 10 more days before Giemsa stain
and colony counting. To assess the ionizing radiation sensitivity, cells
were exposed to graded doses of gamma rays using a60Co-based
© Higher Education Press and Springer-Verlag Berlin Heidelberg 2011 419
Roles of mouse Rad1 in cells
Protein & Cell
irradiator, and incubated for another 10 days to allow colony
formation. To determine the sensitivity to 254-nm UV light, the
mediumwas removed,and thecells wereexposedto gradeddosesof
the UV light, and then the fresh medium was added to the cells, which
were incubated for 10 more days before colony number was
assessed. Survival percentage was calculated as 100 × [(number
of colonies in treated dishes/number of cells seeded in treated
dishes)/(number of colonies in mock-treated control dishes/number of
cells seeded in mock-treated control dishes)]. Mean values were
derived from three independent replicates, and the standard
deviations were calculated.
Assays for cell cycle checkpoint functions
To evaluate G2/M checkpoint control, 106cells were plated on 10-cm
dishes and incubated at 37°C in 5% CO2overnight. Two sets of cells
were exposed to 10Gy of gamma rays, with one set mock treated as
a control. Immediately after irradiation, colcemid (final concentration
of 50ng/mL) was added to one irradiated set of cells, which were
subsequently incubated for various times at 37°C. Cells were
processed, stained with propidium iodide (PI) and analyzed by an
FACSCalibur flow cytometer (Becton Dickinson) using an established
method (Hang and Fox, 2004).
S/M checkpoint function was examined using published proce-
dures (Hu et al., 2008). Briefly, ES cells were grown to 70%
confluence, and 1mmol/L HU was added to the medium to achieve
a drug concentration of 1mmol/L. Cells were incubated at 37°C in 5%
CO2for various times, processed and suspended in PBS. The cells
were probed with rabbit anti-phospho-histone H3 (Upstate), then
FITC-conjugated anti-rabbit antibodies (Jackson ImmunoResearch
Laboratories, INC), and stainedwith PIbefore flowcytometric analysis.
Intra-S phase checkpoint function was also evaluated by radio-
resistant DNA synthesis (RDS) assay in the BrdU labeling experiment
(Hang and Fox, 2004). Briefly, cells were grown to 70% confluence.
The medium was removed and cells were exposed to 20J/m2UV
light.Afterwards,pre-warmed mediumwas addedback todishes,and
cells were re-incubated at 37°C. At various times after UV treatment,
10μmol/L BrdU was added to the medium and cells were pulse-
labeled for 10min. After processed, probed with FITC-conjugated
anti-BrdU antibody, and stained with PI, cells were subjected to flow
An alkaline comet assay for detecting DNA damage was carried
out with the CometAssay kit as described by the manufacturer
(TREVIGEN). Briefly, comet assay slides were loaded with a mixture
of 10μL of ES cell suspension (5 × 105cells/mL) and 90μL of low-
temperature melt agarose at a final concentration of 0.75%. After
solidification, slides were lysed at 4°C in darkness for 1h in lysis
solution. The slides were soaked and subjected to electrophoresis in
alkaline solution, washed and stained with SYBR Green (0.1μg/mL).
The comet images were captured using a fluorescence microscope
(Nikon). The tail moment was analyzed using Euclid comet analysis
software (Euclid Analysis, St. Louis, MO).
Cells grown on coverslips were fixed with 4% paraformaldehyde in
PBS for 15min at room temperature. The coverslips were washed in
PBS twice, incubated in PBS containing 0.5% Triton-X100 for 15min,
then in PBS containing 5% BSA and 0.1% Triton-X100 for 1 h,
washed in PBS again, and incubated with anti-phospho-H2AX
(Upstate) primary antibody (1:100 dilution) in PBS containing 5%
BSA and 0.1% Triton-X100 for 1h at 37°C. Afterwards, coverslips
were washed twice for 5min each in PBS and incubated with FITC-
conjugated anti-mouse antibody (1:100 dilution in PBS containing 5%
BSA and 0.1% Triton-X100) for 1h at 37°C. Finally, the coverslips
were counterstained with DAPI (10 ng/mL). The images were
captured using a fluorescence microscope (Nikon).
Homology-directed recombination assay
ES cell clone with the integrated homologous recombination reporter
DR-GFP was generated as described previously (Pierce et al., 2001).
70μg of the hprtDRGFP plasmid digested with KpnI/SacI was
transfected into 2 × 107cells in 0.8mL of PBS using an electroporator
at 800Vand 10μF. Then cells were plated onto 5 plates, selected by
puromycin (1.2μg/mL) for 7 days and then by 2μmol/L 6-thioguanine
for another 7 days, and the remaining colonies were isolated. The I-
SceI expression vector pCBASce was transfected using a Lipofecta-
mine plus protocol. 105ES cells were plated onto a 6-well dish, and
transfected with 1 μg I-SceI plasmid using the Lipofectamine plus
mixture on the next day. Cells were incubated for 48h, and then
analyzed by FACSCalibur cytometer (Becton Dickinson).
This work was supported by the National Natural Science Foundation
of China (Grant No. 30900813 to ZSH) and the Knowledge Innovation
Program of Chinese Academy of Sciences to HH (Grant No. KSCX2-
BrdU, bromodeoxyuridine; ES, embryonic stem; DSBs, double-strand
breaks; HR, homologous recombination; HU, hydroxyurea; LIF,
leukemia inhibitory factor; MEF, mouse embryonic fibroblasts;
NHEJ, non-homologous end joining; PI, propidium iodide; RA,
retinoic acid; UV, ultraviolet
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