Oncogene (2003) 22, 2110–2120
& 2003 Nature Publishing Group All rights reserved 0950-9232/03 $25.00
www.nature.com/onc
The mammalian mismatch repair protein MSH2 is required for correct
MRE11 and RAD51 relocalization and for efficient cell cycle arrest induced
by ionizing radiation in G2 phase
Annapaola Franchitto1,3,4, Pietro Pichierri1,3,4, Rita Piergentili1, Marco Crescenzi2,
Margherita Bignami2 and Fabrizio Palitti*,1
1
Laboratorio di Citogenetica Molecolare e Mutagenesi – DABAC, Università degli Studi della Tuscia, Via S, Camillo de Lellis, 01100
Viterbo, Italy; 2Laboratorio di Tossicologia Comparata ed Ecotossicologia, Istituto Superiore di Sanità, Viale Regina Elena 299,
00161 Roma, Italy
In yeast, MSH2 plays an important role in mismatch
repair (MMR) and recombination, whereas the function of
the mammalian MSH2 protein in recombinational repair
is not completely established. We examined the cellular
responses of MSH2-deficient mouse cells to X-rays to
clarify the role of MSH2 in recombinational repair. Cell
survival, checkpoint functions and relocalization of the
recombination-related proteins MRE11 and RAD51 were
analysed in embryonic fibroblasts derived from MSH2+/+
and MSH2/ mice, and in MSH2-proficient and deficient
mouse colorectal carcinoma cells. Loss of MSH2 function
was found to be associated with reduction in cell survival
following radiation, absence of either MRE11 or RAD51
relocalization and a higher level of X-ray-induced
chromosomal damage specifically in G2-phase cells.
Finally, MSH2/ cells showed an inefficient early
G2/M checkpoint, being arrested only transiently after
irradiation before progressing into mitosis. Consistent
with the premature release from the G2-phase arrest,
activation of CHK1 was transient and CHK2 was not
phosphorylated in synchronized MSH2-null cells. Our
data suggest that an active MSH2 is required for a correct
response to ionizing radiation-induced DNA damage in
the G2 phase of the cell cycle, possibly connecting DSB
repair to checkpoint signalling.
Oncogene (2003) 22, 2110–2120. doi:10.1038/sj.onc.1206254
Keywords: MSH2; mismatch repair; cell cycle arrest;
DSB repair
Introduction
The DNA mismatch repair (MMR) system is a highly
conserved post replicative editing process that prevents
*Correspondence: F Palitti; E-mail: [email protected]
3
Current address: CNRS, UPR2169, Instabilité Génétique et Cancer,
Institut André Lwoff, 7 Rue Guy-Moquet, 94801 Villejuif, France
4
The first two authors contributed equally
Received 17 July 2002; revised 19 November 2002; accepted 21
November 2002
mutations (for a review see Kolodner, 1996; Modrich
and Lahue, 1996). Germline mutations in MMR genes
are associated with predisposition to colorectal and
other cancers (for a review see Jiricny and NystromLahti, 2000). While the role of MMR in the correction
of replication errors is well established, its involvement
in the processing of DNA damage induced by chemical
and physical agents is less clear. Thus, whereas
inactivation of MMR always leads to tolerance to
methylating agents (Karran and Bignami, 1994), either
moderate levels of resistance (Aebi et al., 1996;
Anthoney et al., 1996) or hypersensitivity to other types
of DNA damage has been observed in MMR-defective
cells (Fiumicino et al., 2000; Pichierri et al., 2001). For
example, MSH2/, MLH1/ and PMS2/ mouse
embryo fibroblasts (MEFs) show a modest increase in
survival compared to wild-type cells after exposure to
ionizing radiation (IR), while conflicting results were
observed in other human cellular models (Aquilina et al.,
1999; Davis et al., 1998; DeWeese et al., 1998; Fritzell
et al., 1997; Xu et al., 2001b; Zeng et al., 2000). In
addition, the MMR proteins have been involved in the
apoptotic response (Bellacosa, 2001; Berry and Kinsella,
2001; Kaina et al., 2001). The mammalian MSH2
protein forms heterodimers with either MSH3 or
MSH6 and these complexes initiate MMR by recognition of base–base mismatches and short insertion/
deletion loops (for a review see Jiricny, 1998; Kolodner
and Marsischky, 1999). In Saccharomyces cerevisiae,
Msh2 and Msh3 also participate directly in recombinational repair (Alani et al., 1994). The yeast Msh2–Msh3
complex facilitates removal of nonhomologous ends
that are formed during gene conversion (Sugawara et al.,
1997) or during repair by the single-strand annealing
pathway. A role for the Msh2 protein in the suppression
of recombination between divergent sequences has also
been reported both in yeast and mammalian cells (Chen
and Jinks-Robertson, 1999; Ciotta et al., 1998; de Wind
et al., 1995; Worth et al., 1994; Zhang et al., 2000).
IR induces a large variety of DNA lesions, including
double-strand breaks (DSBs), single strand breaks
(SSBs), base and sugar damage (Hutchinson, 1985).
DSBs are considered to be potentially the most lethal
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A Franchitto et al
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lesions (Bradley and Kohn, 1979). In S. cerevisiae, the
predominant DSB repair pathway is the homologymediated recombinational repair (HR) (Kanaar et al.,
1998). HR is also an important repair pathway in
mammalian cells (Liang et al., 1998; Sonoda et al., 1998;
Takata et al., 1998). The initial step in DSBs repair by
HR involves processing of DNA ends to produce 30
single-strand tails that are the substrate for homologous
pairing and strand invasion (Kanaar et al., 1998; White
and Haber, 1990). The identity of the nuclease in the
processing step is still uncertain, but MRE11 is a
candidate for this activity (Paull and Gellert, 1998;
Trujillo et al., 1998). The RAD51 and RAD54 proteins
act during the pairing and the strand exchange steps by
forming a nucleoprotein filament along the 30 singlestranded tails. Recombination intermediates produced
in this way are then processed further in reactions that
involve the resolution of Holliday junctions, DNA
synthesis and nick ligation (Baumann and West, 1998).
In higher eukaryotes, DSBs are repaired via two
pathways: the so-called nonhomologous end-joining
(NHEJ) and HR (Kanaar et al., 1998). Vertebrate cells
preferentially repair DSBs by HR in the late-S and G2
phases of the cell cycle when an undamaged sister
chromatid is available, while NHEJ appears to be
confined mainly to the G1/early-S phases (Takata et al.,
1998). Among the proteins implicated in DSB repair,
MRE11 seems crucial for both HR and NHEJ (Haber,
1998) whereas RAD51 is essential for HR (Kanaar et al.,
1998).
To investigate the role for mammalian MSH2 in
DSBs repair following IR, we studied whether the
absence of the MSH2 protein results in an altered
response to DNA damage. To this aim, we have used
mouse embryonic fibroblasts (MEFs) derived from
MSH2 knockout mice and mouse colorectal carcinoma
cells either proficient or defective in MSH2 (Humbert
et al., 1999).
We report that the absence of MSH2 leads to reduced
survival and is also associated with aberrant RAD51and MRE11-focus formation in cells irradiated in
G2 phase, but not in S phase. Moreover, a higher level
of chromosomal damage was found in MSH2/ cells
irradiated in the G2 phase of the cell cycle. Interestingly,
loss of MSH2 results in premature release from the
early-G2/M arrest following IR, with inability to
activate properly the checkpoint kinases CHK1 and 2.
We suggest that active MSH2 protein is required for
both proper early checkpoint function and efficient
DSBs repair after IR in mammalian cells.
an additional 12 h. At 4 h after release from the double
thymidine block, 80% of the cell population was in the late-S/
G2 of the cell cycle. Distribution in the different stages of the
cell cycle was assessed by standard cytofluorimetric techniques,
evaluating the DNA content after propidium iodide (PI)
staining of ethanol-fixed cells.
X-ray irradiation
Irradiation was carried out at 371C with a Gilardoni X-ray
apparatus (MGL 300/6-D) operating at 300 kV and 6 mA, at a
dose rate of 2 Gy/min. After irradiation, cells were recovered in
complete medium for the indicated time until harvested.
Determination of cell survival and apoptotic cell death
To measure cell survival, cells were seeded in complete medium
(100 cells/6 cm dish) and treated with X-rays 18 h later. After 1
week, the surviving colonies were stained with Giemsa and
counted. The results are presented as a fraction of the relevant
control (surviving fraction). For the analysis of the IR-induced
apoptotic cell death, MEFs were seeded onto coverslips,
irradiated with 4 or 8 Gy and processed at different
postirradiation times for the analysis of apoptosis by a TdTmediated end-labelling kit as described by the manufacturer
(Roche Biochemicals).
Determination of the early-G2 arrest
Progression of cells into mitosis from the G2 phase was
assessed by analysis of the mitotic indices. Cells were treated
either with X-ray (2 or 4 Gy) or UV (20 J/m2) and cultured for
different time periods in the presence of 0.02 mg/ml colcemid to
accumulate mitotic cells. Slides prepared at each time point
were stained with PI and mitotic indices were determined by
counting at least 1000 cells per culture. No differences in the
mitotic index were detected in untreated samples from either
MSH2-proficient or MSH2-deficient cells. Moreover, no
differences in the response to colcemid were observed among
the different cell lines used (our unpublished results).
Western blot analysis
Materials and methods
Cell extracts (20 mg) were denatured and separated on 10 or
7% SDS–polyacrylamide gels according to standard procedure. Proteins were transferred to PVDF membranes (BioRad) and immunoblotting was carried out according to
standard procedures. Blots were separately incubated with
primary antibodies against CDC2 (mouse monoclonal, Oncogene Research), CHK1 (rabbit polyclonal, Santa Cruz) and
CHK2 (mouse monoclonal, BD-Pharmingen) or MRE11
(mouse monoclonal, AbCam). To verify the expression of
the MSH2 protein, mouse monoclonal anti-MSH2 antibodies
(BD-Transduction Laboratories) were used. Appropriate
species-specific HRP-conjugate IgGs were used as secondary
antibodies (Amersham). The blots were developed using the
ECL detection system (Amersham). Equal loading and
transfer were assessed reprobing the blots with anti-b-tubulin
antibody (Santa Cruz) and by Ponceau-red staining of the blots.
Cell cultures and synchronization
Analysis of MRE11 and RAD51 foci
All the cell lines were cultured and handled as already
described (Pichierri et al. , 2001). For biochemical analyses,
cells were enriched in late-S/G2 prior to X-ray irradiation by a
double thymidine block. Cells were exposed to 2 mm thymidine
for 12 h. The cultures were then washed, allowed to recover in
fresh medium for 6 h and then exposed to 2 mm thymidine for
Cells were cultured on coverslips, mock-treated (0 h) or
exposed to X-rays (8 Gy) and sampled 1, 2, 4, 8 and 16 h
afterwards. In order to analyse MRE11 foci in untreated or
irradiated cells shortly after treatment, fixation in a 4%
paraformaldehyde-buffered solution was carried out after an
in situ fractionation procedure as described (Mirzoeva and
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Petrini, 2001). Cells were then immediately processed, as
previously described (Pichierri et al., 2001), for immunochemical detection of MRE11 or RAD51 using rabbit polyclonal
anti-RAD51 or MRE11 antibodies (Oncogene Research) and
Alexa546-conjugated mouse anti-rabbit secondary antibody
(Molecular Probes). Alternatively, irradiated cultures were
processed for simultaneous visualization of RAD51 foci and
human proliferating cell nuclear antigen (PCNA) (Santa Cruz
Biotechnology). Secondary antibodies (Molecular Probes) (1 h
incubation at 371C) were selected to provide the appropriate
combinations of species specificity (donkey anti-rabbit or mouse) and colour discrimination (conjugated to either
fluorescein or Alexa546). Cells were counterstained with
DAPI. For each experimental point, at least 200 nuclei were
examined and RAD51 or MRE11 foci scored by eye at a
magnification of 100 using a Zeiss epifluorescence microscope. Only nuclei showing >5 foci were considered as
positive. Images were captured with a CCD camera and
processed by using IPLab image analysis software. Parallel
samples, incubated either with goat normal serum or with the
secondary antibody, allowed one to discriminate possible
artefacts.
Recovery of DNA synthesis after X-ray irradiation
Cell cultures were irradiated with X-rays (4 Gy) and analysed
at different time points (1, 3, 6, and 8 h). To discriminate cells
in active DNA synthesis, BrdUrd (30 mg/ml) was added 1 h
before harvesting and BrdUrd incorporation in interphase
nuclei was evaluated as previously described (Franchitto et al.,
1998). To evaluate the percentage of labelled nuclei, at least
500 interphase cells were scored. Only nuclei displaying
uniform BrdUrd labelling in the entire nuclear volume were
considered as actively replicating. The percentage of cells
undergoing DNA synthesis at each time point was calculated
as the fraction of treated cells versus mock-treated controls.
Chromosome preparations and analysis
In order to analyse chromosomal damage induced by X-ray
irradiation, cultures were treated with X-rays (2 Gy) and
harvested between 3 and 6 h. To label cells in the S phase of the
cell cycle, BrdUrd (30 mg/ml) was added to the cultures
immediately after irradiation together with colcemid (0.2 mg/
ml) until harvesting. Metaphases were prepared according
to standard procedures from colcemid-arrested cells and used
for the immunodetection of the BrdUrd incorporation to
determine the phase of the cell cycle at which they were treated (i.e. labelled, S phase cells; unlabelled, G2-phase cells)
(Palitti et al., 1999).
The frequency of chromosomal aberrations was scored in
both BrdUrd-labelled and unlabelled metaphases. A total of
100 labelled or unlabelled cells were analysed in detail for
chromatid-type aberrations according to the criteria described
by Savage (1976).
Colo5#2 in which correction of the phenotype was
achieved by transfer of a normal human chromosome #2
bearing the wild-type hMSH2 allele (Humbert et al.,
1999). As shown in Figure 1, MSH2 expression was only
detected in the MSH2-proficient cells (Colo26) but not
in Colo5 or in the MSH2/ MEFs.
Both MEFs and colorectal carcinoma cells defective
in MSH2 were slightly more sensitive (B1.5-fold) to
X-ray irradiation than their MSH2-proficient counterparts (Figure 2a, b). This sensitivity was most evident at
the highest doses of X-rays (4 and 8 Gy). As expected,
the revertant Colo5#2 cells showed cell survival closely
similar to the parental Colo26 cells.
These data indicate that, in mammalian cells, loss of
MSH2 confers a slight hypersensitivity to X-rays,
especially at higher dose levels.
Correct MRE11 relocalization in the G2 phase of the cell
cycle after IR requires the MSH2 protein
Radiation sensitivity is often associated with DNA
repair defects, mainly NHEJ or HR (Kanaar et al.,
1998). MRE11 can associate with damaged DNA and is
involved in its repair either by NHEJ or HR (Kanaar
et al., 1998). This factor takes part in multiprotein
complexes that can be visualized by immunofluorescence as discrete nuclear foci (Maser et al., 1997). We
investigated whether loss of MSH2 could affect MRE11
focus formation. In order to study the early response of
the MRE11 protein following DNA damage, we used an
in situ fractionation protocol. Accordingly, three distinct
localization patterns of MRE11 foci can be detected:
pattern I (large aggregates) found in the majority of the
unirradiated cells, pattern II (evenly distributed small
foci) observed shortly after irradiation and pattern III
(large patches) found at later times postirradiation
(Mirzoeva and Petrini, 2001). Since the formation of
pattern II coincides temporally with DSBs repair, as also
Results
Loss of MSH2 confers hypersensitivity to IR
We compared the effects of X-rays on the survival of
primary MEFs derived from MSH2+/+ or MSH2/
mice. As a complementary cellular system, we used an
MMR-proficient mouse colon tumour (Colo26), its
MSH2-defective derivative Colo5 and the revertant
Oncogene
Figure 1 Western blot analysis of MSH2 expression. Total
proteins (20 mg) for each experimental point were loaded onto a
10% SDS–PAGE gel and subjected to Western/immunoblotting.
Blots were probed with antibody raised against MSH2. The
* indicates aspecific bands
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A Franchitto et al
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Figure 2 Reduced cell survival of MSH2-deficient cells following IR. MEFs from MSH2 þ / þ and MSH2/ mice (a) or MSH2proficient (Colo26, Colo5#2) and MSH2-deficient (Colo5) mouse colorectal carcinoma cells (b) were exposed to increasing doses of
X-rays and analysed for clonogenic survival. Each symbol represents the mean of triplicate independent experiments; the s.e.m. is
shown by error bars In some cases, the error bars are smaller than the symbol
demonstrated by colocalization with the H2AX histone
(Mirzoeva and Petrini, 2001; Paull et al., 2000; Rogakou
et al., 1998), we focused our analysis on this specific
MRE11 localization. Both MSH2+/+ and MSHZ/
untreated cells displayed similar numbers of nuclei
bearing type-I and -II foci. (Figure 3a, b and our
unpublished observation). After 8 Gy the percentage of
cells containing MRE11 type-II foci increased in
MSH2+/+ cells in a time-dependent manner, whereas
in MSHZ/ cells the percentage of these foci did not
change significantly up to 8 h postirradiation (F3a, b). In
addition, the MRE11 type-II foci that do form in the
absence of MSH2 are always associated with PCNA
staining (our unpublished observation). The differences
in the percentage of nuclei containing foci between the
two genotypes cannot be ascribed to changes in the total
amount of MRE11 protein, as indicated by Western
blotting analysis (Figure 3c). Moreover, it is unlikely
that the enhanced apoptotic response of MSH2/
compared to MSH2+/+ MEFs (Figure 3d) is responsible
for the altered MRE11 focal localization of these cells.
Apoptosis after irradiation is observed at much later
times than MRE11 relocalization (16 versus 1–4 h). In
addition, the similar timing in the induction of apotosis
in the two cell lines, even by staining with the early
apoptotic marker Annexin V (Boersma et al., 1996) 3d
and data not shown), is not consistent with a selective
depletion of the MRE11-positive population of
MSH2/ cells (Boersma et al., 1996).
Given that the MRE11/RAD50/NBS1 complex is
involved in the activation of the S-phase checkpoint
after IR (Carney et al., 1998), we evaluated the
possibility that IR differently affects the progression of
cells through the S phase in MSH2+/+ and MSH2–/–
cells. A dramatic decrease in cells replicating their DNA
was observed between 3 and 6 h after irradiation but no
significant difference between MSH2+/+ and MSH2–/–
MEFs was observed in the inhibition of DNA synthesis
(Figure 4). The irradiated cells also resumed DNA
synthesis in a similar manner, further indicating that the
progression of damaged cells through the S phase is not
notably affected by loss of MSH2.
These data suggest that the MSH2 protein is required
for efficient relocalization of MRE11 protein after IR
treatment of G2-phase cells, whereas it is dispensable for
a correct activation of the S-phase checkpoint.
RAD51 focus formation in the G2 phase of the cell cycle is
affected by the absence of MSH2 protein
A defect in MRE11 activity could theoretically impair
both NHEJ and HR. However, a role for MSH2 in DSB
repair has been envisaged only in HR. Thus, to
investigate whether the observed incorrect MRE11
relocalization in MSH2-defective cells could be related
to DSB repair by HR, we analysed subnuclear
relocalization of the RAD51 recombinase, a key enzyme
in this process.
Compared to wild-type MEFs, untreated MSH2/
cells showed a significantly higher spontaneous RAD51
focus-forming activity (Figure 5a). After 8 Gy of IR
exposure, the number of nuclei showing RAD51 foci
increased over time and reached the highest values 8 h
after treatment in both MSH2+/+ and MSH2/ cells.
However, focus formation was somewhat reduced in
MSH2/ cells at 4 and 8 h postirradiation (Figure 5a).
Given that HR is active in late-S and G2 phases of the
cell cycle, experiments were undertaken to determine
whether X-rays induce RAD51 foci in both phases.
S-phase cells were identified through PCNA immunofluorescence under fixation conditions that, solubilizing
unbound PCNA, visualize only the fraction bound to
the replication complexes. In wild-type cells, at 4 and 8 h
postirradiation, about two-thirds of the nuclei showing
RAD51 foci were also PCNA-positive, while the
remaining were PCNA-negative. MSH2/ cells showed
as many RAD51 foci as wild-type MEFs in PCNApositive nuclei but, in sharp contrast, no foci were
detected in MSH2/ cells in PCNA-negative nuclei
(F5b, c). As already described by others, the RAD51
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Figure 3 Formation of type-II MRE11 foci following IR. MSH2+/+and MSH2/ MEFs were mock-treated (0 h) or exposed to 8 Gy
of X-rays and subjected to immunofluorescent staining of MRE11 at different recovery times. The data are the mean7s.e.m. of three
independent experiments. (a). MRE11 re-localization in MSH2+/+and MSH2/ cells (b). In untreated cells, type-I MRE11 foci are
predominant in both MSH2+/+and MSH2/ cells (indicated by arrowhead in (a)). Following IR, MSH2-proficient but not MSH2deficient cells show type-II MRE11 foci (indicated by the arrowhead in (c)). Analysis of the MRE11 levels shows no difference in
protein expression between MSH2+/+and MSH2/ cells (c). Total proteins (20 mg) for each experimental point from mock-treated or
8 Gy-irradiated MSH2+/+and MSH2/ MEFs were loaded onto a 7% SDS–PAGE gel and subjected to Westem/immunoblotting
analysis. Blots were probed with the MRE11 antibody. Equal loading and transfer were assessed by ponceau-red staining of the blots.
Enhanced apoptotic cell death after IR treatment in MSH2/ MEFs (d). The horizontal reference line represents the mean value of the
spontaneous level of apoptosis in the two cell lines. The data are the mean from three independent experiments. Standard errors are not
shown but were always o10% of the respective mean value
and MRE11 proteins did not colocalize either in G2- or
in S-phase cells after irradiation (Mirzoeva and Petrini,
2001), and the absence of the MSH2 protein did not
alter this pattern (data not shown). Thus, as S-phaseassociated RAD51 foci are similar in both cell lines
studied, the observed reduction in RAD51 foci in
MSH2/ cells is to be ascribed solely to the absence
of foci in the G2 phase.
Loss of MSH2 is associated with increased levels of
chromatid and isochromatid breaks in the G2 phase
of the cell cycle following irradiation
To verify whether the defects in the response to IRinduced DNA damage in the G2 phase of the cell cycle
observed in MSH2-defective cells is accompanied by a
higher level of chromosomal damage, MEFs were
exposed to 2 Gy of X-rays, a dose that corresponds to
Oncogene
B80% of survival in both genotypes (Figure 2). Since
HR is thought to take place in late-S or G2 phases of the
cell cycle, cells were pulse-labelled with BrdUrd immediately after irradiation, and chromosomal damage
was analysed in both BrdUrd-negative (unlabelled,
G2-phase cells) and -positive (labelled, S-phase cells)
metaphases (Tables 1 and 2, respectively). Because
MSH2/ cells did not correctly arrest in G2-phase
(Figure 6; see below), chromosomal damage in
MSH2+/+ and MSH2/ cells was compared in populations showing similar percentages of labelled mitotic
cells (i.e. treated with colcemid for different lengths of
time; Palitti et al., 1999).
We found a statistically significant increase in the
frequency of chromosomal damage in the MSH2/
MEFs, in comparison with wild-type cells. In particular,
we observed a sharp enhancement (Bthree-fold) of
isochromatid breaks and a more moderate increase in
MSH2 and recombinational repair in G2 phase
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Figure 4 Analysis of radioresistant DNA synthesis in MSH2proficient and -deficient cell lines. Untreated or X-irradiated (4Gy)
MSH2+/+ (closed circles) and MSH2/ (open triangles) MEFs
were harvested at the indicated recovery times. Cells were processed
for the immunodetection of BrdUrd incorporation as described in
‘Materials and methods’. The data are the mean7s.e.m. of three
independent experiments
chromatid breaks (B1.5-fold) after irradiation (Table 1).
Evaluation of the chromosomal damage in MSH2+/+
and MSH2/ cells at a second pair of sampling times
(6 and 5 h postirradiation, respectively) showed no
further increase in the number of chromatid breaks,
while the larger induction of isochromatid breaks in
MSH2/ cells was conserved (data not shown).
Similarly, upon X-irradiation lack of MSH2 protein
in the tumour cell line Colo5 was associated with an
increased number of isochromatid breaks in comparison
with the parental, MSH2-expressing Colo26. Meaningfully, expression of hMSH2 by introduction of the
human chromosome 2 in Colo5 cells reduced the
frequency of breaks to that observed in Colo26 cells.
These data strongly indicate that the higher level of
chromosomal damage observed in MSH2-defective cells
is because of loss of the MSH2 function.
Similar chromosomal damage analyses were performed in cells treated in the late S-phase of the cell
cycle (Table 2). As in the previous experiments, we
compared populations containing similar numbers of
labelled cells. No difference in the frequency of
chromatid or isochromatid breaks was found between
MSH2+/+ and MSH2/ cells, despite a slight increase
Figure 5 Reduced induction of RAD51 foci in MSH2-deficient cells following IR. Immunofluorescence of RAD51 and PCNA after
IR. (a) MEFs were seeded onto coverslips, irradiated with 8 Gy and fixed at different postirradiation times. The data are the
mean7s.e.m. of three independent experiments. The percentage of RAD51-positive nuclei showing PCNA staining is plotted versus
recovery time (C). Dark bars: RAD51- and PCNA-positive cells; light bars: RAD51-positive and PCNA-negative cells. The data are
representative of three different experiments showing similar results. Standard errors are not shown but were always o10% of the
respective mean value
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Table 1 Analysis of the chromosomal damage induced by X-rays in the G2-phase of the cell cycle in MSH2-deficient and proficient unlabelled
cells
Aberrations per 100 cells
Cell line
Genotype
Treatment
Abnormal
cells (%)
Labelled
cells (%)
Chromatid
breaks
MEFs
MSH2+/+
Control
2 Gy+5 h colcemid
5
80
NA
15
5
205
MEFs
MSH2/
Control
2 Gy+4 h colcemid
7
90
NA
11
Colo26
MSH2+/+
Control
2 Gy+4 h colcemid
4
63
Colo5
MSH2/
Control
2 Gy+3 h colcemid
Colo5#2
MSH2+/+
Control
2 Gy+3 h colcemid
Isochromatid
breaks
Chromatid
exchanges
Total aberrations
(-gaps)
Aberr.ns/cell
(mean7s.e)
0
14
0
24
5
243
0.0570.02
2.4370.15
6
285
1
41**
1
10
8
336
0.0870.03
3.3670.19**
NA
10
4
198
0
10
0
13
4
231
0.04+0.02
2.3170.11
4
70
NA
13
2
248
2
31**
0
8
4
287
0.0470.02
2.8770.16*
5
68
NA
8
4
201
0
12
1
8
5
221
0.0570.04
2.2170.12
**Statistically significant Po0.01 (t-test), compared to the wild type
*Statistically significant Po0.05 (t-test), compared to the wild type
Table 2
Analysis of the chromosomal damage induced by X-rays in the late-S phase of the cell cycle in MSH2-deficient and proficient labelled cells
Aberrations per 100 cellsa
Cell line
Genotype
Treatment
Abnormal
cells (%)
Labelled
cells (%)
Chromatid
breaks
MEFs
MSH2+/+
2 Gy+6 h colcemid
Control
6
43
NA
32
8.4
127
MEFs
MSH2/
2 Gy+5 h colcemid
Control
9
55
NA
39
10
140
Isochromatid
breaks
Chromatid
exchanges
Total aberrations
(-gaps)
Aberrations/cell
(mean7s.e.)
0
2
0
15.6
8.4
144.6
0.0870.01
1.4470.10
0
4.2
2
35.6*
12
187
0.1270.03
1.7970.12
a
A total of 500 labelled metaphase cells were scored for each experimental point. *Statistically significant Po0.05 (t-test), compared to the
wild-type
NA, not applicable; spontaneous chromosomal damage was scored in either labelled or unlabelled cells without any significant difference so the
data are referred to 500 metaphase cells either unlabelled and labelled
Figure 6 MSH2-deficient cells arrest only transiently after irradiation in the G2 phase of the cell cycle. MSH2-proficient or -defective
cell lines were treated with 2 Gy (a) or 4 Gy (b) of X-rays and recovered for the indicated times. (c) Arrest in the G2 phase of the cell
cycle is unaffected in MSH2/ cells following UV radiation. MEFs from MSH2+/+or MSH2/ mice were either mock-treated or
exposed to UVC radiation (20 J/m2) and recovered for the indicated times. Mitotic indices were calculated as described in ‘Materials
and methods’; the data are the mean7s.e.m. of three independent experiments
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MSH2 and recombinational repair in G2 phase
A Franchitto et al
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(Btwofold) in the number of chromatid exchanges
obtained in MSH2-defective cells.
In conclusion, these data indicate that loss of MSH2
function is associated with increased levels of X-rayinduced isochromatid and chromatid breaks specifically
during the G2 phase of the cell cycle.
MSH2 is required to sustain G2 arrest induced by IR
We observed defective relocalization of the two DSB
repair enzymes MRE11 and RAD51 specifically in the
G2 phase of the cell cycle. DNA repair and checkpoint
response are two interlinked processes (Lowndes and
Murguia, 2000), and loss of certain MMR proteins has
been correlated to defective G2 accumulation (Davis
et al., 1998; Yan et al., 2001). Thus, we investigated
whether the defective MRE11 and RAD51 relocalization observed in MSH2/ cells is because of defects in
triggering the G2 checkpoint in cells irradiated in the
G2 phase. Recently, Xu et al. (2002), demonstrated that
the G2 accumulation observed by flow cytometry at late
times after irradiation is a pathway completely different
from the early G2/M checkpoint response elicited in G2phase treated cells (Xu et al., 2002). For instance, the
early G2/M checkpoint response is ATM- and BRCA1dependent, whereas the late G2 accumulation is not (Xu
et al., 2001a, 2002). Since we observe defective MRE11
and RAD51 relocalization shortly after treatment
(o10 h), we hypothesized a defect in the early rather
than in the late G2/M response (Yan et al., 2001). Thus,
we analysed the ability of MSH2/ cells irradiated in
G2 to progress into mitosis by assessment of the mitotic
index. Irradiation with 2 and 4 Gy induced an efficient
G2 arrest in all the MSH2-proficient cell lines, as shown
by the reduction in the mitotic index shortly after
irradiation (F6a, b). The higher dose induced a more
prolonged inhibition of cell cycle progression. A similar
reduction in the mitotic index was observed in MSH2deficient cells only 1 h after treatment (F6a, b). At later
times, however, the observed G2 arrest was released in
MSH2-deficient cells. Thus, our data partially agree
with those of Yan et al. (2001) on the G2 accumulation
of MMR-defective cells, suggesting that in the absence
of MSH2, the early G2/M checkpoint is properly
activated but prematurely released. The behaviour
of MSH2-deficient cells appeared to be specifically
attributable to loss of the gene, since the revertant
Colo5#2 cells showed G2 arrest duration similar to
that observed in the parental cells (F6a, b). Conversely,
UV treatment induced an efficient G2 arrest, independent of the presence of a functional MSH2 gene
(Figure 6c), suggesting that the role of MSH2 in the
early G2/M checkpoint depends on the type of DNA
damage.
Consistently with the cytological data, in late-S/G2
enriched MSH2+/+ cells CDC2 was inhibited after 4 Gy,
as indicated by the presence of its phosphorylated,
slowly migrating isoform and by the disappearance of
the unphosphorylated form at 90 min postirradiation
(Figure 7b). Conversely, MSH2/ cells did not show
any apparent difference in the amount of the CDC2
Figure 7 Failure of maintenance of CHK1 phosphorylation and
absence of CHK2 activation in MSH2-deficient cells following IR.
(a) Analysis of cell cycle distribution of MSH2+/+and MSH2/
MEFs in asynchronous cultures (a, b), immediately after release
from the double thymidine block (c, d) and after 4 h from the
release when the majority of cells (B80%) were in late-S/G2 (e, f).
(b) Synchronized cells were either mock-treated or exposed to 4 Gy
of X-rays 4h after the release from the double thymidine block,
when in late-S/G2. Blots were probed with antibody raised against
CDC2 and sequentially reprobed with Tubulin antibodies. (c)
Absence of MSH2 leads to uncorrect CHK1 and CHK2
phosphorylation after IR. -P: phosphorylated isoform. Equal
loading and transfer were assessed by ponceau-red staining of the
blots. The results shown are representative of those observed in
three independent experiments
unphosphorylated isoform between mock-treated and
irradiated samples.
Maintenance of the CDC2 inhibitory phosphorylation is dependent on the two checkpoint kinases CHK1
and hCDS1/CHK2 (Fumari et al., 1999; Matsuoka et al.,
1998; Sanchez et al., 1997). It has been reported that
CHK1 initiates the G2 arrest following IR, whereas
CHK2 is essential to maintain this arrest (Hirao et al.,
2000). Thus, we investigated whether CHK1 and CHK2
were properly activated in MSH2-deficient cells. As
expected, IR induced the rapid (30 min) phosphorylation of both CHK1 and CHK2 in MSH2-proficient cells,
whereas a transient activation of CHK1 and a complete
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lack of CHK2 phosphorylation were observed in
MSH2-deficient cells (Figure 7c).
These results are consistent with the ability of MSH2deficient cells to arrest, but not to maintain, the G2
block after IR and suggest that, in the absence of
MSH2, the G2 checkpoint proteins do not properly
sustain the DNA damage signal.
Discussion
We observed that MSH2-defective mouse cells are more
radiation sensitive than their wild-type counterparts. An
elevated radiosensitivity can be the consequence of
either an impaired checkpoint response or a defect in
repair of IR-induced DNA damage.
IR induces different types of DNA lesions, among
these DSBs, which are repaired through the NHEJ or
the HR pathways (Kanaar et al., 1998). In vertebrate
cells, each of these pathways operates preferentially in a
specific phase of the cell cycle: NHEJ in G1/early-S and
HR in late-S/G2 (Takata et al., 1998). We found that
two of the key proteins in HR, MRE11 and RAD51,
were not correctly relocalized after DNA damage in the
absence of MSH2, consistently with a role for this
mammalian MMR protein in recombination (Alani
et al., 1994; Chen and Jinks-Robertson, 1998, 1999;
Datta et al., 1996; Elliott and Jasin, 2001; Evans et al.,
2000). This defective relocalization was observed in the
G2 phase but not in the S phase of the cell cycle,
suggesting that the role for MSH2 in the relocalization
of MRE11 and RAD51 is somewhat confined to the
G2 phase of the cell cycle. Moreover, loss of MSH2
function results in a higher yield of chromosomal
damage in the G2 phase of the cell cycle, in particular
isochromatid breaks. Specific types of chromosomal
damage are associated with inactivation of different HR
genes. Inactivation of RAD54 or RAD51 in chicken
DT40 cells increases their susceptibility to radiationinduced chromatid breaks, because of a failure of the
earlier stages of HR (Sonoda et al., 1998; Takata et al.,
1998). In contrast, MRE11 mutations are associated
with elevated induction of isochromatid breaks, possibly
deriving from incomplete repair after joint-molecule
formation (Yamaguchi-lwai et al., 1999). Thus, our
observation of a higher frequency of X-ray-induced
isochromatid breaks in MSH2-deficient cells actually
suggests that HR starts, but cannot proceed to completion, as in MRE11-null cells.
Since MRE11 performs an important role in DNA
damage signalling (D’Amours and Jackson, 2002 and
references therein), it is possible that in the absence of
MSH2 the cellular response to DNA damage is not
properly activated and HR does not correctly take
place, forcing cells to use NHEJ to repair DSBs.
Consistently, it has been reported that NHEJ could
serve as backup system for DSB repair when HR is
impaired (Fukushima et al., 2001).
Such a failure to correctly perform HR-mediated DSB
repair in the absence of MSH2 could also correlate with
the observed inability to maintain the G2 arrest. Indeed,
we found that MSH2-deficient cells arrested correctly at
the early G2 checkpoint after IR, but were unable to
maintain the arrest and re-entered the cell cycle within
1 h after treatment. Consistent with premature release
from the early G2/M checkpoint, both the checkpoint
kinases CHK1 and CHK2 were not properly activated
in MSH2/ cells, even though CHK1 phosphorylation
was detected, as in wild-type cells, immediately after
irradiation. CHK1 and CHK2 kinases are both required
to activate the G2 checkpoint after IR (Matsuoka et al.,
1998; Sanchez et al., 1997). CHK1 appears to be
essential in triggering the checkpoint, whereas CHK2
is needed to maintain the arrest (Hirao et al., 2000). Our
observations suggest that MSH2 is not necessary to
impose this arrested state, arguing against a direct role
in the G2 checkpoint, but is required to maintain this
arrested state, possibly acting somewhat indirectly in
later steps of the G2 checkpoint activation. We did not
observe such behaviour following UV irradiation. This
suggests that MSH2 plays a role in response to DNA
lesions induced by IR but not UV irradiation.
Figure 8 Model for the role of MSH2 in DSB repair by HR in the G2-phase of the cell cycle. Following DNA damage, CHK1 is
phosphorylated in both MSH2-proficient and -deficient cells. Cells are arrested at the G2/M border to repair DNA damage. IRinduced base damage, which accumulates in MSH2/ cells (Colussi et al., 2002; DeWeese et al., 1998), could interfere with HR
possibly because of increased levels of DNA mismatches. CHK2 is activated in MSH2-proficient cells to sustain G2 arrest, allowing
time for mismatch removal, and MSH2 might be required to recognize mismatches and recruit MRE11. Consistent with this
possibility, a colocalization of MSH2 and MRE11 has been recently reported (Zink et al., 2002). In the absence of MSH2, MRE11 is
not properly relocalized and CHK2 is not activated, leading to premature release of the G2 arrest and elevated yield of isochromatid
breaks and chromatid breaks
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MSH2 and recombinational repair in G2 phase
A Franchitto et al
2119
Based on our observations and data reported in the
literature, we propose a model to explain the incorrect
MRE11 and RAD51 relocalization, the elevated IRinduced chromosomal damage and the premature
release from the G2 checkpoint, associated with loss of
MSH2. According to this model, isochromatid and
chromatid breaks result from the inability of MSH2defective cells to perform an efficient DSB repair via HR
(Figure 8).
In conclusion, our data support the idea that MSH2 is
required both for successful DSB repair in the G2 phase
of the cell cycle and for the maintenance of the early-G2
arrest, but not to sense IR-induced DNA damage and
initiate the early phases of HR. Abrogation of these two
functions in MSH2/ cells might be responsible for
their increased sensitivity towards high IR doses and
susceptibility to chromosomal damage.
Acknowledgements
We thank Dr P Karran for a critical reading of the manuscript
and discussion. The skilful collaboration of Mr Angelo
Schinoppi is grateful acknowledged. This work was partially
supported by MURST grants and by grants from EC (contract
no. FIGH-CT1999-00011).
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