Báo cáo khoa học: Characterization of the role of a trimeric protein phosphatase complex in recovery from cisplatin-induced versus noncrosslinking DNA damage potx

Characterization of the role of a trimeric protein
phosphatase complex in recovery from cisplatin-induced
versus noncrosslinking DNA damage
Cristina Va
´
zquez-Martin, John Rouse and Patricia T. W. Cohen
Medical Research Council Protein Phosphorylation Unit, College of Life Sciences, University of Dundee, UK
The chemotherapeutic agents cisplatin (cis-diamminedi-
chloroplatinum) and oxaliplatin are currently used for
the treatment of tumours in a variety of tissues, such
as testis, ovary, lung, bowel, head, and neck. These
platinum-containing agents form adducts with DNA
that produce intrastrand and interstrand nucleotide
crosslinks [1], and are thought to be effective because
the DNA damage triggers apoptosis of cancerous cells.
In response to these chemotherapeutic agents, ionizing
radiation and chemical mutagens, activation of the
DNA damage response pathways causes arrest of the
cell cycle to allow time for cells to repair the DNA
before the cell cycle resumes. If the DNA cannot be
repaired or the damage bypassed, apoptosis is initi-
ated. Protein phosphorylation plays a key role in the
DNA damage response, but the protein phosphatases
Keywords
cisplatin; histone 2AX;
methylmethanesulfonate; PPH3;
protein phosphatase 4
Correspondence
P. T. W. Cohen, MRC Protein
Phosphorylation Unit, College of Life
Sciences, Sir James Black Centre,

University of Dundee, Dow Street, Dundee
DD1 5EH, UK
Fax: +44 1382 223778
Tel: +44 1382 384240
E-mail:
(Received 29 April 2008, revised 11 May
2008, accepted 23 June 2008)
doi:10.1111/j.1742-4658.2008.06568.x
Cisplatin (cis-diamminedichloroplatinum) and related chemotherapeutic
DNA-crosslinking agents are widely used to treat human cancers. Saccha-
romyces cerevisiae with separate deletions of the genes encoding the
trimeric protein serine ⁄ threonine phosphatase (Pph)3p–platinum sensitivity
(Psy)4p–Psy2p complex, are more sensitive than the isogenic wild-type
(WT) strain to cisplatin. We show here that cisplatin causes an enhanced
intra-S-phase cell cycle delay in these three deletion mutants. The C-termi-
nal tail of histone 2AX (H2AX) is hyperphosphorylated in the same
mutants, and Pph3p is found to interact with phosphorylated H2AX
(cH2AX). After cisplatin treatment is terminated, pph3D, psy4D and psy2D
mutants are delayed as compared with the WT strain in the dephosphoryla-
tion of Rad53p. In contrast, only pph3D and psy2D cells are more sensitive
than WT cells to methylmethanesulfonate, a noncrosslinking DNA-alkylat-
ing agent that is known to cause a Rad53p-dependent intra-S-phase cell
cycle delay. Dephosphorylation of Rad53p and the recovery of chromosome
replication are delayed in the same mutants, but not in psy4D cells. By com-
parison with their mammalian orthologues, the regulatory subunit Psy4p is
likely to inhibit Pph3p catalytic activity. The presence of a weak but active
Pph3p–Psy2p complex may allow psy4D cells to escape from the Rad53p-
mediated cell cycle arrest. Overall, our data suggest that the trimeric Pph3p–
Psy4p–Psy2p complex may dephosphorylate both cH2AX and Rad53p, the
differences lying in the more stable interaction of the Pph3 phosphatase with

cH2AX as opposed to a transient interaction with Rad53p.
Abbreviations
ATM, ataxia telangiectasia mutated; ATR, ataxia telangiectasia and RAD53 related; 4NQO, 4-nitroquinoline 1-oxide; FACS, fluorescent
activated cell sorter; H2AX, histone 2AX; MMS, methylmethanesulfonate; PFGE, pulsed-field gel electrophoresis; Pph, Saccharomyces
cerevisiae protein serine ⁄ theonine phosphatase; Ppp ⁄ PP, protein serine ⁄ threonine phosphatase in mammals and Drosophila; Ppp4c, protein
phosphatase 4 catalytic subunit (also termed PP4, PPX); Psy, platinum sensitivity; WT, wild-type; cH2AX, phosphorylated histone 2AX.
FEBS Journal 275 (2008) 4211–4221 ª 2008 The Authors Journal compilation ª 2008 FEBS 4211
that participate in these pathways are not well delin-
eated. Several studies have implicated complexes of
protein serine ⁄ threonine phosphatase (Pph)3p in the
DNA damage response pathways in the budding
yeast Saccharomyces cerevisiae. Cells lacking Pph3p
(Ydr075w) and an interacting protein Yln201c [2,3],
the yeast orthologues of mammalian protein phospha-
tase 4 catalytic subunit (Ppp4c) and its regulatory
subunit, R3, respectively [4], were reported to be more
sensitive to the chemical mutagen methylmethanesulfo-
nate (MMS), which alkylates DNA [5]. These mutants
were also found to be more sensitive to cisplatin and
oxaliplatin, so Yln201c was designated platinum
sensitivity 2 (Psy2)p [6]. Surprisingly, Ybl046w was not
identified being as sensitive to DNA-damaging agents
in this screen, although it had been identified in Pph3p
complexes by systematic analyses of the yeast proteome
[2,3], and its putative mammalian homologue R2 had
been identified as a core regulatory subunit in com-
plexes with Ppp4c [7]. However, subsequent investiga-
tions showed that strains deleted for Pph3p, Psy2p and
Ybl046w were all sensitive to cisplatin [4,8], and
Ybl046w was designated Psy4p [9].

One of the earliest events in the cellular response to
many DNA-damaging agents is the phosphorylation of
histone 2AX (H2AX) at Ser129 in its C-terminal tail
and the accumulation of the phosphorylated histone
(cH2AX) at the sites of DNA damage [10,11]. In the
deletion mutants pph3D, psy4D and psy2D, Keogh
et al. [12] found that H2AX was hyperphosphorylated
as compared with the wild-type (WT) strain in both
the absence and the presence of ionizing radiation that
caused a G
2
⁄ M cell cycle arrest, implicating Ph3p–
Psy4p–Psy2p in the dephosphorylation of cH2AX and
efficient recovery from the checkpoint. Substitution of
the H2AX Ser129 by Ala restored the ability of the
pph3D strain to turn off checkpoint signalling in a
timely manner and re-enter the cell cycle after DNA
repair of the double-strand break [12]. Although the
studies of Hanway et al. [5] showed that pph3D and
psy2D cells were more sensitive to MMS than were
WT cells, Keogh et al. [12] reported that the pph3D,
psy4D and psy2D cells were only more sensitive than
WT cells to MMS if the cells also carried deletions of
genes involved in recombination and repair of DNA.
While our studies were in progress, O’Neill et al. [13]
found that, during recovery from MMS-induced DNA
damage, Rad53p dephosphorylation and resumption
of DNA synthesis are delayed in pph3D and psy2D
cells as compared with WT cells but not in psy4D cells.
Consistent with this report, earlier studies had reported

that Rad53p, a key controller of the DNA damage
response pathways leading to intra-S-phase cell cycle
arrest, interacted with Psy2p in a yeast two-hybrid
screen [14]. Here we examine the roles of Pph3p, Psy2p
and Psy4p in response to the DNA-crosslinking agent
cisplatin and the noncrosslinking, alkylating agent
MMS, and show that recovery after cisplatin-induced
DNA damage is delayed in pph3D, psy4D and psy2D
mutant cells, whereas it is only delayed in pph3D and
psy2D cells after MMS-induced DNA damage.
Results
Role of Pph3p and its regulatory subunits Psy4p
and Psy2p in the cell cycle arrest induced by
cisplatin and MMS
In order to determine how the members of Pph3p
complex affect the S. cerevisiae cell cycle on treatment
with cisplatin, we subjected the WT diploid BY4743
cells and the pph3D, psy4D and psy2D mutant cells to
analysis on a fluorescent activated cell sorter (FACS)
machine after incubation with 2 mm cisplatin for vari-
ous times. Figure 1A shows that at 90 and 120 min,
the pph3D, psy4D and psy2D mutants were appreciably
affected by the drug, with an increased fraction of cells
accumulating in S-phase, whereas the WT cells were
largely unaffected. Although the evidence for an intra-
S-phase cell cycle delay is less conclusive in psy4D cells
than in pph3D and psy2D cells, examination of growth
on YPD plates demonstrates that deletion of any com-
ponent of Pph3p–Psy4p–Psy2p decreases cell prolifera-
tion in the presence of cisplatin, and that this trimeric

Pph3p complex confers resistance to cisplatin (Fig. 1B)
[4,8,9].
MMS slows progression through S-phase in WT
S. cerevisiae by methylation of DNA on N
7
-deoxygua-
nine and N
3
-deoxyadenine [15,16]. The differing
reports as to whether pph3D, psy4D and psy2D mutants
were more sensitive than WT cells to MMS [5,12] led
us to compare the sensitivity of the mutants to increas-
ing concentrations of MMS, and our results are in line
with those of Hanway et al. [5]. We demonstrated that
pph3D and psy2D mutants were more sensitive than the
WT strain when inoculated onto YPD plates contain-
ing 0.03% MMS, whereas the psy4D mutant was no
more sensitive than the WT strain to 0.03% MMS on
YPD plates (Fig. 1C). We also treated the cells in
liquid culture (0.03% MMS) and plated them on YPD
plates, obtaining the same results (data not shown). In
addition, we investigated the sensitivity to another
DNA-damaging agent, 4-nitroquinoline 1-oxide
(4NQO), which also affects S-phase. The pph3D and
psy2D mutants were more sensitive than the WT strain
to the UV mimetic 4NQO (20 lgÆmL
)1
on plates),
Cisplatin-induced DNA damage response C. Va
´

zquez-Martin et al.
4212 FEBS Journal 275 (2008) 4211–4221 ª 2008 The Authors Journal compilation ª 2008 FEBS
whereas the psy4D mutant showed a similar sensitivity
to that of the WT strain (data not shown). These
results appear to support a role for Pph3p–Psy2p in
conferring resistance to MMS and 4NQO, and are in
contrast to the effects of cisplatin, which imply a role
for Pph3p–Psy4p–Psy2p in conferring resistance to
cisplatin (Fig. 1B) [8,9].
S. cerevisiae cells often respond to DNA damage by
activating a signal transduction pathway that leads to
phosphorylation of Rad9p by Mec1p. Rad9p phos-
phorylation allows recruitment of Rad53p to Mec1–
Rad9, facilitating Rad53p phosphorylation by Mec1.
Rad53p autophosphorylation then leads to an active
Rad53p that is multiply phosphorylated [17], and the
phosphorylation can be detected by SDS ⁄ PAGE and
immunoblotting as bands that migrate more slowly
than Rad53p. As the protein kinase Rad53p was
reported to interact with Psy2p [18], we investigated
whether cisplatin treatment caused the activation of
Rad53p in WT, pph3D, psy4D and psy2D cells, but we
found that Rad53p was not phosphorylated in
response to 2 mm cisplatin [9], although this concentra-
tion could induce a cell cycle delay (Fig. 1A). Use of
higher concentrations can be a problem due to
cisplatin insolubility, but we have now been able to
demonstrate phosphorylation of Rad53p after treat-
A
WT

DNA content: 2C 4C 2C 4C 2C 4C 2C 4C
pph3 psy4 psy2
min
120
90
60
30
Untreated
Cisplatin
B
1
2
3
4
5
6
WT
MT
pph3 psy4 psy2
pph3 psy4 psy2
Cisplatin
concentration
0m
M
5mM
1
2
3
4
5

6
WT
MT
10-fold yeast
dilution
0%
0.01%
0.03%
1
2
3
4
5
6
WT
MT
1
2
3
4
5
6
WT
MT
MMS (v/v)
concentration
1
2
3
4

5
6
WT
MT
C
Fig. 1. Pph3p complexes confer resistance
to DNA-damaging agents. (A) The diploid
WT strain BY4743 and mutants pph3D,
psy4D and psy2D were grown to mid-expo-
nential phase in liquid culture and incubated
in the presence of cisplatin (2 m
M) or left
untreated. Samples for FACS analysis were
taken at the indicated time points. (B, C)
The sensitivities of the same WT and
mutant (MT) cells to cisplatin and MMS
were examined. Serial dilutions of indepen-
dent colonies (WT 1, 2 and 3) on all plates
are from the control BY4743 (Y20000)
strain. Serial dilutions of independent colo-
nies (MT 4, 5 and 6) are from strains pph3D
(Y34010, left columns), psy4D (Y33072,
middle columns), and psy2D (Y32011, right
columns). The cisplatin and MMS concentra-
tion in each row of plates is indicated on
the right. Plates were incubated at 30 °C for
48 h.
C. Va
´
zquez-Martin et al. Cisplatin-induced DNA damage response

FEBS Journal 275 (2008) 4211–4221 ª 2008 The Authors Journal compilation ª 2008 FEBS 4213
ment of yeast cells with 3 m m cisplatin (Fig. 2A, top
left panel). On examining the recovery after different
times, we could see a slight decrease in the Rad53p
phosphorylation in the WT strain after 4 h, a pro-
nounced decrease at 6 h, and a return to the unphos-
phorylated basal state by 9 h. In contrast, Rad53p was
phosphorylated at 4 h and remained at least partially
phosphorylated in pph3D, psy4D and psy2D mutants
during recovery up to 9 h. The experiment was per-
formed five times.
In response to MMS treatment, DNA damage-
induced phosphorylated forms of Rad53p occurred in
the WT strain and in pph3D, psy4D and psy2D mutant
cells (Fig. 2B, top left panel). On examining the recov-
ery after different times, we saw a pronounced
decrease in Rad53p phosphorylation in the WT and
psy4D strains at 5–7 h. In contrast, phosphorylated
forms of Rad53p remained in the pph3D and psy2D
strains during recovery between 5 h and 7 h. This
experiment was performed four times.
Our data suggest that a complex of Pph3p and
Psy2p may dephosphorylate Rad53p after it has been
activated and phosphorylated in response to MMS,
but that cisplatin-induced activation and phosphoryla-
tion of Rad53p may require a complex of Pph3, Psy4p
and Psy2p for dephosphorylation to occur as rapidly
as in WT cells. Considering that Pph3p dephosphory-
lates Rad53p, we investigated the interaction between
Rad53p and Pph3p–Psy4p–Psy2p by coimmunoprecipi-

tation. In untreated AY925 cells, or cells treated with
0.03% MMS to induce Rad53p phosphorylation,
Rad53p was not coimmunoadsorbed with HA
3
–Pph3p
and Psy4p–MYC
13
(Table 1, and data not shown),
indicating that this interaction may be transient or too
weak to withstand the isolation protocol.
Pph3p–Psy4p–Psy2p dephosphorylates cH2AX
The form of H2AX phosphorylated at Ser129 (termed
cH2AX), which is induced in response to DNA-dam-
aging agents, is normally removed before resumption
of the cell cycle, and Keogh et al. [12] have presented
evidence that Pph3p–Psy4p–Psy2p is involved in this
process. In untreated pph3D, psy4D and psy2D cells, we
found that the phosphorylation of H2AX was mark-
edly elevated as compared with the barely detectable
levels of cH2AX in WT cells (Fig. 3A,B). Treatment
with cisplatin or MMS elevated cH2AX in WT cells
and led to a further increase in the pph3D, psy4D and
psy2D cells (Fig. 3A, cisplatin treatment; Fig. 3B,
MMS 2 h recovery). After removal of cisplatin and
MMS, the c H2AX levels returned to the near-zero
basal levels in the WT cells after several hours but
remain elevated in the pph3D, psy4D and psy2D cells
(Fig. 3A, 8 h; Fig. 3B, 12 h). These results indicate
that the three subunits of Pph3p–Psy4p–Psy2p acting
Recovery 5 h Recovery 6 h

Recovery 7 h Recovery 10 h
Rad53
Rad53P
Rad53
Rad53P
UN MMS
Recovery 2 h Recovery 4 h
Rad53
Rad53P
Rad53
Rad53P
Rad53
Rad53P
WT
pph3
psy4
psy2
pph3
psy4
psy2
WT
WT
pph3
psy4
psy2
pph3
psy4
psy2
WT
WT

pph3
psy4
psy2
pph3
psy4
psy2
WT
WT
pph3
psy4
psy2
pph3
psy4
psy2
WT
WT
pph3
psy4
psy2
pph3
psy4
psy2
WT
WT
pph3
psy4
psy2
pph3
psy4
psy2

WT
WT
pph3
psy4
psy2
pph3
psy4
psy2
WT
Rad53
Rad53P
Recovery 2 h
UN Cisplatin
Recovery 4 h
Rad53
Rad53P
Recovery 6 h Recovery 9 h
A
B
Fig. 2. Relationship between Rad53p and
the Pph3p complex. The diploid WT strain
BY4743 and mutants pph3D, psy4D and
psy2D were grown to mid-exponential
phase in liquid culture and left untreated
(UN) or incubated in the presence of 3 m
M
cisplatin (A) or 0.03% MMS (B) for 90 min.
Cells were filtered, washed free of cisplatin,
and incubated at 30 °C in YPD, and samples
were taken at the indicated times during

recovery. Trichloroacetic acid extracts were
prepared and subjected to immunoblot anal-
ysis with antibodies against Rad53p, which
detect the unphosphorylated Rad53p
(92 kDa) and more slowly migrating phos-
phorylated forms.
Cisplatin-induced DNA damage response C. Va
´
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4214 FEBS Journal 275 (2008) 4211–4221 ª 2008 The Authors Journal compilation ª 2008 FEBS
together normally dephosphorylate cH2AX and are
essential for its complete dephosphorylation.
Taking into account a role for Pph3p–Psy4p–Psy2p
in dephosphorylation of cH2AX, we investigated
whether there was any interaction between the phos-
phatase and the histone. Employing lysates from the
S. cerevisiae cells AY925 PSY4–MYC
13
and
AY925HA
3
–PPH3, cH2AX was found in the anti-HA
immunopellets (Fig. 3C bottom panel) and in the anti-
MYC immunopellets (data not shown), but not in the
immunopellets from the control cells that did not
express the tagged proteins (Fig. 3C, top panel).
HA
3
–Pph3p interacted with increasing amounts of
cH2AX generated in the presence of increasing doses

of MMS (Fig. 3C) and even with the low levels of
cH2AX that were sometimes present in yeast cell ly-
sates in the absence of DNA-damaging agents (data
not shown). Most of the cH2AX is bound to the phos-
phatase even after treatment with 0.5% MMS
(Fig. 3C, bottom panel). Given that endogenous
Pph3p–Psy4p–Psy2p is also present in the cells, it
appears likely that all of the cH2AX is bound to the
phosphatase. No increases in the levels of the catalytic
subunit HA
3
–Pph3p or the regulatory subunit Psy4p–
MYC
13
(data not shown) were observed after DNA
damage induced by MMS.
UN MMS
Recovery 2 h Recovery 6 h Recovery 12 h
H2A/H2B
H2AX
H2A/H2B
H2AX
Recovery 2 h Recovery 4 h
H2A/H2B
H2AX
WT
pph3
psy4
psy2
pph3

psy4
psy2
WT
WT
pph3
psy4
psy2
pph3
psy4
psy2
WT
WT
pph3
psy4
psy2
pph3
psy4
psy2
WT
WT
pph3
psy4
psy2
pph3
psy4
psy2
WT
WT
pph3
psy4

psy2
pph3
psy4
psy2
WT
pph3
psy4
psy2
WT
UN CDDP
H2A/H2B
H2AX
Recovery 6 h Recovery 8 h
H2A/H2B
H2AX
A
B
C
Fig. 3. The Pph3p complex is required for
dephosphorylation of cH2AX. The diploid
WT strain BY4743 and mutants pph3D,
psy4D and psy2D were grown to mid-expo-
nential phase in liquid culture and left
untreated (UN) or incubated in the presence
of 2 m
M cisplatin (CDDP) (A) or 0.03%
MMS (B) for 90 min. Cells were filtered,
washed free of cisplatin, and incubated at
30 °C in YPD, and samples were taken at
the indicated times during recovery. Trichlo-

roacetic acid extracts were prepared and
subjected to immunoblot analysis with anti-
bodies against cH2AX (H2AXphospho-
Ser129). Lower panels are blots probed for
total H2A ⁄ H2B as a control. (C) The Pph3p
complex associates with cH2AX. Lysates
from control WT AY925 cells and AY925
HA
3
–Pph3p cells untreated or treated with
MMS at the indicated concentrations were
immunoadsorbed with antibodies to HA and
probed for cH2AX. Lysate (L), 50 lg; the
supernatant (S, same volume as lysate)
and pellet (P, from 2 mg of lysate) were
obtained by centrifugation following the
immunoadsorption.
C. Va
´
zquez-Martin et al. Cisplatin-induced DNA damage response
FEBS Journal 275 (2008) 4211–4221 ª 2008 The Authors Journal compilation ª 2008 FEBS 4215
Pph21p and Pph22p are protein phosphatases that
are highly related to Pph3p, and in some cases show
overlapping function with Pph3 [19]. S. cerevisiae
cells with deletions of the genes encoding both
Pph21p and Pph22p have extremely low viability. As
the most abundant isoform is Pph21p, we examined
pph21D cells in order to determine whether Pph21p
may be partly responsible for the dephosphorylation
of cH2AX in yeast. We treated WT and pph21D

cells with 2 mm cisplatin or 0.03% MMS, and also
examined the recovery after washing the cultures
extensively. The levels of c H2AX phosphorylation
revealed no differences in the dephosphorylation of
cH2AX in pph21D cells as compared with the WT
cells, either before or after treatment or during
recovery (Fig. 4).
Role of Pph3p and its regulatory subunits Psy4p
and Psy2p in recovery of chromosome replication
following MMS-induced DNA damage
cH2AX is believed to play a central role in the
recruitment and ⁄ or retention of DNA repair factors
at the sites of DNA damage [20]. In order to investi-
gate whether a Pph3p complex is required for the
recovery of chromosome replication following removal
of DNA-damaging agents, we employed pulsed-field
gel electrophoresis (PFGE). Cells were arrested in G
1
with a-factor, released into S-phase, and then treated
with MMS for 90 min. The drug was washed away
and cells were allowed to recover. At various times,
chromosomes were prepared from WT, pph3D, psy4D
and psy2D cells, and separated by PFGE. These gels
resolved a characteristic ladder of bands correspond-
ing to the 16 S. cerevisiae chromosomes, visualized
after ethidium bromide staining (Fig. 5). Treatment of
cells with MMS resulted in loss of chromosome bands
(Fig. 5, WT, pph3D, psy4D and psy2D, 0 h) due to the
presence of forks and replication bubbles that prevent
entry of the chromosomes into the gel [21,22]. Treat-

ment with MMS also sometimes resulted in the
appearance of a ‘smear’ of low molecular mass DNA
species that may represent some chromosome degra-
dation (Fig. 5, psy4D, 0 h). When WT cells were
washed free of the DNA-damaging agents and
allowed to recover, the intact chromosomes started to
reappear after 2 h of recovery and were clearly visible
at 4 h (Fig. 5, WT), indicating that the S-phase arrest
had been overcome and chromosome replication had
UN
CDDP
MMS
UN
CDDP
MMS
WT
pph21
Treatment for 90 min
H2A/H2B
H2AX
UN
CDDP
MMS
UN
CDDP
MMS
WT
pph21
Recovery 2 h
H2A/H2B

H2AX
H2A/H2B
H2AX
UN
CDDP
MMS
UN
CDDP
MMS
WT
pph21
Recovery 6 h
Fig. 4. Pph21p (PP2Ac ortholog) is not
involved in cH2AX dephosphorylation in
yeast. The WT strain BY4743 and the
pph21D mutant were grown to mid-expo-
nential phase in liquid culture and incubated
in the presence of cisplatin (CDDP) (2 m
M)
or MMS (0.03%), or left untreated (UN), for
90 min. Cells were filtered, washed free of
the drug, and incubated at 30 °C in YPD,
and samples were taken at the indicated
times during recovery. Trichloroacetic acid
extracts were prepared and subjected to
immunoblot analysis with antibodies against
cH2AX. Lower panels: blots were stripped
and immunostained for total H2A ⁄ H2B as a
control.
Fig. 5. The Pph3p complex is required for recovery of chromosome

replication. (A) The WT haploid strain BY4741 and mutants pph3D,
psy4D and psy2D were grown to mid-exponential phase, arrested
in G
1
with a-factor, released into S-phase, and treated with 0.05%
MMS for 90 min. Cells were filtered, washed extensively, and incu-
bated in YPD at 30 °C for 6 h. Samples for PFGE were taken after
a-factor treatment (20 lgÆmL
)1
) (control), after MMS treatment and
following removal of MMS after recovery for 2, 4 and 6 h.
Cisplatin-induced DNA damage response C. Va
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4216 FEBS Journal 275 (2008) 4211–4221 ª 2008 The Authors Journal compilation ª 2008 FEBS
resumed and gone to completion. When pph3D and
psy2D cells were allowed to recover from exposure to
MMS, intact chromosomes had barely reappeared
after 6 h of recovery, indicating that these mutants
were slower in the recovery of chromosome replica-
tion (Fig. 5: pph3D,6h;psy2D, 6 h). In contrast,
recovery in psy4D cells was similar to that in WT
cells (Fig. 5, psy4D , MMS 4 h). Thus, a correlation
was observed between delayed completion of chromo-
some replication and prolonged MMS-induced
Rad53p phosphorylation in pph3D and psy2D cells.
Similar results were obtained in three independent
experiments.
Further analysis of chromosome replication follow-
ing cisplatin-induced damage suggested that pph3D,

psy4D and psy2D cells were all slightly delayed as
compared with WT cells, but the small difference [at
concentrations of cisplatin (3 mm) near its maximum
solubility] was difficult to confirm (data not shown).
Discussion
In S. cerevisiae, Pph3p has been shown to interact
with regulatory subunits Psy4p and Psy2p, and these
three proteins and their interactions have been found
to be conserved through evolution to Drosophila and
mammals [4]. We have previously demonstrated that
deletion of any component of yeast Pph3p–Psy4p–
Psy2p causes hypersensitivity to the antitumour drug
cisplatin, indicating that all three proteins may oper-
ate as a functional unit in vivo, playing a role in the
cisplatin response [9]. Cisplatin interferes with DNA
function by causing intrastrand and interstrand cross-
linking of nucleotide bases and, in replicating cells,
DNA damage usually induces an intra-S-phase cell
cycle arrest. Accordingly, we show in this article that
treatment of pph3D, psy4D and psy2 D mutant cells
with cisplatin causes enhanced accumulation of cells
in S-phase as compared with WT cells, although the
effect is less conclusive in the case of the psy4D cells.
Nevertheless, the data suggest that a correlation is
observed between the slow growth of pph3D, psy4D
and psy2D cells in the presence of cisplatin [9] and
accumulation of cells in S-phase. Delayed S-phase
progression is usually associated with activation of
the intra-S-phase checkpoint mediated by Rad53
phosphorylation, and indeed, treatment of cells with

3mm cisplatin for 90 min resulted in phosphorylation
of Rad53p (Fig. 2A), although slightly lower concen-
trations did not activate Rad53p [9]. Notably, recov-
ery from cisplatin-induced Rad53p phosphorylation
was delayed in all three mutants: pph3D, psy4D and
psy2D.
At sites of DNA damage, the Ser129-phosphorylated
H2AX derivative, cH2AX, forms foci for the recruit-
ment of factors involved in repair of DNA damage
and maintenance of the cell cycle arrest. H2AX was
found to be hyperphosphorylated in pph3D, psy4D and
psy2D strains in both the absence and the presence of
ionizing radiation [12], and in the present study, in the
absence and the presence of cisplatin. In addition, we
showed that Pph3p directly or indirectly binds to
cH2AX, indicating that Pph3p–Psy4p–Psy2p forms a
stable complex with H2AX when Ser129 is phosphory-
lated and is therefore likely to be the phosphatase
complex dephosphorylating the histone C-terminal tail.
Keogh et al. [12] have provided evidence that cH2AX
is removed from the site of DNA damage before it is
dephosphorylated. If this is the case, removal from the
action of the ataxia telangiectasia mutated (ATM) ⁄
ataxia telangiectasia and RAD53 related (ATR)
kinases at the site of DNA damage may decrease the
kinase ⁄ phosphatase ratio and allow the phosphatase to
dephosphorylate cH2AX.
In mammalian cells, PP2A isoforms, the orthologues
of S. cerevisiae Pph21p and Pph22p, have been
reported to dephosphorylate cH2AX [23], and in some

cases, e.g. in the mammalian target of rapamycin path-
way, Pph21p and Pph22p have overlapping functions
with Pph3p [24]. It was therefore important to examine
whether Pph21 ⁄ 22p might play a role in the dephos-
phorylation of cH2AX. Cells with deletion of the most
abundant isoform, Pph21p, exhibit depletion of phos-
phatase activity to 35% of the total attributable to
Pph21 and Pph22 [25]. In addition, these pph21D cells
showed a significantly lower budding index and slightly
slower growth than WT cells on nonfermentable
carbon sources. However, the pph21
D cells showed no
hyperphosphorylation of cH2AX and no delay in
recovery from cisplatin and MMS as compared with
WT cells, supporting the idea that in S. cerevisiae,
cH2AX is dephosphorylated by Pph3p and not by
Pph21p and its closely related isoform Pph22p.
In contrast to the effects of cisplatin, which cross-
links DNA strands, our studies in which yeast cells
were treated with MMS, which mainly alkylates DNA
but can cause double-strand breaks [26], showed
delayed dephosphorylation of Rad53p and delayed
recovery of chromosome replication only in pph3D and
psy2D cells as compared with WT cells, but not in the
psy4D mutant. The results are in line with the competi-
tive growth assays that identified the pph3D and psy2D
cells as MMS hypersensitive [5]. Recently, O’Neill
et al. [13] found delayed recovery from MMS-induced
Rad53 phosphorylation and decreased progression of
replication forks along the DNA in pph3D and psy2D

C. Va
´
zquez-Martin et al. Cisplatin-induced DNA damage response
FEBS Journal 275 (2008) 4211–4221 ª 2008 The Authors Journal compilation ª 2008 FEBS 4217
cells. These authors conclude that a dimeric complex,
Pph3p–Psy2p, is responsible for the dephosphorylation
of Rad53p, and they further suggest that Rad53p
dephosphorylation at the replication fork is necessary
to allow the resumption of DNA synthesis. Rad53p
has been shown to interact with the lagging strand rep-
lication apparatus regulating the phosphorylation of
DNA polymerase a-primase complex [27], and Psy2p
was found to interact with proteins at stalled replica-
tion forks in a yeast two-hybrid screen [28].
Our studies indicate that different Pph3 phosphatase
complexes or more likely different phosphatases are
responsible for the dephosphorylation of Rad53p after
cisplatin and MMS-induced DNA damage. Phosphory-
lation of Rad53p is detected by a mobility shift after
gel electrophoresis, and multiple phosphorylation sites
in Rad53p are suggested by the several bands that can
sometimes be separated [13,29]. Different phosphoryla-
tions of Rad53p may be triggered by the two DNA-
damaging agents, and the different phosphorylation
sites may then be dephosphorylated by different phos-
phatases. The Mg
2+
-dependent phosphatases, Ptc2p
and Ptc3p, have been implicated in the dephosphoryla-
tion of Rad53p after a G

2
⁄ M arrest in response to
irreparable double-strand breaks in the DNA [29].
Keogh et al. [12] have suggested that dephosphoryla-
tion of Rad53p during recovery from a repairable
double-strand break depends on the prior dephos-
phorylation of cH2AX by Pph3p–Psy4p–Psy2p [12].
Our results on recovery from cisplatin-induced DNA
damage are consistent with Rad53p phosphorylation
at a particular site being maintained by cH2AX, and
when the latter is dephosphorylated, Rad53p may be
dephosphorylated by Ptc2p and Ptc3p (Fig. 6).
The question of whether Pph3p–Psy2p or Pph3p–
Psy4p–Psy2p is involved in the dephosphorylation of
Rad53p after DNA damage by MMS is interesting.
An alternative explanation is presented by considering
the mammalian complex Ppp4c–R2–R3, which is
orthologous to Pph3p–Psy4p–Psy2p. The isolation of
endogenous Ppp4c–R2 revealed that R2 inhibited
Ppp4c, and suggested that R2 may be a core regula-
tory subunit that facilitates binding of further regula-
tory subunits to Ppp4c [7]. The interaction of R3 with
Ppp4c was then shown to require prior preassembly of
Ppp4c and R2 [8]. These observations suggest that if
the complexes are conserved through evolution, Psy4p
may be present in the complex that dephosphorylates
Rad53p. In addition, by comparison with R2, the yeast
orthologue Psy4p may be an inhibitory regulatory sub-
unit for Pph3p, so that in psy4D cells, the active
Pph3p, weakly associated with Psy2p, may dephos-

phorylate Rad53p. In psy4D cells, phosphorylated
Rad53p would therefore not be present at replication
forks to stall DNA synthesis. Our MMS sensitivity
studies (Fig. 1B) could also be explained by this mech-
anism. If we consider Psy4p as an inhibitory regulatory
subunit of Pph3p, psy4D cells could escape from the
Rad53p checkpoint, and grow similarly to WT cells;
pph3D and psy2D cells would show slow growth
because of the activation of the checkpoint.
The stable interaction of Pph3p and Psy2p with
cH2AX may require the presence of Psy4p. The inter-
action of Rad53p with a Pph3p complex, which we
could not detect by coimmunoadsorption, would
appear to be transient. The different strengths of the
interactions of the phosphatase complex with its sub-
strates may underlie the nonessential nature of Psy4p
for dephosphorylation of Rad53p by Pph3p in psy4D
cells, although we cannot completely exclude the exis-
tence of functional dimeric complexes in WT cells.
Overall, our studies are consistent with a role for
Pph3p–Psy4p–Psy2p in the dephosphorylation of both
cH2AX and Rad53p. The complex may have an addi-
tional function at stalled replication forks, but the data
do not necessitate such a role, as dephosphorylation of
both cH2AX and Rad53p is likely to be a prerequisite
for chromosome replication to resume. A working
model for the roles of Pph3p–Psy4p–Psy2p in the
recovery from DNA damage induced by the crosslink-
ing anticancer drug cisplatin and the noncrosslinking
agent MMS is presented in Fig. 6. Our data suggest

that the sites phosphorylated on Rad53p and dephos-
phorylated by different phosphatases may be depen-
dent on the type of the DNA damage. However,
different levels of DNA damage cannot be entirely
excluded, because cisplatin does not readily enter the
yeast cell, and therefore it is possible that the overall
amount of MMS-induced DNA damage may be higher
than that caused by cisplatin.
Experimental procedures
Yeast strains and general methods
All methods for the manipulation of yeast and preparation
of media were performed according to standard protocols
[30]. The growth conditions for the yeast strains and drug
sensitivity studies were as described previously [9]. The
strains used in this study are listed in Table 1. The S. cere-
visiae strain AY925, in which Psy4p bears a C-terminal
MYC
13
epitope tag and Pph3p an N-terminal HA
3
tag,
was constructed by a PCR-based method as described in
[9]. The haploid and diploid strains with deletions of genes
PPH3, PSY4 and PSY2 encoding ORFs YDR075w,
YBL046w and YNL201c, respectively, were from Euroscarf
Cisplatin-induced DNA damage response C. Va
´
zquez-Martin et al.
4218 FEBS Journal 275 (2008) 4211–4221 ª 2008 The Authors Journal compilation ª 2008 FEBS
(European Saccharomyces cerevisiae Archive for Functional

Analysis), Institute for Microbiology, Johann Wolfgang
Goethe-University Frankfurt, Germany.
Yeast extracts, immunoblotting, and
immunoprecipitation
Extracts for immunoblot analysis were prepared either as
described previously [9] or by a slightly modified trichlo-
roacetic acid-lysis method [31]. Briefly, in the trichloro-
acetic acid method, the cells were washed with 20%
trichloroacetic acid (v ⁄ v) and were then disrupted with
0.5 mL of 20% trichloroacetic acid (v ⁄ v) ⁄ 0.7 · 10
7
cells
by vortexing for 1 min in the presence of glass beads in
a mini-bead beater. The lysates, separated from the
beads, were centrifuged for 5 min at 13 000 g. The pellets
were resuspended in 200 lL of sample buffer adjusted to
0.3 m Tris ⁄ HCl with 1 m Tris-HCl pH 8.8, boiled for
10 min, and clarified by centrifugation for 5 min at
13 000 g. Proteins in the extracts were subjected to
SDS ⁄ PAGE and transferred to nitrocellulose membranes.
In coimmunoprecipitation experiments, aliquots of lysates
(2 mg of protein) prepared in the absence of trichloroacetic
acid from cells expressing Psy4p–MYC
13
and HA
3
–Pph3p
were incubated with either anti-c-MYC or anti-HA agarose
beads (Sigma-Aldrich, Poole, UK) on a shaking platform at
4 °C for 2 h. After centrifugation for 5 min at 13 000 g, the

beads were washed two times in lysis buffer containing
0.15 m NaCl and twice in 50 mm Tris ⁄ HCl (pH 7.5) and
0.1 mm EGTA. The beads were boiled for 5 min in SDS
sample buffer, and released proteins were subjected to
SDS ⁄ PAGE (4–12% polyacrylamide) and immunoblotting
with either of the monoclonal antibodies anti-MYC (Roche
Diagnostics, Indianapolis, IN, USA) or anti-HA (produced
in the Division of Signal Transduction Therapy, University
of Dundee). Rad53p immunoblots were performed using a
mixture of two antibodies (yN-19 and yC-19, Santa Cruz
Biotechnology Inc., Santa Cruz, CA, USA). Antibodies to
histones H2A ⁄ H2B and phosphoSer129-H2AX were from
Abcam (Cambridge, UK).
FACS analysis
Cells (1 · 10
7
) were resuspended in 70% (v ⁄ v) ethanol and
left for at least 2 h at 4 °C. The cells were then washed in
50 mm Tris ⁄ HCl (pH 7.8), treated with 0.2 mgÆmL
)1
RNaseA (Sigma-Aldrich) at 37 °C overnight, and washed
with 200 mm Tris ⁄ HCl (pH 7.5), 211 mm NaCl, and 78 mm
MgCl
2
; propidium iodide was then added to give a concen-
tration of 50 lgÆmL
)1
in the same buffer at least 1 h prior
Pph3
Psy2

Psy4
H2AX
DNA damage
Rad-53
Ptc2/Ptc3
Cisplatin
H2AX-
P
(
H2AX)
ATM/ATR
kinases recruited
Rad53-
Cell cycle
dela
y
Recovery
P1
Pph3
Psy2
Psy4
P2
MMS
H2AX-
P
(
H2AX)
DNA damage
ATM/ATR
kinases recruited

Rad53-
Cell cycle
dela
y
Pph3
Psy2
Psy4
H2AX
Rad-53
Recovery
Fig. 6. Schematic for the roles of Pph3p–Psy4p–Psy2p in recovery from cisplatin- and MMS-induced DNA damage. Pph3p–Psy4p–Psy2p
forms a stable complex with cH2AX via Psy4p. During recovery from cisplatin- or MMS-induced DNA damage, cH2AX may be removed from
the site of action of the ATM ⁄ ATR kinases, allowing Pph3p–Psy4p–Psy2p to dephosphorylate cH2AX. In the case of the cisplatin-induced
DNA damage response, the site(s) phosphorylated (P1) on Rad53p are dephosphorylated by the phosphatases Ptc2 and Ptc3. In the case of
the MMS-induced DNA damage response, the site(s) phosphorylated (P2) on Rad53p are distinct and are dephosphorylated by a transient
interaction with Pph3p–Psy4p–Psy2p. Psy4p is not absolutely essential for this dephosphorylation, allowing a weakly interacting Pph3p–
Psy2p complex to dephosphorylate the P2 site(s) in the psy4D cells.
C. Va
´
zquez-Martin et al. Cisplatin-induced DNA damage response
FEBS Journal 275 (2008) 4211–4221 ª 2008 The Authors Journal compilation ª 2008 FEBS 4219
to FACS analysis in a Becton Dickinson FACSort machine,
managed by R. Clarke (University of Dundee, UK).
Analyses of chromosomes by PFGE
Cells were grown to early log phase (A
600 nm
of 0.5) in YPD
at 30 °C and arrested in G
1
by addition of a-factor

(20 lgÆmL
)1
). When budded cells accounted for < 5% of
the population (confirmed by FACS analysis), the cells were
released from the G
1
arrest by filtration and extensive wash-
ing, and this was followed by incubation in prewarmed
YPD for 30 min to allow entry into S-phase before addition
of MMS (0.05%) or cisplatin (3 mm). After 90 min in MMS
or cisplatin, cells were filtered, washed extensively with YPD
containing 5% (w ⁄ v) sodium thiosulfate, and incubated in
YPD at 30 °C. At the times indicated, 1 · 10
8
cells were
removed and fixed in 70% ethanol at 4 °C overnight before
preparation of chromosomes, exactly as described in the
CHEF DRII instruction manual (BioRad, Hemel Hemp-
stead, UK). PFGE was carried out using the BioRad
CHEF DRII apparatus at 14 °C in a 1% agarose (pulsed
field-certified BioRad) gel in 89 mm Tris, 89 mm boric acid
and 2 mm EDTA (pH 8) for 24 h at 6 VÆcm
)1
using a 120°
included angle with a 6.8–158 s switch time ramp. Gels were
stained with 1 lgÆmL
)1
ethidium bromide for 30 min and
washed for 2 h in water before the DNA was visualized.
Acknowledgements

We thank the Medical Research Council, UK for
financial support.
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Y02011 BY4741 psy2D::kanMX4 Euroscarf
BY4743 (Y20000,
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MATa ⁄ MATa; his3D1 ⁄ his3D1;
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met15D0 ⁄ MET15;
LYS2 ⁄ lys2D0;
ura3D0 ⁄ ura3D0
Euroscarf
Y34010 BY4743pph3D::kanMX4 ⁄
pph3D::kanMX4
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psy4D::kanMX4
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psy2D::kanMX4
Euroscarf
Y33831 BY4743pph21D::kanMX4 ⁄
pph21D::kanMX4
Euroscarf
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