A truncated DNA-damage-signaling response is activated after DSB formation in the G1 phase of Saccharomyces cerevisiae

Abstract

In Saccharomyces cerevisiae, the DNA damage response (DDR) is activated by the spatio-temporal colocalization of Mec1-Ddc2 kinase and the 9-1-1 clamp.
In the absence of direct means to monitor Mec1 kinase activation in vivo, activation of the checkpoint kinase Rad53 has been taken as a proxy for DDR activation. Here, we identify serine 378 of
the Rad55 recombination protein as a direct target site of Mec1. Rad55-S378 phosphorylation leads to an electrophoretic mobility
shift of the protein and acts as a sentinel for Mec1 activation in vivo. A single double-stranded break (DSB) in G1-arrested cells causes phosphorylation of Rad55-S378, indicating activation of
Mec1 kinase. However, Rad53 kinase is not detectably activated under these conditions. This response required Mec1-Ddc2 and
loading of the 9-1-1 clamp by Rad24-RFC, but not Rad9 or Mrc1. In addition to Rad55–S378, two additional direct Mec1 kinase
targets are phosphorylated, the middle subunit of the ssDNA-binding protein RPA, RPA2 and histone H2A (H2AX). These data suggest
the existence of a truncated signaling pathway in response to a single DSB in G1-arrested cells that activates Mec1 without
eliciting a full DDR involving the entire signaling pathway including the effector kinases.

Full text

A truncated DNA-damage-signaling response is
activated after DSB formation in the G1 phase
of Saccharomyces cerevisiae
Ryan Janke
1
, Kristina Herzberg
1
, Michael Rolfsmeier
1
, Jordan Mar
1
,
Vladimir I. Bashkirov
1
, Edwin Haghnazari
1
, Greg Cantin
2
, John R. Yates III
2
and
Wolf-Dietrich Heyer
1,3,
*
1
Department of Microbiology, University of California, Davis, CA 95616-8665,
2
Department of Cell Biology,
SR-11, Scripps Research Institute, La Jolla, CA 92307 and
3
Department of Molecular and Cellular Biology,
University of California, Davis, CA 95616-8665, USA
Received October 7, 2009; Revised December 17, 2009; Accepted December 19, 2009
ABSTRACT
In Saccharomyces cerevisiae, the DNA damage
response (DDR) is activated by the spatio-temporal
colocalization of Mec1-Ddc2 kinase and the 9-1-1
clamp. In the absence of direct means to monitor
Mec1 kinase activation in vivo, activation of the
checkpoint kinase Rad53 has been taken as a
proxy for DDR activation. Here, we identify serine
378 of the Rad55 recombination protein as a direct
target site of Mec1. Rad55-S378 phosphorylation
leads to an electrophoretic mobility shift of the
protein and acts as a sentinel for Mec1 activation
in vivo. A single double-stranded break (DSB) in
G1-arrested cells causes phosphorylation of
Rad55-S378, indicating activation of Mec1 kinase.
However, Rad53 kinase is not detectably activated
under these conditions. This response required
Mec1-Ddc2 and loading of the 9-1-1 clamp by
Rad24-RFC, but not Rad9 or Mrc1. In addition to
Rad55–S378, two additional direct Mec1 kinase
targets are phosphorylated, the middle subunit of
the ssDNA-binding protein RPA, RPA2 and histone
H2A (H2AX). These data suggest the existence of a
truncated signaling pathway in response to a single
DSB in G1-arrested cells that activates Mec1
without eliciting a full DDR involving the entire sig-
naling pathway including the effector kinases.
INTRODUCTION
The DNA damage response (DDR) is a complex signal
transduction network that functions to regulate the
cellular response to genotoxic stress (1). In
Saccharomyces cerevisiae, the phosphoinositol-3-kinase-
like (PIK) kinase Mec1 occupies a central role in the
DDR. The other PIK-kinase, Tel1, plays a minor role in
the DDR in wild-type cells (2). Unlike ATM in mamma-
lian cells, where phosphorylation of residue serine 1981 is
indicative of ATM activation (3), there is no direct assay
to monitor activation of ATR or Mec1 kinase in vivo.
Mec1 is targeted to ssDNA covered by the
ssDNA-binding protein RPA through its DNA-binding
subunit Ddc2, an ortholog of ATRIP in mammalian
cells (4). ssDNA can accumulate as a result of replication
fork stalling or during processing of DNA damage, such
as the resection of a double-strand DNA break (DSB).
DDR activation requires the colocalization of
Mec1-Ddc2 and the 9-1-1 complex, a PCNA-like clamp
composed of the Rad17, Mec3 and Ddc1 proteins in S.
cerevisiae (5,6). Biochemical reconstitution experiments
showed that efficient Mec1 kinase activity requires the
proper loading of the 9-1-1 complex by its clamp loader
Rad24-RFC onto partial duplex DNA (7). Both sensor
complexes, Mec1–Ddc2 and 9-1-1, recognize the DNA
damage independently of each other and their
colocalization greatly enhances Mec1 activation (6). The
DNA damage signal is relayed through the damage-
specific mediator Rad9 or the replication stress-specific
*To whom correspondence should be addressed. Tel: +1 530 752 3001; Fax: +1 530 752 3011; Email: wdheyer@ucdavis.edu
The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors.
Present addresses:
Kristina Herzberg, Hoffmann & Eitle, Munich, Germany.
Michael Rolfsmeier, Washington State University, Pullman, WA 99163, USA.
Jordan Mar, University of California, Berkeley, CA 94720, USA.
Vladimir I. Bashkirov, Applied Biosystems, Foster City, CA 94404, USA.
Edwin Haghnazari, DiscoveRx Corp. Fremont, CA 94538, USA.
2302–2313 Nucleic Acids Research, 2010, Vol. 38, No. 7 Published online 8 January 2010
doi:10.1093/nar/gkp1222
ßThe Author(s) 2010. Published by Oxford University Press.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/
by-nc/2.5), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

mediator Mrc1 to the effector kinases, Chk1, Dun1 and
Rad53 (1).
Activation of the Rad53 effector kinase is mediated by
the Rad9 adaptor protein recruiting Rad53 as a substrate
for Mec1 kinase (8). Subsequent extensive
autophosphorylation of Rad53 is indicative of activation
and can be monitored through an electrophoretic mobility
shift or an auto-kinase assay (9). Rad53 activation has
been taken as a general proxy for DDR activation in
S. cerevisiae (9–16). Genotoxic stress during different
phases of the cell cycle poses distinct challenges. For
example in G1, the absence of a sister chromatid
impedes the use of recombinational repair, although in
diploids the homolog can serve as a template (17). In
S-phase, DNA repair requires coordination with DNA
replication, and the DDR involves suppression of late
firing origins (1). Likewise in G2, mitosis and the onset
of anaphase need to be coordinated with DNA repair. At
the molecular level, this complexity is reflected in differ-
ences in the activation of Rad53 kinase in response to
various forms of DNA damage in the G1 versus other
phases of the cell cycle. In G1-arrested cells, Rad53 is
not activated in response to oxidative DNA damage or a
single DSB induced by HO-endonuclease (15,16,18).
Rad53 activation in G1 requires much higher concentra-
tions of alkylation damage than in S or G2 (19). For UV,
Rad53 activation requires damage processing by the
nucleotide excision repair pathway specifically in G1, but
not in S-phase (20). The damage is either repaired silently
or the damage remains unrepaired until entry into S when
the damage is processed. Hence, activation of the DDR in
G1-arrested budding yeast cells appears to be governed by
different parameters than in other phases of the cell cycle.
However, Rad53 activation does not measure the activa-
tion of the DDR at the sensor level but represents a stage
in the signaling cascade, where already significant signal
transduction and signal amplification has taken place (1).
In mammalian cells, localized activation of the sensor
kinase (ATM) is amplified to a pan-nuclear response in
the activation of the effector kinases CHK1 and CHK2
(the mammalian homolog of yeast Rad53) during the
DDR (21). The sensor kinases, ATM, ATR and their
yeast paralogs have many phosphorylation substrates
besides signaling components (22,23). For lack of tools
to monitor kinase activity in vivo, it is unclear whether
the sensor kinases (primarily Mec1 in budding yeast) are
activated under genotoxic stress conditions that fail to
activate Rad53 kinase.
Homologous recombination is a major pathway in the
repair of DSBs, gaps, and interstrand crosslinks, as well as
in the restart of stalled or broken replication forks (24).
Rad51 protein catalyzes the key reactions of homology
search and DNA strand invasion. Rad55–Rad57 are
two Rad51 paralogs in budding yeast with a specialized
role in either formation or stabilization of the Rad51
filament (25,26). Formation of an active Rad51 filament
on ssDNA dedicates the substrate to recombinational
repair, making Rad55–Rad57 an ideal regulatory target
to modulate recombination. Indeed, Rad55 is a terminal
target of the DDR after DNA damage or replication fork
blockage (27). Phosphorylation of an N-terminal cluster
of serines (Rad55-S2,8,14) is important for full
function of Rad55, and a non-phosphorylatable mutant
(Rad55-S2,8,14A) leads to increased sensitivity to
genotoxic stress (28). Rad55 phosphorylation after DNA
damage causes an electrophoretic mobility shift, that
is unchanged in the Rad55-S2,8,14A mutant protein, sug-
gesting that a different phosphorylation site controls the
mobility shift (27,28).
In this study, we identify Rad55–S378 as the amino-acid
residue that controls the phosphorylation event(s) leading
to the electrophoretic mobility shift after DNA damage.
Rad55–S378 occurs in an SQ amino-acid sequence
context, the preferred target site for PIK kinases, and a
combination of in vivo and in vitro experiments identified
S378 as a direct Mec1 site. Using the Rad55–S378 con-
trolled mobility shift as a sentinel for Mec1 activation
in vivo, our data show that Mec1 can be activated under
conditions where Rad53 is not detectably activated. In
G1-arrested cells expressing the HO-endonuclease, Mec1
but not Rad53 was activated, as demonstrated by
phosphorylation of Rad55–S378, histone H2A (g-H2A)
and of RPA2. This response depended on both Mec1–
Ddc2 kinase complex and loading of the 9-1-1 clamp
by Rad24-RFC. Our findings suggest the existence
of a truncated DNA-damage-signaling pathway in
G1-arrested cells that involves activation of Mec1 kinase
but does not lead to activation of the full DDR involving
activation of the effector kinases Dun1, Rad53, or Chk1.
MATERIALS AND METHODS
Strains and plasmids
All S. cerevisiae strains used in this study are in the W303
background and listed in Supplementary Table S1. The
plasmid pJH132 was kindly provided by Dr Haber. It
contains the GAL10::HO fusion gene in a URA3 ARS1
CEN4 vector and allows galactose-regulated expression of
the HO endonuclease. Alternatively, we used pWDH408,
which has a TRP1 marker instead of URA3. The
rad55-S378A mutant allele was generated through
site-directed mutagenesis using the QuikChange System
(Stratagene) and the mutation was confirmed via DNA
sequencing of the entire RAD55 open reading frame.
It was recloned from a YCp50 vector into the modified
pBlueScriptKS vector, which already contained 335 bp of
upstream and 395 bp of downstream RAD55 sequences
between HindIII and XbaI sites. The genomic integration
of the rad55-S378A mutant allele was performed via
RAD55 allele replacement by co-transformation of
strain WDHY2009 with pYES-LEU2 and a linear DNA
fragment bearing the rad55-S378A gene with 50and 30
flanks obtained from pBlueScriptKS-rad55-S378A
through SacI-XhoI digestion. Leu
+
transformants were
replica plated on 5-fluoroorotic acid (5-FOA)-containing
medium to select for 5-FOA-resistant Ura

integrants.
The success of the allele replacement was screened by
PCR and confirmed by DNA sequencing of the chromo-
somal region comprising the RAD55 open reading frame
and the flanking sequences.
Nucleic Acids Research, 2010, Vol. 38, No. 7 2303

Cultures, G1 arrest, HO expression, methyl
methanesulfonate (MMS) treatment
For studies with G1-arrested cultures all strains were
transformed with plasmids pJH132 or pWDH408. The
cultures were grown in synthetic dropout media SD-ura
or SD-trp containing 3% raffinose as the carbon source.
In early log-phase (OD
600
= 0.3–0.4) 100 ng/ml a-factor
was added to the cultures and the cells were allowed to
arrest in G1 for 2 h. The arrest was confirmed using light
microscopy with at least 90% shmoo development.
Subsequently, cultures were split into three subcultures:
one was left untreated, 2% galactose was added to one
for induction of the HO-endonuclease, and 0.1% MMS
was added to the third subculture. All three subcultures
were incubated for another 2 h. The yeast strains used
for these experiments contained the bar1-Dmutation.
The product of the BAR1 gene is a secreted protease that
cleaves a-factor and is only expressed by MATa cells. The
‘barrier’ activity associated with this protease facilitates the
recovery of MATa cells from a-factor arrest. Strains with
the bar1-Dmutation are extremely sensitive to a-factor and
are slower to recover from G1 arrest. The use of this
mutation allows for more stable G1 arrest of the cells
and a more sparing use of a-factor in the culture media.
Antibodies
Rad55 protein was immunoprecipitated and immuno-
blotted using specific antibodies raised in rabbits and
rats, respectively (29). The phosphorylation status of
Rad55–S378 was analyzed using an affinity-purified
phosphoS378-specific antibody raised in rabbits against
an oligopeptide containing the phosphorylated residue
(PhosphoSolutions, Aurora, CO). Rad53 was detected
with a commercially available antibody raised in goats
(Santa Cruz Biotechnology, CA). The HA-tagged
proteins Sae2, Ddc2, Ddc1, Mre11 and Chk1 were
identified by commercially available anti-HA antibody
(murine HA.11, Covance Research Products, Inc.).
Original strains containing the HA-tagged proteins were
kindly provided by Dr Longhese. The Myc-tagged Mrc1
protein was identified using murine monoclonal anti-myc
antibodies (Santa Cruz Biotechnology, CA). The
anti-3-phosphoglycerate kinase (3-PGK) antibody was
purchased from Abcam (Cambridge, MA). RPA2, Rad9
and Dun1 were identified using protein-specific polyclonal
antibodies. The anti-RPA2 antibody was kindly provided
by Dr Brush, and the anti-Rad9 antibody by Dr Stern.
The anti-g-H2AX antibody was kindly provided by
Dr Bonner and used as published (30).
TCA-mediated protein precipitation
Twenty OD
600
units of cells were harvested, washed once
with water, once with 20% trichloroacetic acid (TCA),
resuspended in 100 ml TCA and frozen at 20C. After
thawing the cells at room temperature (RT), 100 mlof
glass beads were added and the suspension vortexed for
4 min. The supernatant was transferred to a fresh tube,
the beads washed twice with 100 ml 5% TCA and the
supernatants were pooled with the first one. All solutions
used in this procedure contained the protease inhibitors
leupeptin (2 mM), pepstatin A (1 mM), benzamidine
(1 mM), and PMSF (1 mM), and the phosphatase inhib-
itors Na
3
VO
4
, (0.1 mM) and NaF (1 mM). The
precipitated proteins were pelleted at 3000 rpm for
10 min at RT, the supernatant discarded and the pellet
resuspended in 100 ml 1.5 La
¨mmli sample buffer
(94 mM Tris pH 6.8, 3% SDS, 15% glycerol, 7.5%
b-mercaptoethanol, 0.0015% bromphenolblue). Due to
the acidic pH the sample turned yellow and needed to
be neutralized by adding 50 ml 2 M Tris. After boiling
the protein sample for 3 min, cell debris was pelleted
and discarded. Samples could be stored at 20C. For
analysis, total protein extract corresponding to three
OD
600
units of cells was loaded on suitable SDS–
polyacrylamide protein gels. Proteins were transferred
onto nitrocellulose and immunodetected using standard
Western blotting techniques.
Rad55 immunoprecipitation and immunoblotting
This method was performed as described in (29) with the
following minor modifications: the cleared protein extract
was incubated with anti-Rad55 antibody overnight at 4C
and the immuno-complexes were precipitated using
Protein G-Sepharose beads for 8 h.
Purification of Rad55–Rad57 and Rad55–S378A–Rad57
from S. cerevisiae
Purification was performed as described in (29). The
overexpression vector is based on pJN58 (31) and
contains the [GST]-RAD55 or [GST]-rad55-S378A
and [His6]-RAD57 fusion genes, whose expression is
controlled by the bidirectional GAL1-10 promoter.
Expression is induced by addition of galactose to the
medium. The consecutive dual affinity chromatography
purification strategy described in (29) efficiently selects
for the heterodimer and yields both subunits at equal
levels.
In vitro kinase assay
In vitro kinase assay was performed as described (29).
Briefly, in Mec1 kinase assays the reactions were
assembled on ice containing 2 mM HEPES (pH 7.4),
10 mM NaCl, 2 mM MnCl
2
, 0.2 mM DTT, 20 mCi [g-
32
P]
ATP, 40 mM cold ATP, 1 mg PHAS-1 or 2 mg purified
Rad55–Rad57 or Rad55–S378A–Rad57 heterodimer as a
substrate, and 20 ml (bed volume) of Protein A Sepharose
beads with immunoprecipitated HA-Mec1 or
kinase-deficient HA-Mec1-kd in a total volume of 30 ml.
Expression and immunoprecipitation using anti-HA
antibodies (HA.11, BAbCO) and Protein A sepharose
CL-4B (GE) of HA-Mec1 and HA-Mec1-kd were per-
formed immediately prior to the reaction as described
(32). The reactions were incubated at 30C for 30 min,
mixed with 7 ml5La
¨mmli (300 mM Tris pH 6.8, 10%
SDS, 50% glycerol, 25% b-mercaptoethanol, 0.005%
bromphenolblue) sample buffer, denatured and the
supernatants loaded onto a 4–16% gradient SDS–PAGE
gel, which was stained and dried after running. The dried
gel was used to expose autoradiography film. In Rad53
2304 Nucleic Acids Research, 2010, Vol. 38, No. 7

kinases assays the reactions contained 50 mM Tris–HCl
(pH 7.5), 10 mM MgCl
2
, 10 mM MnCl
2
, 1 mM DTT,
1.5 mCi [g-
32
P] ATP, 0.25 mM cold ATP, 2 mg affinity
chromatography-purified Rad55–Rad57 or Rad55–
S378A–Rad57 heterodimer as a substrate, and 1 mg
affinity chromatography-purified Rad53 or Rad53-kd.
The reactions were incubated and processed as described
for the Mec1 kinase assay.
Rad53 in situ autophosphorylation assays
Rad53 in situ autophosphorylation assays were performed
exactly as described earlier (9).
RESULTS
Phosphorylation controlled by serine 378 elicits the
electrophoretic mobility shift of Rad55 protein after
DNA damage
Rad55 protein was identified as a terminal target of the
DDR by virtue of a DNA damage-induced electrophoretic
mobility shift that depended on Mec1 kinase (27).
A cluster of N-terminal phosphorylation sites on Rad55
(serines 2, 8 and 14) are important for full function of
Rad55 protein in recombinational DNA repair (28).
However, phosphorylation of serines 2, 8 and 14 does
not contribute to the electrophoretic mobility shift, as
the Rad55-S2,8,14A mutant protein displayed the same
mobility shift as wild-type protein (28). Previous work
established that the Rad55 electrophoretic shift after
DNA damage was caused by phosphorylation using
phosphatase experiments (27). Deletion/substitution
analysis suggested an involvement of the C-terminus of
the 406 amino acid Rad55 protein, where every deletion
substitution affecting S378 eliminated the shift, whereas
any deletion/substitution not including S378 either had
no or a partial effect (29) (Figure 1, data not shown).
We focused on S378 also because it occurs in the preferred
site context of PIK kinases (33) (Figure 1A). Substitution
of S376 and S378 to the non-phosphorylatable residue
alanine essentially abolished the DNA damage-induced
Rad55 shift (Figure 1B, lanes 1–4). To corroborate this
finding, we integrated the rad55-S378A mutant into its
native genomic locus exactly replacing the wild-type
gene and found that the DNA damage-induced shift was
virtually eliminated in this mutant (Figure 1B, lanes 5–8).
To further establish that serine 378 is phosphorylated after
DNA damage in vivo, we generated phospho-specific
antibodies for this residue to show that S378 is specifically
phosphorylated after treatment with MMS (Figure 1C).
The signal by the anti-Rad55-phosphoS378 antibody is
specific for genotoxic stress conditions and abolished
in the rad55-S378A strain (Figure 1). Together with
results from dot blots using phosphorylated and
non-phosphorylated peptides (not shown), these controls
show that the antibody is phospho-specific and not just
serine-specific. We conclude that S378-dependent
phosphorylation controls the Rad55 mobility shift after
genotoxic stress.
PIK kinases like Mec1 prefer an S/TQ sequence context
(33). The Rad55 S378 residue occurs in this context
(SQ; Figure 1A). Evidence discussed below shows that it
represents a direct Mec1 target site (Figures 2 and 3). S378
is directly preceded by 7 serine or threonine residues
(Figure 1A), and it appears likely that the electrophoretic
shift is caused by the phosphorylation of multiple residues
in this area. Substitution of serine/threonine residues with
alanine in this area partially diminish the electrophoretic
mobility shift after DNA damage (29). Efforts to map
these phosphorylation sites by general and targeted mass
spectrometry methods were unsuccessful, likely because of
the occurrence of multiple phosphorylation events on
several residues in this area, a common problem in the
identification of phosphorylation sites (data not shown).
We conclude that S378 itself is phosphorylated and likely
affects phosphorylation of several preceding amino-acid
residues that elicit an electrophoretic mobility shift of
Rad55 after DNA damage. Hence, the Rad55 mobility
shift is an excellent sentinel for S378 phosphorylation.
The function and consequences of S378 phosphorylation
are not known presently, we use it here as a tool to monitor
Mec1 activity in vivo. Further experiments will be needed
to identify the function of S378 phosphorylation, which
are complicated by the complex phosphorylation pattern
of Rad55–Rad57 with eight phosphorylation sites
(unpublished results).
Rad55–S378 controlled phosphorylation is largely
dependent on Mec1 in vivo
The DNA-damage-induced mobility shift of Rad55 is
largely dependent on Mec1 (27). A low level of shifted
material remains in a mec1 deletion or kinase-deficient
mec1-kd strain (Figure 1D). We tested whether this
residual phosphorylation depends on Tel1, the second
PIK-kinase in budding yeast. While a tel1 single deletion
mutation has no effect on Rad55–S378 phosphorylation
(Figure 1D, (27)), the residual Rad55–S378
phosphorylation in the mec1-Dmutant strains appears
completely eliminated in the mec1 tel1 double mutant.
This suggests that Tel1 can phosphorylate Rad55 in an
S378-dependent manner, albeit making a minor contribu-
tion even in mec1-Dcells.
Mec1 but not Rad53 directly phosphorylates Rad55 in a
S378-dependent manner in vitro
Rad55–S378 occurs in the preferred target motif of
PIK-kinases including Mec1 (33). We sought to determine
whether Mec1 directly phosphorylates Rad55 in
an S378-dependent manner using in vitro kinase experi-
ments. Purified wild-type Rad55–Rad57 heterodimer or
Rad55–S378A–Rad57 mutant heterodimer were used as
a substrate for Mec1 kinase. The corresponding
enzymatically inactive version of Mec1 (Mec1-kd) was
used as a control to demonstrate the dependency of the
reactions on Mec1 kinase activity. The activities of the
wild-type and mutant Mec1 kinases were tested using
the model substrate PHAS1 (Figure 2A), showing
vigorous activity of wild-type Mec1 kinase and very low
activity by the mutant Mec1-kd kinase. Mec1 kinase effi-
ciently phosphorylates Rad55 protein in vitro (Figure 2B,
lane 1). Mec1-kd mutant kinase shows a significant
Nucleic Acids Research, 2010, Vol. 38, No. 7 2305

(2-fold) reduction of Rad55 phosphorylation (Figure 2B,
lane 3). The Rad55–S378A mutation significantly reduced
(2-fold) phosphorylation by wild-type Mec1 kinase
(Figure 2B, lane 2), suggesting that S378 is a primary
Mec1 site on Rad55 protein.
We have previously shown that Rad53 phosphorylates
Rad55 protein (28). In order to exclude that Rad55–S378
is also a Rad53 target site, in vitro kinase assays were
performed with Rad53. Phosphorylation of wild-type
Rad55–Rad57 and Rad55–S378A–Rad57 mutant
heterodimer by Rad53 did not significantly differ
(Figure 2C, upper panel, compare lane 1 with lane 2).
These results imply that while Rad53 does phosphorylate
Rad55 protein in vitro, it is not dependent on S378. The
mutant kinase Rad53-kd is incapable of phosphorylating
either substrate (lanes 3 and 4). Rad53 kinase activity was
verified by its autophosphorylation activity (lanes 1 and
2). As expected, no activity was observed for Rad53-kd
(Figure 2C, third panel, lanes 3 and 4).
From the in vitro kinase experiments we conclude
that both Mec1 and Rad53 phosphorylate Rad55–Rad57
in vitro. However, only Mec1 kinase phosphorylates
Rad55 in an S378-dependent fashion. Together with the
in vivo data described in Figure 1, these data identify
Rad55–S378 as a direct target residue of Mec1 kinase
that can be monitored by an electrophoretic mobility shift.
α-Rad55-S378-
α-Rad55
MMS -+-+
Rad55 Rad55-S378A
P
A
369 T L S T S S S S C S Q V F N 382
378
B
C
-
+
-
Rad55 Rad55-S378A
MMS - +-+
Rad55 Rad55-
S376A,S378A
+
Rad55
Rad55- P
3-PGK
12 34
56 78
12 34
Plasmid-encoded Chromosomal integration
Chromosomal integration
D
Wild type
mec1-kd mec1-Dtel1-Dmec1-Dtel1-D
MMS
-+--- -
++++
Rad55
Rad55- P
12345678910
Figure 1. Rad55 is phosphorylated at serine residue 378 in vivo in response to DNA damage in a Mec1-dependent fashion. (A) Region of the Rad55
amino-acid sequence surrounding serine residue 378. Serine 378 and glutamine 379 are highlighted. (B) Cells expressing plasmid-borne wild-type
Rad55 (lanes 1, 2), Rad55–S376A, S378A (lanes 3, 4) or wild-type Rad55 (WDHY2172: lanes 5, 6) and Rad55–S378A (WDHY2528 rad55-S378A:
lanes 7, 8) from the chromosomally integrated mutant gene were treated with 0.1% MMS for 2 h (lanes 2, 4, 6, 8) or left untreated (lanes 1, 3, 5, 7).
The Rad55 electrophoretic mobility shift was monitored using immunoprecipitation-immunoblotting using anti-Rad55 antibodies (27) and equal
amounts of extract (lanes 1–4) or by direct immunoblotting using anti-Rad55 antibodies (lanes 5–8). Lanes 1, 2 and 3, 4 came from different parts of
one gel. 3-PGK served as a loading control for the direct immunoblotting in lanes 5–8. (C) Wild-type (WDHY2172: lanes 1, 2) or chromosomally
integrated mutant (WDHY2528 rad55-S378A: lanes 3, 4) S. cerevisiae cells were treated with 0.1% MMS for 2 h (lanes 2, 4) or left untreated (lanes
1, 3). Rad55 protein was immunoprecipitated from equal amounts of cell extracts and analyzed by immunoblotting with antibodies specific for the
phosphorylated residue serine 378 (a-Rad55-S378-P, upper panel) and with polyclonal anti-Rad55 antibodies (a-Rad55, lower panel). (D) Wild-type
(DES460) and mutant (MD85 mec1-kd, DES459 mec1-D,WDHY1227 tel1-D,WDHY1515 mec1-Dtel1-D) cells were treated with 0.1% MMS for 2 h
(lanes 2, 4, 6, 8, 10) or left untreated (lanes 1, 3, 5, 7, 9). Rad55 protein was immunoprecipitated from equal amounts of cell extracts and analyzed by
immunoblotting using polyclonal anti-Rad55 antibodies. The electrophoretic mobility retardation of Rad55 protein is an indication for
phosphorylation of Rad55–S378.
2306 Nucleic Acids Research, 2010, Vol. 38, No. 7

Rad55–S378 is phosphorylated in a Mec1-dependent
fashion in response to a single double strand break
during G1 arrest
Previous analyses of the DDR in G1-arrested
(by afactor) budding yeast cells showed that a single
DSB created by the HO endonuclease failed to induce
a cell-cycle delay or activation of Rad53 kinase
(15,16,34,35). This has led to the conclusion that the
DDR kinase-signaling pathway is not activated under
these conditions (15,16). However, the activity of the
major checkpoint kinase Mec1 was not directly monitored
in these experiments. We noted that induction of a single
DSB by the HO-endonuclease in G1-arrested cells led to
Rad55–S378 phosphorylation, as indicated by the
electrophoretic shift, in a Mec1 kinase-dependent fashion
(Figure 3A, lower panel). This suggested that Rad55–S378
was phosphorylated by Mec1 in G1-arrested cells
experiencing a single DSB. Consistent with all previous
observations (15,16,34,35), we confirmed that Rad53
A
B
Rad55
Rad53
32P
α-[GST]
Coomassie
Rad53 Rad53-kd
--
--++
++
Coomassie
Mec1 Mec1-kd
PHAS-1
C
32P
Coomassie
Rad55-Rad57
Rad55-S378A-Rad57 --
--++
++
Mec1 Mec1-kd
Rad55
32PRad53-kd
Rad53
Rad53-kd
Rad55
12
123 4
1234
Rad55
PHAS-1
Rad55-Rad57
Rad55-S378A-Rad57
32P
Figure 2. Rad55 is phosphorylated in a S378-dependent manner by
Mec1 but not Rad53 kinase in vitro. In vitro kinase assays. (A) The
synthetic substrate PHAS-1 was incubated with immunoprecipitated
HA-tagged wild-type (Mec1: lane 1) or kinase-deficient Mec1 kinase
(Mec1-kd: lane 2) and analyzed using SDS–PAGE. The gel was
Coomassie stained and subsequently dried. The dried gel was subjected
to autoradiography. The Coomassie-stained protein bands served as a
loading control. (B) Affinity chromatography purified [GST]-Rad55-
[His6]-Rad57 wild-type protein (Rad55–Rad57: lanes 1, 3) or [GST]–
Rad55–S378A–[His6]–Rad57 mutant protein (Rad55–S378A–Rad57:
lanes 2, 4) were used as a substrate for immunoprecipitated wild-type
HA-Mec1 (Mec1: lanes 1, 2) or the catalytic-deficient mutant version
(Mec1-kd: lanes 3, 4). The reactions were analyzed as in (A). The same
amounts of kinase and substrate were used in all reactions (Figure 3B),
and the discrepancies between lanes 1/2 and 3/4 in the Coomassie panel
are due to differences in staining/destaining. (C) Affinity chromatogra-
phy purified wild-type GST-Rad53 kinase (Rad53: lanes 1, 2) or the
catalytic-deficient mutant version (Rad53-kd: lanes 3, 4) were incubated
with purified [GST]–Rad55–[His6]–Rad57 (Rad55-Rad57: lanes 1, 3) or
[GST]–Rad55–S378A–[His6]–Rad57 (Rad55–S378A–Rad57: lanes 2, 4).
The reaction was analyzed as in (A). Rad53 kinase activity was
observed through Rad53 autophosphorylation, which became
apparent after autoradiography (
32
P). The anti-GST immunoblot
served as a loading control for [GST]–Rad53 and [GST]–Rad53–kd.
Note that there is no electrophoretic shift of the GST–Rad55 fusion
protein in response to phosphorylation by Rad53 kinase.
h 0 0.5 1 2 0 0.5 1 2 0 0.5 1 2
32P
α-Rad53
B
A
C
α-3-PGK
α-Rad53
HO
control HO induction 0.1% MMS
α-Rad53
α-Rad55
wild type mec1-Δ
−+ + ++−
-HO
0.1%
MMS
Rad55-
Rad55 P
Rad53
α-3-PGK
12345 6
12 3
1234 5678 9101112
Rad53-
Rad53
P
Rad53
kinase
Figure 3. Rad55-S378 is phosphorylated after HO-mediated DSB
induction in G1-arrested cells in the absence of detectable Rad53 acti-
vation. (A) Mec1 controls Rad55-S378 phosphorylation in G1-arrested
cells. Rad55 was immunoprecipitated from equal amounts of cell
extracts of G1-arrested wild-type (WDHY2172: lanes 1–3) or mec1-D
(WDHY2173: lanes 4–6) cells after HO-endonuclease had been induced
(lanes 2, 3, 5, 6) or not (lanes 1, 4) and analyzed by immunoblotting
(lower panel). Rad53 was analyzed by immunoblotting in total cell
extracts (upper panel). The apparent variation in the Rad53 protein
level is not systematic and likely without significance. (B) Rad53 is
not detectably activated in response to a single DSB during G1-arrest
in wild-type cells (WDHY2172). Samples were taken from cultures of
untreated cells (control: lanes 1–4), after induction of HO endonuclease
(HO induction: lanes 5–8) and after addition of 0.1% MMS (lanes
9–12) at indicated times after G1 arrest was established by addition
of a-factor. The upper panels show in situ Rad53 activity assays.
In the middle panels, Rad53 was analyzed by immunoblotting of
total cell extracts. The lower panels show corresponding loading
controls. (C) Rad53 is activated in response to a single DSB in
cycling wild-type cells (WDHY2172). Samples were taken from
cycling cultures of untreated cells (lane 1), cells with one DSB inflicted
by HO endonuclease (lane 2) and MMS-treated cells (lane 3) after 2 h
of treatment. The upper panel shows the Rad53 immunoblot and the
lower panel the corresponding loading controls (B).
Nucleic Acids Research, 2010, Vol. 38, No. 7 2307

kinase is not detectably activated under these conditions
(Figure 3A and B).
While in cycling cells most of the Rad55 pool
experienced phosphorylation at S378 (Figure 1D), in
G1-arrested cells with a single DSB a smaller but still
sizable proportion of the Rad55 pool was shifted
(Figures 3A and 5). Considering that each cell experiences
only a single DSB, one might have expected that
only a small fraction of the Rad55 pool experiences
phosphorylation. However, repair proteins show
dynamic exchange between the nuclear pool and the
DNA damage site (36), which leads to an accumulation
of the phosphorylated protein species during the 2 h of
HO nuclease expression.
The experiment in Figure 3A showed no Rad53 activa-
tion at 2 h after DSB induction in G1-arrested cells. To
corroborate this initial observation and to exclude the
possibility that that we missed a transient activation of
Rad53, we performed time course experiments with
a-factor arrested cells. Fully consistent with previous
observations (15,16,19), Rad53 is not activated in
untreated control cells but is quickly activated, within
30 min or less, in cells treated with MMS (Figure 3B,
lanes 1–7, 15–20). In G1-arrested cells expressing
HO-endonuclease, Rad53 was not activated at any time
point (Figure 3B, lanes 5–8), as judged by two assays: An
electrophoretic shift indicates Rad53 autophosphorylation
indirectly, whereas the in-gel activity assay directly
measures Rad53 kinase activity (9). Both assays gave
completely congruent results, showing that at the time of
Rad55–S378 phosphorylation Rad53 is not detectably
activated. As a further control, we confirmed that Rad53
is readily activated in cycling cells expressing
HO-endonuclease or by treatment with MMS (Figure
3C). We conclude that in a-factor (G1)-arrested budding
yeast cells the DNA-damage-signaling pathway is
activated at the sensor kinase level (Mec1), but that this
signal does not lead to detectable activation of the effector
kinase Rad53.
Histone H2A and RPA2 are phosphorylated in response
to a single DSB during G1 arrest
Mec1 kinase phosphorylates many targets, and we asked
what other Mec1 target proteins are phosphorylated
in G1-arrested cells after induction of a single DSB
by HO-endonuclease. As negative control, cells were left
untreated during arrest. As positive control, cells were
treated with 0.1% MMS, which is expected to induce
full activation of the entire DNA-damage-signaling
cascade from Mec1 to Dun1, Rad53 and Chk1
(Figure 4, lanes 1 and 3).
Phosphorylation of histone H2AX at S139 (g-H2AX) is
a sensitive marker for DSB formation that depends on
activation of PIK-kinases in the DDR (37). Mec1 is the
dominant kinase for this response in yeast and directly
phosphorylates H2A on S129 (the equivalent residue on
yeast H2A). We tested whether g-H2A levels increase after
formation of a single HO-induced break in G1-arrested
cells. As shown in Figure 4A, the g-H2A level increased
significantly (3-fold) in response to a single HO-mediated
DSB, whereas the increase was almost 8-fold in response
to MMS, as quantified by densitometry and normalized
for the loading control (3-PGK in Figure 4A). We
conclude that a single DSB elicits a limited g-H2A
response in G1-arrested cells. This is consistent with
previous observations in G1-arrested cells with an
unrepairable DSB (38).
RPA2, the middle subunit of the hetero-trimeric
ssDNA-binding protein RPA, is directly phosphorylated
by Mec1 kinase in response to DNA damage, eliciting an
electrophoretic shift of the RPA2 protein (39). RPA2 is
phosphorylated, as indicated by the mobility shift, to the
same extent in G1-arrested cells with a single DSB or
when treated with MMS (Figure 4B top left, lanes 2 and
3). We conclude that RPA2 is another substrate that is
phosphorylated by Mec1 in response to a single DSB in
G1-arrested cells. The extent of RPA2 phosphorylation
was surprising, but likely reflects the dynamic exchange
between the free and DNA-bound pools (36), which
leads to an accumulation of the phosphorylated protein
species during the 2 h of HO expression.
Sae2 is a nuclease that works in conjunction with the
Rad50–Mre11–Xrs2 complex in DSB resection (40). Sae2
phosphorylation after DNA damage is largely dependent
on Mec1 and can be monitored by an electrophoretic
mobility shift (41). Upon induction of HO-endonuclease
Sae2 protein did not undergo an electrophoretic shift
indicating that Sae2 was not phosphorylated under these
conditions (Figure 4B, lane 2). In G1-arrested cells treated
with MMS, Sae2 protein exhibited the expected
electrophoretic shift (lane 3). Mre11 also is
phosphorylated in response to DNA damage (42), and
an MMS-induced electrophoretic shift is detected in
MMS-treated G1-arrested cells (Figure 4B, lane 3).
However, Mre11 phosphorylation is independent of
Mec1, in fact induced in cells lacking Mec1, and Tel1
has been identified as the most probable in vivo kinase
(42). Expression of HO-endonuclease failed to induce
Mre11 phosphorylation indicated by the lack of a
mobility shift (lane 2), suggesting that Tel1 kinase is not
detectably induced under these conditions.
Ddc2 targets Mec1 to RPA-covered ssDNA, and Ddc1
is a component of the 9-1-1 complex (4). The
spatio-temporal colocalization of both complexes,
Mec1-Ddc2 and the 9-1-1 complex, is required for the
activation of the DNA-damage-signaling cascade in
cycling cells (5). Mec1-dependent Ddc2 phosphorylation,
as monitored by a mobility shift, is evident after DNA
damage and occurs in unperturbed cells at the end of
S-phase (43). Since Mec1 can phosphorylate Ddc2
in vitro and in vivo phosphorylation does not depend on
any other checkpoint factor (9-1-1 clamp, Rad24 clamp
loader, Rad53) (43), it is likely that Ddc2 is a direct Mec1
target. Mec1-dependent Ddc1 phosphorylation also
occurs after DNA damage or during an unperturbed
S-phase (44). Unlike Ddc2, Ddc1 phosphorylation
depends on the 9-1-1 clamp and its Rad24 clamp loader
(44). Ddc1 and Ddc2 are both phosphorylated in
G1-arrested cells after MMS exposure as indicated by
their mobility shifts (Figure 4B, lanes 3). Expression
of HO-endonuclease in G1-arrested cells fails to elicit
2308 Nucleic Acids Research, 2010, Vol. 38, No. 7

this response, suggesting that neither Ddc2 nor Ddc1 are
phosphorylated by Mec1 under these conditions.
Rad9 and Mrc1 are adaptor proteins that mediate the
kinase response from the top-level sensor kinase Mec1 to
the effector kinases Rad53, Dun1 and Chk1. Both proteins
undergo Mec1-dependent phosphorylation after DNA
damage induction at S/TQ cluster domains (8,45), and
this is confirmed in G1-arrested cells treated with MMS
(Figure 4B, lanes 3). Neither protein exhibits a mobility
shift in response to a single DSB in G1-arrested cells,
suggesting that they are not phosphorylated under these
conditions. As expected from the Rad9 and Mrc1 results,
neither Dun1 nor Chk1 kinase shows evidence for a
mobility shift, indicating they are not activated. Both
kinases are activated in response to MMS in G1-arrested
cells (Figure 4B, lanes 3). The absence of detectable
activation of Rad53 kinase in response to a single
HO-mediated DSB in G1-arrested cells was established
previously (15,16,34,35) and confirmed here (Figures 3,
4A and 5).
We conclude that in response to a single DSB induced
by HO-endonuclease in a-factor arrested (G1) cells a
truncated signaling response is induced that activates
Mec1 to phosphorylate some direct targets, such as
Rad9-
Rad9
Mrc1-
Mrc1
Chk1-
Chk1
Dun1-
Dun1
-HO MMS
RPA2-
RPA2
Sae2-
Sae2
Ddc2-
Ddc2
Ddc1-
Ddc1
Mre11-
Mre11
P
Rad53-
Rad53
123
P
P
P
P
P
P
P
P
P
B
A
-HO MMS
γ−H2A
3-PGK
123
123
-HO MMS
-HO MMS
Figure 4. Histone H2A and RPA2 are phosphorylated in G1-arrested cells after induction of a single DSB. (A) Cultures of wild-type cells
(WDHY2172) were arrested in G1 and left untreated (lanes 1), HO-endonuclease was induced (lanes 2) or 0.1% MMS (lanes 3) was added for
2 h. Levels of phosphorylated histone H2A (g-H2AX) were determined using a modification-specific antibody and quantified against the 3-PGK
loading control by densitometry. One example of the anti-Rad53 immunoblots of the corresponding samples in A and B is shown, and in all cases
tested Rad53 was induced by MMS and not induced by expression of HO. (B) Cultures of wild-type cells (WDHY2172) or cells with HA-tagged
versions of Ddc1 (WDHY2460), Sae2 (WDHY2462), Mre11 (WDHY2502), Ddc2 (WDHY2517), or Chk1 (YLL839) or a Myc-tagged version of
Mrc1 (WDHY2180) were treated as in (A). The indicated proteins were analyzed by immunoblotting in total cell extracts using suitable antibodies
(see ‘Materials and Methods’ section). Electrophoretic mobility retardation indicates phosphorylation of the corresponding protein.
Rad55 Rad53
MMSHO
_MMSHO
_
Wild type
ddc2
rad24
rad50
rad9
mrc1
Rad55-
Rad55
Rad55-
Rad55
Rad55-
Rad55
Rad55-
Rad55
Rad55-
Rad55
Rad55-
Rad55
Rad53-
Rad53
Rad53-
Rad53
Rad53-
Rad53
Rad53-
Rad53
Rad53-
Rad53
Rad53-
Rad53
123 456
P
P
P
P
P
P
P
P
P
P
P
P
Figure 5. Genetic dependency of Rad55–Ser378 phosphorylation in
response to a single double strand break in G1-arrested cells.
Cultures of wild-type (WDHY2172) and mutant strains (WDHY2534
ddc2-D, WDHY2523 rad24-D, WDHY2511 rad50-D, WDHY2452
rad9-D, WDHY2449 mrc1-D) were left untreated (lanes 1),
HO-endonuclease was induced (lanes 2) or the cells were treated with
0.1% MMS for 2 h (lanes 3) during G1-arrest. Rad55 was immunopre-
cipitated from equal amounts of cell extracts and analyzed by
immunoblotting using polyclonal anti-Rad55 antibodies (left panels,
panels 1–3). Rad53 was detected by immunoblotting in total cell
extracts of the corresponding samples (right panels, lanes 4–6).
Nucleic Acids Research, 2010, Vol. 38, No. 7 2309

Rad55, histone H2A and RPA2, but does not lead to full
activation of the entire DDR-signaling cascade as
indicated by the absence of phosphorylation of Mec1
targets in the signaling pathway such as Ddc2, Ddc1,
Mre11, Mrc1, Rad9, Dun1, Chk1 and Rad53.
Ddc2 and loading of the 9-1-1 complex are required for
the truncated Mec1-signaling pathway in G1-arrested cells
In order to define the genetic requirements for the
truncated signaling pathway in response to a single
HO-mediated DSB in G1-arrested cells, we analyzed the
Rad55–S378 phosphorylation status in G1-arrested cells
after HO induction in mutants affecting various aspects
of the signaling pathway at the sensor and adaptor level.
Ddc2, like its mammalian homolog ATRIP, targets
Mec1 kinase (mammalian ATR) to RPA-coated ssDNA
and is absolutely required for DNA damage checkpoint
induction (4). The spatio-temporal colocalization of the
Mec1–Ddc2 complex with the 9-1-1 complex activates
the DDR (5). The 9-1-1 complex is loaded in a
Mec1-independent fashion by a DNA damage-specific
clamp loader, the Rad24-RFC via an interaction with
RPA bound to ssDNA (5,7,46). This dual requirement
for DNA damage sensing provides significant specificity
in the DDR. Yeast lacking Ddc2 or Rad24 completely
failed to phosphorylate Rad55 at S378 in response to a
single DSB induced by HO in G1-arrested cells, as
evidenced by the lack of an electrophoretic shift
(Figure 5, second and third panel, lanes 2). Likewise, in
response to MMS no Rad55-S378 phosphorylation was
observed (lanes 3). As expected, both ddc2 and rad24
mutants significantly curtail Rad53 activation after
DNA damage induction by MMS (lanes 6). These impor-
tant results establish that the truncated signaling response
identified in G1-arrested cells in response to a single
HO-mediated DSB exhibits the same sensor requirements
as the traditional, full-blown DDR.
The Rad50–Mre11–Xrs2 complex is involved in the
resection of the DSB (40,47), and we asked whether this
complex is required for Rad55–S378 phosphorylation in
G1-arrested cells in response to a single DSB (Figure 5).
Surprisingly, Rad55 was constitutively phosphorylated in
cells lacking Rad50 protein, as indicated by the observa-
tion that about half of the Rad55 pool underwent a
mobility shift (lane 1). The amount of Rad55–S378
phosphorylation did not increase after induction of HO
endonuclease (lane 2), but led to a complete mobility shift
after addition of MMS. These data show that rad50 cells
experience genotoxic stress that leads to constitutive acti-
vation of Mec1 without noticeable Rad53 activation (lanes
4 and 5). While the constitutive Rad55 phosphorylation is
clearly Rad50-independent, it is difficult to make a con-
clusion about the DSB-induced Rad55 phosphorylation.
Rad9 and Mrc1 are adaptor proteins that mediate the
signaling response from Mec1 to the effector kinases
Dun1, Rad53 and Chk1 (48). Similar to rad50,rad9 and
mrc1 cells show constitutive Rad55–S378 phosphorylation
that is not significantly enhanced by induction of
HO endonuclease in G1-arrested cells (Figure 5, bottom
panels, lanes 1 and 2). As expected for a direct Mec1
target, Rad9 and Mrc1 are not required for Rad55–S378
phosphorylation in response to MMS in G1-arrested cells.
Again similar to rad50 cells, Rad53 is not constitutively
activated in rad9 or mrc1 cells (lanes 4). Also in rad54D
cells, Rad55 was constitutively phosphorylated on S378 in
a Mec1-dependent fashion in the absence of Rad53 acti-
vation (Supplementary Figure S1, data not shown). The
constitutive limited Mec1 activity in rad50, mrc1, rad9 and
rad54 mutants suggests that these cells experience low level
genotoxic stress. This is reminiscent of the constitutive
SOS induction found in mutants in certain repair and rep-
lication genes (e.g. recG, rep, polA)inEscherichia coli (49).
It is possible that this is replication-associated and that
Rad55 phosphorylation was triggered in the cell cycle(s)
before the G1-arrest. Rad55 protein phosphorylated at
S378 is stable for at least 8 h after induction of a single
DSB (data not shown).
We conclude that the truncated DNA-damage-signaling
pathway in G1-arrested cells in response to a single DSB
has the same sensor requirements (Mec1-Ddc2, 9-1-1/
Rad24 clamp loader), as the classic DDR, but does not
transmit the signal in the Mec1-Rad53-Chk1-Dun1
cascade beyond the Mec1 sensor kinase.
DISCUSSION
A truncated DNA-damage-signaling pathway in response
to a single DSB in G1-arested cells leads to limited
activation of the DDR
Using Rad55–S378 phosphorylation as a sentinel of
Mec1 kinase activity in vivo we discovered a truncated
DNA-damage-signaling pathway that is active in
G1-arrested wild-type cells suffering limited DNA
damage (a single DSB) and is constitutively activated in
a number of mutants in genes involved in DNA metabo-
lism (RAD50,MRC1,RAD9,RAD54). Activation of
Mec1 kinase depended on both sensor components
of the canonical DDR, the Mec1–Ddc2 complex
and loading of the 9-1-1 complex by the Rad24-RFC.
It appears that Mec1 kinase activation was restricted to
the site of damage, targeting besides the DNA repair
protein Rad55, histone H2A, and RPA2, which is bound
to processed DSBs and where RPA provides the binding
site for the Mec1–Ddc2 complex and the Rad24-RFC.
However, the signaling cascade appears truncated after
the sensor level, as there is no detectable activation of
the effector kinases, most notably Rad53, and no
evidence for phosphorylation of the adaptor proteins
Rad9 and Mrc1. It is unlikely that the function of
Rad53 kinase is replaced by the paralogous protein
Mek1, which is known to function only during meiosis,
where it substitutes for Rad53 in the meiotic checkpoint
(50). These data suggest that the DDR is not an ON/OFF
switch, but capable of an intermediate level of activation
(Supplementary Figure S2).
The bacterial SOS response fulfills a similar function as
the eukaryotic DDR in enhancing survival and genomic
stability. The SOS response involves the regulation of the
LexA transcriptional repressor that controls a suit of
about 40 genes with functions in DNA repair (e.g. recA,
2310 Nucleic Acids Research, 2010, Vol. 38, No. 7

uvrA,uvrB,ruvA), DNA damage tolerance and
mutagenesis (recA,umuC,umuD,DinB), replication
restart (polB), cell division (sulA) and SOS autoregulation
(lexA,recA,recX,dinI) (51). The DDR in eukaryotes is
a kinase-signaling network that controls similar
effector pathways in a mechanistically different way (1).
However, the biological functions of both pathways in
ensuring survival and genomic stability are highly
similar. The SOS response has been a paradigm for a
complex regulatory network. The level of DNA damage
determines whether cells induce the full or a partial
transcriptional program by regulation of the LexA
repressor level and through the different architectures of
the LexA-regulated promoters (52). This leads to the
different levels of the SOS response through a temporal
pattern of transcriptional induction leading from
early/low level responses (uvrA,uvrB,uvrD) to additional
responses as the level of DNA damage increases (RecA
accumulation, cell-cycle arrest through induction of
sulA, and induction of umuDC-dependent translesion
synthesis) (51).
There is evidence for a threshold in activating the DDR
(measured as activation of Rad53) in the G1 and S-phases
of the cell cycle (34,53). Given the mechanisms of the func-
tionally similar SOS response in bacteria it appears
unlikely that the eukaryotic DDR functions solely as a
threshold-triggered ON/OFF switch. Data presented
here and in Barlow et al. (35) provide evidence for the
induction of the DDR in G1-arrested wild-type cells that
is different from the canonical-signaling response in that it
does not detectably activate Rad53 kinase. What could be
the physiological function of such a limited activation of
the DDR in yeast? A deliberate partial response preempts
a full DDR with an undesirable cell-cycle delay in
response to DNA damage that is easily addressed during
the S/G2 phase. The number of Mec1 targets that have
been identified under these conditions is limited to Rad55,
histone H2A and RPA2, and more work is needed to
identify additional G1 targets to uncover further effector
processes that might be regulated under these conditions.
We speculate that RPA2, histone H2A, Rad55
phosphorylation may affect DSB processing, repair
pathway or target (homolog) choice, or DNA replication.
What are the mechanisms that control the transition
from the limited activation of the DDR in G1-arrested
cells after a single DSB to a full response upon S-phase
entry? DSB processing is significantly more efficient in
S/G2 cells than in G1-arrested cells and controlled by
CDK phosphorylation of Sae2 (16,54). The accumulation
of ssDNA leads to extensive RPA–ssDNA complexes that
recruit more Mec1–Ddc2 kinase molecules and possibly
9-1-1 clamps (Supplementary Figure S2). This likely
explains the threshold identified in G1 cells, where one,
two, or three DSBs did not trigger Rad53
phosphorylation, but the addition of a fourth DSB
caused Rad53 activation (34). Physical assays detect
limited DSB processing in G1-arrested cells (34,55). A
proportion of such cells also contained RPA1 foci,
another indication of DSB processing (35). Our observa-
tion that RPA2 is phosphorylated under these conditions
is consistent with Mec1 being active at a processed DSB.
However, this mechanism does not explain why in G1 cells
with a single DSB Mec1 kinase signaling is not transmitted
to the effector kinases. It is interesting to note that Ddc1
and Ddc2 are not phosphorylated under these conditions
(Figure 4B). Both proteins are direct targets of Mec1 and
phosphorylated during a normal S-phase and after DNA
damage induction in a Rad53-independent fashion
(43,44,56). The critical difference between these studies
and our work is that Ddc1 and Ddc2 phosphorylation
were observed under conditions (S-phase + UV,
G2+UV) that led to full induction of the signaling
cascade including Rad53 activation (43,44), unlike the
limited induction in G1 cells with a single DSB used
here. Dpb11 is an essential replication protein that
functions in the S-M checkpoint to activate Mec1
directly or in conjunction with the 911 clamp (57,58).
Ddc1 Phosphorylation recruits Dpb11, a mechanism
conserved in fission yeast (59,60). The absence of Ddc1
phosphorylation in G1-arrested cells with a single DSB
and the association of Dpb11 with the replication fork
suggest that Dpb11 is not involved. We speculate that
G1-specific mechanisms restrain signaling in response
to a single DSB such as G1-specific phosphatases or
inhibitors.
Are Rad53, Dun1 and Chk1 truly not activated or are
they activated at a low level that cannot be detected
by standard assays?
Consistent with previous observations (15,16,34,35),
Rad53 (as well as the Dun1 and Chk1) is not detectably
activated by a single HO-mediated DSB in G1-arrested
cells. It is impossible to distinguish whether these kinases
are truly not activated or activated to low level that eludes
detection by the standard assays employed here and in the
other studies. Barlow et al. (35) observed that the
ribonucleotide reductase inhibitor Sml1 was degraded in
20% of G1-arrested cells experiencing a single I-SceI
induced DSB without detectable Rad53 activation,
leading the authors to suggest that Sml1 degradation
was a more sensitive measure of DDR activation than
Rad53 kinase activation. Degradation of Sml1 is triggered
by phosphorylation by Dun1 kinase (61), but it is possible
that in G1-arrested cells also Mec1 kinase targets Sml1. In
addition, there is evidence that Dun1 can be activated in
Rad53-independent fashion (62). Collectively, these obser-
vations provide evidence that DNA-damage-signaling in
G1 cells is different from other phases of the cell cycle,
whether this involves no kinase activation downstream of
Mec1 (as suggested in Supplementary Figure S2) or a low
level of activation of the effector kinases (not detectable by
the presently employed assays) remains to be tested.
SUPPLEMENTARY DATA
Supplementary Data are available at NAR Online.
ACKNOWLEDGEMENTS
The authors are grateful to Bill Bonner, George Brush,
Stephen Elledge, Maria Pia Longhese, Tom Petes,
Nucleic Acids Research, 2010, Vol. 38, No. 7 2311

David Stern, Lorraine Symington and Ted Weinert for
kindly providing them with antibodies, strains and
plasmids. They thank Valley Stewart for discussions
about the SOS response and Shannon Ceballos, Kirk
Ehmsen, Erin Schwartz, William Wright for their
comments on the manuscript and help with the figures.
FUNDING
Training (grant T32ES007059 to R.J.); National Institutes
of Health (grants P41 RR011823 to J.R.Y. and R01
CA92276 to W.D.H.). Funding for open access charge:
National Institutes of Health.
Conflict of interest statement. None declared.
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