DNA Repair 8 (2009) 1452–1461
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DNA Repair
journa l homepage: www.e lsev ier .com/
Brief report
Dissect e m
telome
Chinonye Ste
a Department o
b Laval Univers City, Q
c Department o A
d Ludwig Cente
a r t i c l
Article history:
Received 29 A
Received in revised form 27 August 2009
Accepted 21 September 2009
Available online 31 October 2009
Keywords:
cdc13-1
Cell cycle chec
H2A phosphor
BRCT
Telomere
zing t
Cdc13, induces a cell cycle checkpoint that arrests cells at the metaphase to anaphase transition—much
like the response to an unrepaired DNA double strand break (DSB). Throughout the cell cycle, the multi-
domainadaptorproteinRad9 is required for theactivationof checkpoint effectorkinaseRad53 in response
to DSBs and is similarly necessary for checkpoint signaling in response to telomere uncapping. Rad53
activation in G1 and S phase depends on Rad9 association with modified chromatin adjacent to DSBs,
which is mediated by Tudor domains binding histone H3 di-methylated at K79 and BRCT domains to
histone H2A phosphorylated at S129. Nonetheless, Rad9 Tudor or BRCT mutants can initiate a checkpoint
1. Introdu
In meta
are a delet
tivation of
promotion
a checkpoi
downstream
Signal trans
extensively
conserved
visiae, DSB
(MRE11–RA
where Tel1
∗ Correspon
Tel.: +1 773 83
E-mail add
1568-7864/$ –
doi:10.1016/j.kpoint
ylation response to DNA damage in nocodazole-treated cells. Mutations affecting di-methylation of H3 K79, or
its recognition by Rad9 enhance 5′ strand resection upon telomere uncapping, and potentially implicate
Rad9 chromatin binding in the checkpoint response to telomere uncapping. Indeed, we report that Rad9
binds to sub-telomeric chromatin, upon telomere uncapping, up to 10 kb from the telomere. Rad9 binding
occurred within 30 min after inactivating Cdc13, preceding Rad53 phosphorylation. In turn, Rad9 Tudor
and BRCT domain mutations blocked chromatin binding and led to attenuated checkpoint signaling as
evidenced by decreased Rad53 phosphorylation and impaired cell cycle arrest. Our work identifies a role
for Rad9 chromatin association, during mitosis, in the DNA damage checkpoint response to telomere
uncapping, suggesting that chromatin binding may be an initiating event for checkpoints throughout the
cell cycle.
© 2009 Elsevier B.V. All rights reserved.
ction
zoans, unrepaired DNA double strand breaks (DSBs)
erious form of DNA damage that can lead to inac-
tumor suppressors, activation of oncogenes and the
of carcinogenesis. In response to DSBs, cells initiate
nt response that senses DNA lesions and signals to
effectors to induce cell cycle arrest and DNA repair.
duction in response to DNA damage has been reviewed
and many of the checkpoint signaling proteins are
from yeast to humans [1–3]. In Saccharomyces cere-
induction recruits the Mre11–Rad50–MRXrs2 complex
D50–NBS1 in humans) and Tel1 (ATM) to breaks [1,4]
may phosphorylate histone H2A (H2AX) at S129 [5,6].
ding author at: JFK R320, 924 East 57th St., Chicago, IL 60637, USA.
4 0250; fax: +1 773 702 4394.
ress: skron@uchicago.edu (S.J. Kron).
Next, the multi-domain adaptor protein Rad9 (53BP1, MDC1,
BRCA1) localizes to chromatin adjacent to DSBs [7–10] and DNA
ends undergo 5′–3′ resection in the late S and G2/M phases. The
Rfa1–Rfa2–Rfa3 heterotrimer (RPA) binds to ssDNA and recruits
the Rad24 (RFC) clamp loader to assemble the Rad17–Mec3–Ddc1
clamp (Rad9–Rad1–Hus1) at the junction of ssDNA and dsDNA [11].
The RFA, Rad24 and Rad17 complexes recruit Ddc2 (ATRIP) and
the Mec1 (ATR) kinase to breaks [4,12–15]. Mec1 phosphorylates
Rad9, which recruits Rad53 (CHK2) to DSBs and leads to Mec1-
dependent phosphorylation and auto-phosphorylation of Rad53
[16–18]. Finally, Rad53 kinase activity propagates a DNA damage
signal that leads to cell cycle arrest and DNA repair.
The nucleosome, the basic unit of chromatin, is an octameric
DNA–protein complex consisting of histones H3, H4, H2A, H2B and
146 bps of DNA [19,20]. Histones can be modified by ubiquiti-
nation, phosphorylation, methylation and acetylation [21]. These
chromatin modifications alter the interaction among histones,
DNA and other proteins, and thereby play significant roles in the
DNA damage response [1,22–27]. For example, the mammalian
see front matter © 2009 Elsevier B.V. All rights reserved.
dnarep.2009.09.010ion of Rad9 BRCT domain function in th
re uncapping
C. Nnakwea,d, Mohammed Altafb, Jacques Côtéb,
f Pathology, The University of Chicago, Chicago, IL 60637, USA
ity Cancer Research Center, Hotel-Dieu de Quebec (CHUQ), 9 McMahon Street, Quebec
f Molecular Genetics and Cell Biology, The University of Chicago, Chicago, IL 60637, US
r for Metastasis Research, The University of Chicago, Chicago, IL 60637, USA
e i n f o
pril 2009
a b s t r a c t
In Saccharomyces cerevisiae, destabililocate /dnarepai r
itotic checkpoint response to
phen J. Kronc,d,∗
ue. G1R 2J6, Canada
elomeres, via inactivation of telomeric repeat binding factor

C.C. Nnakwe et al. / DNA Repair 8 (2009) 1452–1461 1453
phosphoinositide-3-kinase-related protein kinases (PIKKs) ATM,
ATR and DNA-PK, phosphorylate H2A variant H2AX in response
to DNA damage [28–30]. In budding yeast, histone H2A is phos-
phorylated by Mec1 and Tel1 kinases to form a chromatin domain
that extend
modifiers in
phorylation
but also m
teins, includ
lesions [36]
homolog C
H2A via tan
Moreover,
inducing ag
G1 and S ph
The biol
protect chro
When the te
component
ture resemb
response du
yeast that b
key role in t
[46]. The te
stranded te
dissociates
with -fact
mutants do
tion of Cdc2
undergo 5′
activating R
metaphase
in gene exp
at non-perm
radiation [4
a powerful
G2/M check
Seminal
buddingyea
When chal
mutants de
ity, fail to a
phase of th
instead div
perature sh
structure ch
carboxyl te
implicated
interact wit
this interac
[9,10,61]. C
chromatin i
di-methyla
for Rad9 chr
Tudor dom
H3 at K79 a
the conserv
Tudor dom
checkpoint
rad9-Y798Q
the cdc13-1
mulation at
While R
to telomere
signaling da
targeted the
role of H2A phosphorylation in checkpoint signaling at uncapped
telomeres. Comparative protein modeling was used to identify
residues in the Rad9 BRCT domains that define a phospho-H2A
binding pocket. As expected, mutation of these residues resulted
ckpo
owe
ts dis
mere
alysi
terac
toge
tion
eckp
mito
teria
ester
Wes
and
ed. C
nd r
Cl pH
es w
× g
nated
to ni
anti
ous
ouse
rn bl
osp
ed us
ated
ative
c13-
53 a
the
LQLK
leas
ed h
rocol
wer
late
ed [
as a
as c
ean
rom
oma
cribe
ght c
terile
was
was
or 30
and t
mlYP
2 h
at 2s up to 50 kb from DNA lesions and recruits chromatin
cluding NuA4, Ino80 and SWR [23,31–33]. H2A phos-
may lead to changes in chromatin structure [34,35]
ediates recruitment and retention of checkpoint pro-
ing Rad9 homologs 53BP1, MDC1 and BRCA1, at DNA
. Recruitment of MDC1, like Schizosaccharomyces pombe
rb2, is mediated by direct binding to phosphorylated
dem BRCA1 carboxyl-terminal (BRCT) domains [37,38].
hta1, 2-S129A point mutants are sensitive to DSB-
ents, deficient in non-homologous end joining and have
ase checkpoint defects [9,10,35].
ogy of telomeres, specialized chromatin domains that
mosome ends, has been reviewed extensively [39–43].
lomere is “uncapped” by the loss of one or more protein
s that bind to G1–3T::C1–3A repeats, the resulting struc-
les one end of a natural DSB and activates a checkpoint
ring mitosis. Cdc13 [44], a telomere-specific protein in
inds to single stranded G1–3T::C1–3A overhangs, plays a
he recruitment of telomerase [45] and telomere capping
mperature sensitive cdc13-1 P371S allele binds single
lomeric repeats at permissive temperatures (<25 ◦C) but
at restrictive temperatures (>26 ◦C). When held in G1
or and shifted to non-permissive temperature, cdc13-1
not activate Rad53 [47]. However, with the accumula-
8 activity in late S or G2/M phases, uncapped telomeres
–3′ resection to generate long regions of ssDNA [46],
ad53 and a sustained mitotic checkpoint arrest at the
to anaphase transition. Thus, the characteristic changes
ression and cell cycle progression in cdc13-1 mutants
issive temperature resemble the response to ionizing
6,48–50]. As such, the cdc13-1 mutation has served as
tool to elucidate molecular mechanisms mediating the
point response to DNA damage [39,44,51–53].
work characterizing the DNA damage checkpoint in
st identifiedRad9asakeymediator in signaling [54,55].
lenged with genotoxic agents or ionizing radiation,
leted for the RAD9 gene display DNA damage sensitiv-
ctivate Rad53, and display checkpoint defects at each
e cell cycle. Similarly, rad9 cdc13-1 cells fail to arrest,
iding to form microcolonies of inviable cells after tem-
ift [44,51,56–60]. Rad9, 53BP1 and Crb2 share a domain
aracterized by tandem BRCT and Tudor domains at the
rminus. Although the Rad9 BRCT domains have been
in oligomerization [61,62], the Rad9 BRCT domains also
h phosphorylated H2A peptides in vitro and disrupting
tion leads to G1 and intra-S phase checkpoint defects
onsistent with dual recognition of chromatin by Rad9,
mmunoprecipitation studies demonstrate that H3 K79
tion and H2A S129 phosphorylation are both necessary
omatin association [9,10,61]. The Rad9, 53BP1 and Crb2
ains have been shown to bind di-methylated histone
nd/or H4 K20 in vitro [7,63]. Yeast strains deficient in
ed H3 K79 methyl-transferase Dot1 or expressing the
ain mutant rad9-Y798Q display G1 and intra-S phase
defects after ionizing radiation [8]. In addition, rad9,
Tudor domain mutants and dot1 strains harboring
mutation also have an increased rate of ssDNA accu-
uncapped telomeres [51,56,57,59].
ad9 is necessary for checkpoint signaling in response
uncapping, a role for Rad9 chromatin association in
mage to telomeres remains unclear. In this study, we
putativeRad9phospho-H2Abinding site toanalyze the
in che
tion. H
mutan
to telo
tionan
this in
Taken
associa
age ch
during
2. Ma
2.1. W
For
sured
collect
5 min a
Tris–H
Sampl
16,100
fractio
ferred
mouse
9E10 m
anti-m
Weste
H2A ph
detect
conjug
resent
2.2. cd
Rad
rying
(WHW
then re
collect
tent.
Mic
strains
Next, p
describ
scored
scored
cates, m
2.3. Ch
Chr
as des
overni
with s
Factor
culture
37 ◦C f
water
in200
1 h and
20 minint defects in G1 and S phase after ionizing radia-
ver, contrary to nocodazole-based assays, rad9 BRCT
played an attenuated or impaired checkpoint response
uncapping. In addition, chromatin immunoprecipita-
s shows thatRad9binds to sub-telomeric chromatinand
tion is dependent on both chromatin-binding domains.
ther, these results demonstrate that Rad9 chromatin
at sub-telomeric chromatin is important for DNA dam-
oint activation in the response to telomere uncapping
sis.
ls and methods
n blot analysis
tern blot analysis, OD600 of yeast cultures were mea-
volumes corresponding to 2–2.5 OD600 units were
ell pellets were treated with 0.2N NaOH for at least
esuspended in 100l of 1× SDS sample buffer (125 mM
6.8, 20% glycerol, 4% SDS, 1.43 M -mercaptoethanol).
ere incubated at 95 ◦C for 5 min and centrifuged at
for 1 min to clarify the lysate. 15l of cell lysate was
with NUPAGE 3–8% TA gels (Invitrogen) and trans-
trocellulose. Rad53-FLAG blots were probed using M2
-FLAG (Sigma, 1:1000), and Rad9–13Myc blots using
e anti-MYC (1:200, Santa Cruz), and detected with goat
IgG HRP conjugate secondary antibody. Phospho-H2A
otting was performed with rabbit anti-yeast histone
ho-Ser129 (Millipore 1:100 in 1× PBS) and tubulin was
ing 1:500 YL1/2 rat anti-tubulin (Millipore) and HRP
goat anti-rat IgG secondary antibody. Figures are rep-
of two or more replicate experiments.
1 checkpoint assays
ctivation and flow cytometry analysis: Strains car-
cdc13-1 allele were incubated at 25 ◦C in -factor
PGQPNleY) [64] for 1 h, transferred to 37 ◦C for 1 h and
ed into 37 ◦C media for an additional 3 h. Samples were
ourly and analyzed for Rad53 activation and DNA con-
ony analysis: Sonicated and serially diluted cdc13-1
e plated and incubated at 25, 30 or 37 ◦C for 6.5–7 h.
s were transferred to 4 ◦C until scored, as previously
55]. Briefly, microcolonies with ≤four cell bodies were
rrested and microcolonies with >four cell bodies were
ycling. The data shown are the average of three repli-
± SD.
atin immunoprecipitation assays
tin immunoprecipitation (ChIP) assays were performed
d previously [8] with minor modifications. Briefly,
ultures grown in minimal media were pelleted, washed
water and grown in YPD to OD600 0.3–0.4 at 20 ◦C. -
added to the final concentration of 15g/ml and the
grown for 1 h at room temperature and then shifted to
min. Cells were pelleted and washed once with sterile
wice with pre-warmed media. Cells were resuspended
Dandgrownat37 ◦C. Sampleswere collectedat30 min,
time points and cross-linked with 1% formaldehyde for
0 ◦C. Cross-linked chromatin was sonicated (Diagenode