Chemical Genetics Reveals a Specific Requirement for Cdk2 Activity in the DNA Damage Response and Identifies Nbs1 as a Cdk2 Substrate in Human Cells

Article (PDF Available)inPLoS Genetics 8(8):e1002935 · August 2012with42 Reads
DOI: 10.1371/journal.pgen.1002935 · Source: PubMed
The cyclin-dependent kinases (CDKs) that promote cell-cycle progression are targets for negative regulation by signals from damaged or unreplicated DNA, but also play active roles in response to DNA lesions. The requirement for activity in the face of DNA damage implies that there are mechanisms to insulate certain CDKs from checkpoint inhibition. It remains difficult, however, to assign precise functions to specific CDKs in protecting genomic integrity. In mammals, Cdk2 is active throughout S and G2 phases, but Cdk2 protein is dispensable for survival, owing to compensation by other CDKs. That plasticity obscured a requirement for Cdk2 activity in proliferation of human cells, which we uncovered by replacement of wild-type Cdk2 with a mutant version sensitized to inhibition by bulky adenine analogs. Here we show that transient, selective inhibition of analog-sensitive (AS) Cdk2 after exposure to ionizing radiation (IR) enhances cell-killing. In extracts supplemented with an ATP analog used preferentially by AS kinases, Cdk2(as) phosphorylated the Nijmegen Breakage Syndrome gene product Nbs1-a component of the conserved Mre11-Rad50-Nbs1 complex required for normal DNA damage repair and checkpoint signaling-dependent on a consensus CDK recognition site at Ser432. In vivo, selective inhibition of Cdk2 delayed and diminished Nbs1-Ser432 phosphorylation during S phase, and mutation of Ser432 to Ala or Asp increased IR-sensitivity. Therefore, by chemical genetics, we uncovered both a non-redundant requirement for Cdk2 activity in response to DNA damage and a specific target of Cdk2 within the DNA repair machinery.
Chemical Genetics Reveals a Specific Requirement for
Cdk2 Activity in the DNA Damage Response and
Identifies Nbs1 as a Cdk2 Substrate in Human Cells
Lara Wohlbold
, Karl A. Merrick
, Saurav De
, Ramon Amat
, Jun Hyun Kim
, Ste
phane Larochelle
Jasmina J. Allen
, Chao Zhang
, Kevan M. Shokat
, John H. J. Petrini
, Robert P. Fisher
1 Department of Structural and Chemical Biology, Mount Sinai School of Medicine, New York, New York, United States of America, 2 Program in Biochemistry and Program
in Cell and Molecular Biology, Weill Cornell Graduate School of Medical Sciences, New York, New York, United States of America, 3 Molecular Biology Program, Memorial
Sloan-Kettering Cancer Center, New York, New York, United States of America, 4 Department of Cellular and Molecular Pharmacology, University of California San
Francisco, San Francisco, California, United States of America
The cyclin-dependent kinases (CDKs) that promote cell-cycle progression are targets for negative regulation by signals from
damaged or unreplicated DNA, but also play active roles in response to DNA lesions. The requirement for activity in the face
of DNA damage implies that there are mechanisms to insulate certain CDKs from checkpoint inhibition. It remains difficult,
however, to assign precise functions to specific CDKs in protecting genomic integrity. In mammals, Cdk2 is active
throughout S and G2 phases, but Cdk2 protein is dispensable for survival, owing to compensation by other CDKs. That
plasticity obscured a requirement for Cdk2 activity in proliferation of human cells, which we uncovered by replacement of
wild-type Cdk2 with a mutant version sensitized to inhibition by bulky adenine analogs. Here we show that transient,
selective inhibition of analog-sensitive (AS) Cdk2 after exposure to ionizing radiation (IR) enhances cell-killing. In extracts
supplemented with an ATP analog used preferentially by AS kinases, Cdk2
phosphorylated the Nijmegen Breakage
Syndrome gene product Nbs1—a component of the conserved Mre11-Rad50-Nbs1 complex required for normal DNA
damage repair and checkpoint signaling—dependent on a consensus CDK recognition site at Ser432. In vivo, selective
inhibition of Cdk2 delayed and diminished Nbs1-Ser432 phosphorylation during S phase, and mutation of Ser432 to Ala or
Asp increased IR–sensitivity. Therefore, by chemical genetics, we uncovered both a non-redundant requirement for Cdk2
activity in response to DNA damage and a specific target of Cdk2 within the DNA repair machinery.
Citation: Wohlbold L, Merrick KA, De S, Amat R, Kim JH, et al. (2012) Chemical Genetics Reveals a Specific Requirement for Cdk2 Activity in the DNA Damage
Response and Identifies Nbs1 as a Cdk2 Substrate in Human Cells. PLoS Genet 8(8): e1002935. doi:10.1371/journal.pgen.1002935
Editor: Nancy Maizels, University of Washington, United States of America
Received February 15, 2012; Accepted July 17, 2012; Published August 23, 2012
Copyright: ß 2012 Wohlbold et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: The work was supported by a fellowship of the Deutsche Forschungsgemeinschaft to LW (WO1456) and by NIH grant GM056985 to RPF. The funders
had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail:
In eukaryote s, responses to DNA damage or replication errors
must be coordinated with cell division. For example, checkpoint
pathways signal the presence of DNA lesions to the cell-cycl e
machinery, leading to reversible arrest or apoptosis. In reciprocal
fashion, the cyclin-dependent kinases (CDKs) that regulate cell-
cycle progr ession also appear to control aspe cts of the DNA
damage r esponse. For example, CDK activ ity promotes repair of
DNA double-strand breaks (DSBs) by homolog ous recombination
(HR) in yeast [1–3]. In Saccharomyces cerevisiae, CDK phosph ory-
lates th e conserved repair protein Sae2 to prom ote DNA-end
resection and thereby channel DSBs into the HR pathway [4]. In
metaz oans, CDKs have been shown to phosphorylate DNA
repai r and checkpoint proteins (reviewed in [5,6]), but the
consequences of most of those phosphorylations rem ain unclear.
One exception is the Sae2 homolog CtIP, the phosphorylation of
which appears to facilitate resection, and might couple DSB
repai r pathway ch oice to cell-cycle position in mammalian cells
It is also uncertain which CDK/cyclin complexes regulate
responses to DNA damage in metazoans. In yeast, a single CDK
catalytic subunit triggers entry to both S phase and mitosis,
whereas metazoans normally rely on multiple CDKs [9]. The
latter arrangement suggests a potential solution to the problem of
maintaining some CDK activity in the face of inhibitory
checkpoint signals: specialization of individual CDKs to evade
those signals. In mammalian cells, Cdk2 is the nearly exclusive
partner of cyclin E, which is expressed near the G1/S boundary,
and the preferred partner of cyclin A early in S phase. Later in S
phase, cyclin A begins binding Cdk1 [10], to trigger initiation from
late-replicating origins [11] and attenuate S phase-specific gene
expression [12]. Finally, Cdk1 assembles with cyclin B during S
and G2 phases, and is activated late in G2 to promote mitosis.
Despite the temporal restriction and apparent functional special-
ization of CDKs in mammalian cells, discerning non-redundant
functions of specific catalytic subunits has been difficult. Cells
lacking Cdk2 can divide more or less normally, and Cdk2
are viable, but infertile due to a defect in meiosis [13,14].
Moreover, cells lacking all interphase-specific CDKs can prolifer-
PLOS Genetics | 1 August 2012 | Volume 8 | Issue 8 | e1002935
ate, albeit more slowly than wild-type cells, by substituting Cdk1
for the ‘‘correct’’ partners in complexes with cyclins D, E and A
Because of that plasticity, eliminating or reducing expression of
individual CDKs by gene disruption or RNA interference (RNAi)
may not reveal which functions those CDKs perform, perhaps
exclusively, when they are present. For example, Cdk2 is likely to
control the onset of DNA replication, based on its activation
timing [10,11] and the lack of a Cdk1 requirement for S-phase
entry when Cdk2 is present [16]. By the same logic, Cdk2 might
take a lead role in influencing the choice of DSB repair pathway
early in S phase. Consistent with that idea, loss or depletion of
Cdk2 was reported to increase radiation-sensitivity and cause
defects in DNA damage repair and checkpoint signaling [17–19].
It was later suggested, however, that the requirement for CDK
activity in response to DNA damage is a general one, with no
specific need for Cdk2 either to repair damage or to resume the
cell cycle after repair is complete [20]. More recently, an exclusive
function was ascribed to Cdk2 in imposing a G2/M checkpoint
arrest in cells lacking wild-type function of the tumor suppressor
p53 [21].
Precise temporal control over the activity of individual CDKs,
which was needed to uncover the Cdk1 requirement in yeast DSB
repair [1,3], has not heretofore been possible in metazoans; none
of the available small-molecule inhibitors can discriminate
between Cdk1 and Cdk2, and depletion of specific CDKs allows
ectopic CDK-cyclin pairs to take over functions normally
performed by the missing enzyme [15,22,23]. To dissect the
precise roles of different CDKs in human cells, we took a
chemical-genetic approach, in which a bulky amino acid residue in
the active site—the gatekeeper—is mutated to Gly, creating extra
space to accommodate bulky adenine analogs [24]. Analog-
sensitive (AS) Cdk2 is susceptible to inhibition by non-hydrolyz-
able analogs that bind poorly to wild-type kinases, and able to use
bulky ATP analogs (as well as natural ATP) as substrates [25,26].
By a gene targeting strategy we applied previously to human Cdk7
[27], we replaced both wild-type copies of Cdk2 with Cdk2
human cells, and uncovered a requirement for Cdk2 activity in cell
proliferation, which was missed by gene knockout- and RNAi-
based studies [28].
Requirements for Cdk2 activity in the DNA damage response
are also likely to have escaped detection. Here we show that
transient treatment with an allele-specific inhibitor decreased
survival of Cdk2
but not Cdk2
cells after exposure to ionizing
radiation (IR), indicating a specific requirement for Cdk2 activity
in orchestrating an effective DNA damage response. In whole-cell
extracts, Cdk2
labeled Nbs1, product of the gene mutated in the
autosomal recessive Nijmegen Breakage Syndrome (NBS) of
microcephaly, immunodeficiency, and increased incidence of
hematopoietic malignancy (reviewed in [29]). Nbs1 is part of the
essential Mre11-Rad50-Nbs1 complex, which functions in recog-
nition and repair of DSBs (reviewed in [30]). We mapped the
Cdk2-mediated phosphorylation of Nbs1 to Ser432. In vivo, that
phosphorylation occurs during S phase after the Mre11 complex is
recruited to chromatin, and is prevented by general CDK
blockade or delayed and diminished by specific inhibition of
Cdk2. Mutations of Nbs1-Ser432 that prevent phosphorylation
increase sensitivity to cell-killing by IR, and thus phenocopy
selective inhibition of Cdk2. Therefore, by chemical genetics we
have uncovered a specifically Cdk2-dependent pathway within the
DNA damage response of human cells.
Cdk2 activity is requi red for normal IR–resista nce
As previously reported [28], the Phe80-to-Gly gatekeeper
mutation, which rendered Cdk2 AS, also impaired binding to
cyclin A in vivo, but this defect was corrected by treating cells with
bulky adenine analogs. When normal CDK-cyclin pairing was
thus maintained, sustained treatment with 1-(tert-Butyl)-3-(3-
methylbenzyl)-1H-pyrazolo[3,4-d]pyrimidin-4 amine (3-MB-
PP1)—a selective inhibitor of AS, but not wild-type, CDKs—
impaired proliferation of asynchronously growing Cdk2
Acute inactivation of Cdk2, in synchronized populations of
untransformed, human telomerase-expressing retinal pigment
epithelial (RPE-hTERT) cells, impeded passage through the G1
restriction point (when continued cell-cycle progression becomes
independent of mitogen stimulation [31]) and entry into S phase,
and increased the frequency of arrest in the G0 or G1 phase of the
cell cycle [28].
The effects of Cdk2 inhibition on cell proliferation were
reversible; a 48-hr treatment of asynchronous Cdk2
hTERT cells with 10
mM 3-MB-PP1, followed by removal of the
drug, allowed colony formation with no loss of efficiency
(Figure 1A). We were therefore able to test the effect of transient
Cdk2 inhibition on survival after IR-induced DNA damage.
or wild-type cells were pre-treated with DMSO or 3-MB-
PP1 for 24 hr; c-irradiated with 2 or 4 Gy, re-plated and
incubated for an additional 24 hr in the same condition as the
pre-treatment; then washed and returned to fresh, drug-free
medium for 14 d before colonies were counted. Even in the
absence of the drug, the mutant had increased sensitivity to IR
compared to wild-type cells (Figure 1B), suggesting that Cdk2
hypomorphic. This could be due to the cyclin-binding defect and
incomplete compensation by other CDKs, which would be
consistent with results in Cdk2
cells [19]. Transient treatment
with 10
mM 3-MB-PP1 further decreased survival by Cdk2
,10-fold, but did not affect sensitivity of wild-type cells, indicating
that Cdk2 activity is specifically required for resistance to DNA
damage caused by IR.
In the preceding experiment, Cdk2 inhibition was implemented
24 hr before, and maintained for 24 hr after, exposure to IR.
Author Summary
Multiple cyclin-dependent kinases (CDKs) control human
cell proliferation, but it remains unclear how functions of
different CDKs are coordinated during unperturbed cell
division or after dividing cells incur DNA damage. DNA
lesions activate checkpoint signaling pathways to inhibit
CDK activity, arrest the cell division cycle, and thus prevent
loss of genetic information; but an effective response to
damage also requires CDK activity to modify components
of repair and checkpoint pathways. We took a chemical-
genetic approach to ask if a specific CDK, Cdk2, played a
specialized, non-redundant role in protecting genomic
integrity of human cells. By sensitizing Cdk2 to chemical
inhibition, we were able to detect a specific requirement
for its catalytic activity in survival of cells after exposure to
ionizing radiation (IR). We identified Nbs1, product of the
gene mutated in the cancer-predisposing Nijmegen
Breakage Syndrome, as a Cdk2 substrate and showed that
mutant forms of Nbs1 that cannot be modified by Cdk2
are defective in protecting cells from death due to IR–
induced DNA damage. Therefore, our work defines a DNA
damage response pathway that depends on catalytic
activity of a specific CDK in human cells and suggests a
mechanism to promote efficient repair without triggering
inappropriate cell division.
Cdk2 Phosphorylates Nbs1 to Promote IR Resistance
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Enhanced cell-killing by this regimen could indicate a requirement
for Cdk2 to phosphorylate repair or checkpoint effectors, or some
cell-cycle derangement occurring during the pre-treatment period.
To distinguish between these mechanisms, we varied the relative
timing of Cdk2 inhibition and irradiation. Cdk2
cells treated
with 3-MB-PP1 for 24 hr prior to irradiation and return to drug-
free medium were barely sensitized to IR, compared to mock-
treated cells (Figure 1C). In contrast, a 24-hr 3-MB-PP1 treatment
initiated at the time of irradiation increased IR-sensitivity, relative
to mock treatment, to nearly the same extent as did the 48-hr,
‘‘before-and-after’’ exposure (Figure 1D). Therefore, Cdk2 cata-
lytic activity is specifically required after DNA damage occurs to
promote survival of human cells exposed to IR.
Cdk2 phosphorylates Nbs1
To identify targets through which Cdk2 executes its functions in
DNA damage responses, we labeled proteins in whole-cell extracts
of wild-type RPE-hTERT and HCT116 human colon carcinoma
cells with purified Cdk2
/cyclin A and the analog substrate
P]N6-(benzyl)-ATP [32,33]. Proliferation of both cell types is
sensitive to selective inhibition of Cdk2 [28]. RPE-hTERT cells
are largely intact in their responses to DNA damage [34],
however, whereas HCT116 cells are defective in mismatch repair
and can be deficient in Mre11-complex function [35]. Although
overall labeling patterns were similar, several bands were more
heavily phosphorylated in RPE-hTERT, as opposed to HCT116,
extracts—including prominent signals at .100, ,80 and
,35 kDa (Figure 2A). We observed similar, cell type-specific
labeling in extracts of the two lines expressing Cdk2
nously [28]. There was no labeling, however, when wild-type
Cdk2 complexes were substituted for Cdk2
, indicating that all
visible signals represent direct targets of Cdk2 activity.
To test if Cdk2
phosphorylated the Mre11 complex, we
subjected labeled extracts to anti-Mre11 immunoprecipitation
(Figure 2B). Although we did not recover the ,80 kDa species
that approximately matches Mre11 in electrophoretic mobility
(and remains unidentified), a labeled polypeptide the size of Nbs1
(,95 kDa) co-precipitated with Mre11. A labeled band of similar
mobility appeared in anti-Nbs1 immunoprecipitates (Figure 2C),
confirming that Cdk2
selectively phosphorylates Nbs1 in crude
extracts. Labeling intensity correlated with Nbs1 abundance in
different cell types (Figure S1).
Human Nbs1 contains one match, at Ser432, to the consensus
CDK recognition motif S/T-P-X-K/R (where X is any residue).
This site is conserved in metazoans, and both fission yeast Nbs1
and Xrs2—the corresponding subunit of the budding yeast Mre11
complex—contain potential CDK phosphorylation sites [36–38].
To map phosphorylation(s) by Cdk2 in human Nbs1, we
transcribed and translated cDNAs encoding the wild-type protein
(WT) and mutants with Ser432 changed to Ala (S432A) or Asp
(S432D) in reticulocyte lysates, which were then labeled with
/cyclin A and [c-
P]N6-(benzyl)-ATP. Whereas all three
forms of Nbs1 were translated efficiently, only the wild-type
version was labeled (Figure 2D). Next, to ask if Cdk2 phosphor-
ylated Nbs1 in the Mre11 complex with the same dependence on
Ser432, we performed labeling in extracts of NBS-T cells—
transformed fibroblasts derived from a patient with NBS, which do
not express full-length Nbs1—or the same cells expressing full-
Figure 1. Cdk2 activity is required for normal DNA damage resistance. (A) Transient treatment with 10 mM 3-MB-PP1 for 48 hr does not
affect efficiency of colony formation by wild-type or Cdk2
RPE-hTERT cells. (B) Treatment with 10 mM 3-MB-PP1 before and after irradiation (24 hr
each) sensitizes Cdk2
RPE-hTERT cells to killing by IR. (C) Pre-treatment with 3-MB-PP1 for 24 hr does not sensitize Cdk2
cells to IR. (D)
Treatment with 3-MB-PP1 for 24 hr after IR sensitizes Cdk2
cells to killing. Error bars are +/2 standard deviation (SD) of duplicates.
Cdk2 Phosphorylates Nbs1 to Promote IR Resistance
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Figure 2. Nbs1 is phosphorylated by Cdk2 on Ser432 in human whole-cell extracts. (A) Labeling by Cdk2
/or Cdk2
/cyclin A, as
indicated, with [c-
P]N6-(benzyl)-ATP in RPE-hTERT or HCT116 whole cell extracts. (B) Anti-Mre11 immunoprecipitates from labeling reactions
analyzed by autoradiography (top) and anti-Nbs1 and -Mre11 immunoblot (bottom). Arrow indicates band at position of Nbs1, which did not appear
in mock immunoprecipitates lacking antibody (‘‘control’’). (C) Anti-Nbs1 immunoprecipitates from labeling reactions analyzed by autoradiography
(top) and anti-Nbs1 immunoblot (bottom). (D) Reticulocyte lysates programmed with indicated cDNAs were labeled by Cdk2
. Labeling in extract
(left) and anti-Nbs1 immunoprecipitates (right) was detected by autoradiography, and expression of Nbs1 isoforms confirmed by immunoblot
Cdk2 Phosphorylates Nbs1 to Promote IR Resistance
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length Nbs1 after transient transfection. Wild-type, S432A and
S432D Nbs1 variants were expressed at equal levels and recovered
with similar efficiency in anti-Mre11 immunoprecipitates. Only
the wild-type protein was labeled, however (Figure 2E), confirming
that an intact Ser432 residue is required for Cdk2
phosphorylate Nbs1 within the Mre11 complex.
Cell-cycle CDKs phosphorylate Nbs1-Ser432 in vitro and
in vivo
To ask which human CDK/cyclin pair(s) could phosphorylate
Nbs1, we incubated a fragment of human Nbs1 fused to
glutathione-S-transferase [GST-Nbs1(397-742)] with various puri-
fied CDKs (Figure 3A). Cdk2/cyclin A, Cdk1/cyclin A and Cdk1/
cyclin B phosphorylated GST-Nbs1(397-742) with similar efficien-
cies relative to a known, common substrate, histone H1. In
contrast, neither Cdk7 nor Cdk9 could label Nbs1 above
background levels, although both were active towards their known
substrates. We conclude that Nbs1 is a substrate of the cell-cycle
effectors Cdk1 and Cdk2.
Antibodies raised against a phospho-Ser432-containing peptide
recognized GST-Nbs1(397-742) purified from bacteria only after it
was incubated with Cdk2/cyclin A, Mg
and ATP (Figure 3B).
We detected no signal in the Cdk2/cyclin A preparation or when
GST-Nbs1(397-742) was incubated in the absence of CDK,
confirming specificity of the antibody for phosphorylated Nbs1.
The anti-phospho-Ser432 antibody recognized wild-type Nbs1,
but not the S432A or S432D variant, expressed in NBS-T cells
(bottom). (E) NBS-T cells were transiently transfected with empty vector or ones encoding Myc-tagged Nbs1 variants, as indicated. Labeling by Cdk2
was detected by autoradiography of extract (left) and anti-Mre11 immunoprecipitates (right), and equal expression and recovery of Nbs1 isoforms
were confirmed by immunoblot (bottom). Nbs1+ denotes control cells expressing full-length Nbs1 endogenously.
Figure 3. CDKs phosphorylate Nbs1 on Ser432 in vitro and in vivo. (A) Purified recombinant Nbs1 fragment (GST-Nbs1(397-742)) was
incubated in vitro with indicated CDK/cyclin complexes and [c-
P]-ATP. Histone H1, monomeric Cdk2, and the carboxyl-terminal domain (CTD) of
RNA polymerase II, served as control substrates for Cdk1 or -2, Cdk7, and Cdk9, respectively. (B) Incubation of GST-Nbs1(397-742) in vitro with Cdk2/
cyclin A, Mg
and ATP generates an epitope recognized by the anti-Nbs1-S432-P antibody in immunoblots. (C) Wild-type but not Ser432-mutant
Nbs1, transiently expressed in NBS-T cells, is recognized by anti-Nbs1-S432-P antibody. (D) Roscovitine and/or purvalanol A treatment of HCT116 cells
for 15 hr diminishes Nbs1-Ser432 phosphorylation without affecting Nbs1 levels, whereas DRB has no effect. (E) Treatment of Cdk7
but not wild-
type HCT116 cells with 2
mM 3-MB-PP1 for 24 hr decreases phosphorylation of Cdk1 (P-T161), Cdk2 (P-T160), and Nbs1-Ser432.
Cdk2 Phosphorylates Nbs1 to Promote IR Resistance
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(Figure 3C), indicating that Nbs1-Ser432 is phosphorylated in
To ask if CDKs are responsible for Nbs1 phosphorylation in
vivo, we treated HCT116 cells with kinase inhibitors. Roscovitine
and purvalanol A, which inhibit multiple CDKs, including Cdk1
and Cdk2 [39,40], diminished phospho-Ser432 without affecting
total Nbs1 levels, whereas 5,6-dichloro-1-b-D-ribofuranosyl-benz-
imidazole (DRB), a transcriptional poison thought to work through
inhibition of Cdk9 [41], had no effect (Figure 3D). Because none of
these compounds is specific for a single class of kinase, we asked if
selective inhibition of Cdk7—the upstream activator of both Cdk1
and Cdk2 [27]—prevented Nbs1 phosphorylation. Treatment
with 3-MB-PP1 diminished activating phosphorylation of Cdk1
and Cdk2 in Cdk7
but not wild-type HCT116 cells, and also
decreased Nbs1-Ser432 phosphorylation (Figure 3E). Cdk7 is
incapable of modifying Nbs1 directly (Figure 3A); a dependence
on Cdk7 activity therefore suggests that Nbs1-Ser432 is a target of
a CDK (or CDKs) downstream of Cdk7, such as Cdk1 or Cdk2.
Phosphorylation of Nbs1-Ser432 is cell-cycle regulated
If Nbs1-Ser432 were a target of Cdk1 and/or Cdk2 in vivo, we
would expect its phosphorylation state to fluctuate with cell-cycle
position. We tested this prediction in RPE-hTERT cells, which
arrest in a quiescent, G0-like state upon growth to confluence, and
re-enter the cell cycle synchronously upon re-plating at lower
density. Nbs1-Ser432 phosphorylation was low in G0 and
increased 20–25 hr after release, concomitant with increases in
cyclin A accumulation and activating phosphorylation of Cdk2
(Figure 4A, right), and the appearance of a cell population with
.2N DNA content (Figure 4A, left).
The Mre11 complex is recruited to chromatin throughout [42],
and required for successful completion of, S phase [43,44]. To ask
if Ser432 phosphorylation correlated with relocalization of Nbs1 to
chromatin, we separated extracts of RPE-hTERT cells, harvested
at different times after release from G0, into soluble and
chromatin-bound fractions. Nbs1 was predominantly associated
with chromatin by 10 hr after release, and Ser432 phosphoryla-
tion of chromatin-bound Nbs1 increased between 10 and 20 hr
(Figure 4B). Treating cells with roscovitine, either at the time of re-
plating or 16 hr later, prevented increases in cellular DNA content
(Figure 4C, left) and Nbs1 phosphorylation (Figure 4C, right,
compare lanes 2, 5 and 8). In contrast, addition of aphidicolin
blocked DNA replication, but not Cdk2 activation or Nbs1
phosphorylation (Figure 4C, lanes 3 and 6). We conclude that
Nbs1 is normally phosphorylated by CDKs after recruitment of
the Mre11 complex to chromatin in S phase, but that this
phosphorylation does not require ongoing DNA synthesis.
Chromatin association of Nbs1, moreover, does not depend on
Ser432 phosphorylation: in subcellular fractionation experiments,
wild-type, S432A and S432D forms of Nbs1 had indistinguishable
sensitivity to salt extraction from pelleted chromatin (Figure S2).
The timing of Nbs1-Ser432 phosphorylation coincided with the
activation of Cdk2 (Figure 4A), but available inhibitors are
incapable of distinguishing Cdk2- from Cdk1-dependent phos-
phorylations in wild-type cells. The Cdk2
cells [28] allowed us
to ask if Nbs1-Ser432 is a preferred target of Cdk2 in vivo. We
released Cdk2
or wild-type RPE-hTERT cells from G0, in the
presence of either the allele-specific inhibitor 3-MB-PP1; or 6-
benzylaminopurine (6-BAP), an adenine analog that corrects a
cyclin-binding defect of Cdk2
but does not inhibit its activity, and
thereby rescues delayed G1 progression due to the hypomorphic
mutation [28]. Neither drug affected Nbs1 phosphorylation
in wild-type cells (Figure 4D and data not shown). In Cdk2
treated with 6-BAP or in wild-type cells treated with either drug,
Nbs1-Ser432 phosphorylation appeared by 16 hr after release and
increased over the next 8 hr. In contrast, 3-MB-PP1 treatment of
cells diminished Nbs1 phosphorylation levels, relative to
those in 6-BAP-treated cells (Figure 4D). Even in the presence of 3-
MB-PP1, however, a phospho-Ser432 signal appeared in Cdk2
cells between 20 and 24 hr, suggesting that another kinase could
phosphorylate this site in vivo, albeit with delayed kinetics.
Therefore, Nbs1-Ser432 is phosphorylated preferentially by Cdk2,
but might also be a target for another CDK, such as Cdk1, which
is activated later in S phase [10,11].
Nbs1-Ser432 is required for normal resistance to IR
We next investigated possible requirements for Nbs1-Ser432
phosphorylation in vivo in NBS-T cells, which have impaired
checkpoint signaling and reduced ability to repair DSBs by HR,
leading to increased frequency of chromosomal aberrations and
sensitivity to DNA-damaging agents (reviewed in [30]). These cells
express the Nbs1
allele found in the majority of NBS patients,
which encodes a 26 kDa amino-terminal fragment and a 70 kDa
protein (p70) lacking the correct amino-terminus. The larger
fragment retains Mre11-binding ability (and an intact Ser432
residue), and is likely to fulfill essential functions of Nbs1 in
patients with NBS [45]. Depletion of residual Nbs1 by RNAi
resulted in loss of colony forming ability (Figure 5A), indicating
that p70 provides essential functions in NBS-T cells as well.
Because the Nbs1 fragments are difficult to detect with available
anti-Nbs1 antibodies [45], knockdown was verified by measuring
RNA levels (Figure S3A). Loss of viability was due to depletion of
Nbs1 gene products rather than off-target effects, because it could
be corrected by stable expression of RNAi-resistant, wild-type
Nbs1 (Figure 5A). The S432A and S432D versions were likewise
capable of rescue, indicating that Ser432 phosphorylation is not
required for survival. All three forms were expressed at equal
levels, and co-immunoprecipitation of Mre11 and Rad50 revealed
no effect of the mutations on complex formation (Figure 5B).
When irradiated in G2, NBS-T cells are defective in activating
the G2/M checkpoint to restrain mitosis in the presence of DNA
damage [46]. All three Nbs1 variants complemented this defect to
similar extents; the fraction of cells entering mitosis after exposure
to IR was reduced (Figure S3B), and phosphorylation of the
checkpoint kinase Chk2 was restored (Figure S3C). In fact, we
observed low levels of Chk2 phosphorylation even in the absence
of IR exposure in cells expressing Ser432 mutant forms of Nbs1,
possibly suggesting accumulation of DNA damage under normal
growth conditions. Nonetheless, we conclude that phosphorylation
of Nbs1-Ser432 is not required for normal G2/M checkpoint
function. Furthermore, Nbs1-Ser432 phosphorylation was not
itself affected by DNA damage (Figure S3C).
We next asked if Nbs1-Ser432 was required for efficient
recombinational repair, measured as the frequency of gene
conversion of a split green fluorescent protein (GFP) reporter
after introduction of a DSB by the endonuclease I-SceI [47]. NBS-
T cells had low HR frequencies that were further reduced when
endogenous Nbs1 fragments were depleted by RNAi (Figure S3D).
Transient expression of RNAi-resistant Nbs1
, Nbs1
increased gene-conversion frequencies by similar
amounts, indicating that phosphorylation of Nbs1-Ser432 is
dispensable for HR. We also tested whether Nbs1-Ser432
phosphorylation was required for a specific function of the
Mre11 complex in HR: resection from sites of DSBs to generate
single-strand DNA (ssDNA) overhangs for strand invasion. We
exposed NBS-T cells expressing no exogenous Nbs1, Nbs1
or Nbs1
to 4 Gy of IR and measured the number
of cells positive for foci of the ssDNA-binding protein replication
Cdk2 Phosphorylates Nbs1 to Promote IR Resistance
PLOS Genetics | 6 August 2012 | Volume 8 | Issue 8 | e1002935
protein A (RPA) 1 and 6 hr later. There was no significant
difference in RPA focus formation among the four genotypes
(Figure S4), indicating that neither Nbs1-Ser432 nor full-length
Nbs1 was required at the end-resection step. This also indicates
that Nbs1 phosphorylation by CDKs is not required for
recruitment of the Mre11 complex to damage foci, just as it did
not appear to be needed for localization to bulk chromatin (Figure
S2). We also measured Nbs1 focus formation directly and observed
Figure 4. Ser432 is phosphorylated in S phase after Nbs1 recruitment to chromatin. (A) Time course of release from contact inhibition (G0)
of RPE-hTERT cells, monitored by flow cytometry for DNA content and immunoblotting for accumulation of cyclin A, activated (Thr160-
phosphorylated) Cdk2 and Ser432-phosphorylated Nbs1. (B) RPE-hTERT cells were harvested and fractionated at indicated times after release from
G0. Recruitment of Nbs1 to chromatin precedes phosphorylation on Ser432. (C) RPE-hTERT cells treated with DMSO, 4
mg/ml aphidicolin or 20 mM
roscovitine at indicated times after release from G0 were monitored for DNA content (left) and accumulation of cyclin A, activated Cdk2 and
phosphorylated Nbs1 (right). Asterisk denotes anti-phospho-Ser432 cross-reactive ,110 kDa band that is absent in G0 and roscovitine-sensitive, but
unlikely to be an Nbs1 gene product, because it is present in NBS-T cells (Figure S2B) and absent in Nbs1 immunoprecipitates (Figure 3D, 3E). (D)
or wild-type RPE-hTERT cells were released from G0 for indicated times in the presence of 0.5 mM 6-BAP or 10 mM 3-MB-PP1, as indicated,
and tested for total (top) and Ser432-phosphorylated Nbs1 (bottom).
Cdk2 Phosphorylates Nbs1 to Promote IR Resistance
PLOS Genetics | 7 August 2012 | Volume 8 | Issue 8 | e1002935
no difference between cells expressing wild-type or S432A alleles
of Nbs1 after exposure to 10 Gy IR (data not shown). Consistent
with resection being normal in the absence of Nbs1-Ser432
phosphorylation, neither Nbs1-Ser432 substitution mutations
(Figure S5A) nor inhibition of Cdk2
in RPE-hTERT cells
(Figure S5B) affected levels of CtIP, which promotes resection by
interacting with the Mre11 complex [8], and which in turn is
stabilized by its association with Mre11 [48].
Likewise, Nbs1-Ser432 phosphorylation does not seem to be
required for the normal response to replication stress; Ser432
mutant Nbs1 alleles fully complemented the hypersensitivity of
NBS-T cells to chronic hydroxyurea (HU) exposure (Figure S6). In
contrast, NBS-T cells stably complemented with S432A or S432D
alleles of Nbs1 were hypersensitive to killing by IR, compared to
those complemented with wild-type Nbs1, although the parental
NBS-T cells were more sensitive still (Figure 5C). We performed
the same measurement after depleting residual Nbs1 by RNAi. (As
expected because Nbs1 is essential, after knockdown we recovered
too few colonies of non-complemented NBS-T cells for reliable
counting.) Both S432A- and S432D-expressing cells were more
sensitive to killing by IR than were those expressing wild-type
Nbs1 (Figure 5D). This suggests that phosphorylation of Nbs1-
Ser432 contributes to the radioprotective effects of Cdk2 in human
cells. Therefore, chemical genetics uncovered both a specific
requirement for the activity of human Cdk2 in the response to
DNA damage, and a specific target of Cdk2 within the DNA
repair machinery.
The cell-cycle machinery signals to the Mre11 complex
We have directly implicated a specific CDK-substrate interac-
tion in the DNA damage response of human cells. By a chemical-
genetic approach we identified Nbs1 as a target of Cdk2, and
mapped the phosphorylation to a conserved CDK consensus
recognition site. Both Cdk1 and Cdk2 phosphorylated Nbs1 with
similar efficiency in vitro, and drugs that inhibit both CDKs—
either directly (roscovitine or purvalanol A) or indirectly (3-MB-
PP1 in Cdk7
cells)—diminished Nbs1-Ser432 phosphorylation
in vivo more effectively than did inhibition of Cdk2 alone. These
results are consistent with both Cdk2 and Cdk1 acting on Nbs1, in
the temporal order in which they become activated during S phase
[10,11]. Nonetheless, Nbs1-Ser432 phosphorylation was delayed
and diminished by selective Cdk2 inhibition during synchronous
Figure 5. Nbs1-Ser432 is required for normal X-ray resistance but not viability. (A) Cells were treated with Nbs1 or control siRNA and
transiently transfected with empty vector or vectors encoding wild-type, S432A or S432D Nbs1 variants, as indicated, and tested for colony-forming
ability. Error bars indicate + /2 SD of duplicate measurements. (B) Nbs1 was immunoprecipitated from extracts of NBS-T cells, untransfected or stably
expressing wild-type, S432A or S432D Nbs1, and probed for Nbs1, Rad50 and Mre11. (C) IR sensitivity of NBS-T cells expressing indicated Nbs1
isoforms. Cells were irradiated at indicated doses and tested for colony formation 14 d after irradiation. (D) IR sensitivity of cells from (C), transiently
transfected with siRNA targeting Nbs1. (The parental NBS-T cells that express no full-length Nbs1 do not survive this treatment.) Values represent the
means of duplicates +/2 SD.
Cdk2 Phosphorylates Nbs1 to Promote IR Resistance
PLOS Genetics | 8 August 2012 | Volume 8 | Issue 8 | e1002935
exit from G0, indicating a non-redundant function of Cdk2 in
initiating Nbs1 phosphorylation. A preferential interaction be-
tween Cdk2 and the Mre11 complex is also suggested by the
recent report that Cdk2/cyclin A, but not Cdk1, binds directly to a
carboxyl-terminal motif in Mre11 [48].
Mre11, Rad50 and Nbs1 are essential, and participate in
multiple pathways to protect genome integrity in mammalian cells
(reviewed in [30,49]). By depletion of endogenous Nbs1 fragments
in NBS-T cells and complementation with mutant Nbs1 alleles, we
have shown here that: 1) the fragments can support essential and
HR-related functions of the Mre11 complex in human somatic
cells; and 2) Nbs1 phosphorylation by CDKs is not essential for
viability, gene conversion or resistance to replicative stress, but
contributes to normal IR-resistance. Because inhibition of Cdk2
also increased IR-sensitivity, the data suggest that Nbs1 functions
downstream of Cdk2 in a DNA damage response pathway.
The Mre11 complex is recruited to chromatin during S phase
[42], whereupon CDKs phosphorylate Nbs1 (this report), possibly
to regulate Mre11-complex functions in response to DNA lesions
incurred in replication [50]. Mutation of Ser432 to either Ala or
Asp had similar effects on IR-sensitivity, suggesting that dephos-
phorylation of Nbs1 might also be important in vivo (or that Asp
did not mimic phospho-Ser). Nbs1 is also recruited to human
telomeres during late S or early G2 phase [51,52], suggesting
another, possibly cell cycle-regulated function of the Mre11
complex; we were unable, however, to detect consistent effects of
Nbs1 Ser432 mutations on telomere length in complemented NBS-
T cells (unpublished observations).
A specific requirement for Cdk2 in the DNA damage
Taken together, our results support a role for Cdk2-mediated
phosphorylation of Nbs1 in protecting genomic integrity. CDKs
phosphorylate other proteins that work in concert with Nbs1. For
example, the HR factor CtIP is phosphorylated during S/G2 and
interacts directly with Nbs1 to promote repair focus formation in
human cells [53]. Interaction of CtIP with BRCA1, another repair
protein, depends on CtIP-Ser327 phosphorylation by a CDK [54];
and mutation of CtIP-Thr847, a second putative site of CDK
phosphorylation, increases sensitivity to camptothecin [4]. Out-
standing questions include: 1) Which aspects of Mre11-complex
function depend on CDK activity? 2) Do Nbs1-Ser432 mutations
exacerbate phenotypes due to loss of other CDK-mediated
phosphorylations, e.g. on CtIP or BRCA2 [55]? 3) Might Cdk2
inhibition synergize with disruptions in other DNA damage
response pathways to potentiate cell-killing by genotoxic agents?
To regulate DNA replication [56] and the G1/S transcriptional
program [57], different CDKs phosphorylate multiple proteins
within the relevant machineries. Specific CDK/cyclin pairs and
phosphorylation sites can appear genetically redundant. Similar
complexity is likely in DNA damage response pathways. Previous
attempts to define functions of individual CDKs relied on: 1)
relatively non-specific chemical inhibitors; or 2) gene disruptions
or RNAi, which neither allow temporal control over enzymatic
activity, nor prohibit non-physiologic compensation by other CDKs
[15,22]. Here, by a chemical-genetic strategy that preserved
normal CDK-cyclin pairing [28], we uncovered non-redundant
requirements for Cdk2 in human cells exposed to IR. Moreover,
by manipulating Cdk2 activity selectively with small molecules, we
could show that it is needed after damage occurs. This allowed us
to exclude the possibility that increased IR-sensitivity was due to
pre-existing defects in Cdk2 mutant cells, and infer a requirement
for Cdk2 to phosphorylate proteins de novo in response to DNA
lesions [5]. The tools developed here should allow us to identify
additional functions and targets of Cdk2 within DNA damage
response pathways.
A basis for Cdk2 specialization?
In budding yeast, checkpoint signaling arrests cell-cycle
progression without CDK inhibition [58]. Cdk1 activity is
required at multiple steps in HR, and several of its relevant
targets have been identified (reviewed in [5,6]). In mammalian
cells, which rely on CDK inhibition for G2 arrest in response to
DNA lesions, CDK substrates have nevertheless been identified in
DNA damage repair [54,55,59–61] and checkpoint [62,63]
pathways. However, an exclusive, catalytic role for a specific
CDK in protecting genome integrity had yet to be established. It
was recently shown that Cdk2
human cells are defective in
implementing a p53-independent G2/M checkpoint arrest [21].
This suggested a non-redundant role for Cdk2 protein but not
necessarily for its catalytic activity. Cdk2 has a non-catalytic
scaffold function that prevents premature assembly of Cdk1-cyclin
complexes [28]; bypass of a checkpoint could therefore be due to
ectopic activation of Cdk1 in Cdk2
Here we have demonstrated a specific requirement for the
catalytic activity of Cdk2 in survival of human cells exposed to
IR—a DNA-damaging and checkpoint-activating treatment. This
suggests that Cdk2 retains activity in this setting, perhaps by virtue
of its distinctive mode of regulation (Figure 6). In contrast to Cdk1,
which must interact with a cyclin in order to be phosphorylated by
Cdk7, Cdk2 is phosphorylated predominantly as a monomer and
then binds cyclin to become activated [10,27]. Possibly as a
consequence of this pathway insulation, Cdk2 is relatively
refractory, during unperturbed cell cycles, to inhibitory phosphor-
ylation [64]—the principal mechanism by which DNA structure
checkpoints inactivate Cdk1 and restrain mitosis in mammalian
cells [65]. We propose that the ability to evade inhibitory
modification, and inability to trigger mitosis, are specific adapta-
tions that permit Cdk2 to function efficiently and safely in the
presence of DNA damage. In the future, it will be interesting to
test whether Cdk2 activity is required for a p53-independent G2/
M checkpoint pathway in Cdk2
cells, in which aberrant Cdk1-
cyclin binding can be prevented. If so, targeting Cdk2 with specific
inhibitors could be an attractive therapeutic strategy in human
cancers with p53 mutations.
Materials and Methods
Cell culture, extract preparation, and immunological
RPE-hTERT or HCT116 cells were grown in Dulbecco’s
modified Eagle medium (DMEM):F-12 or McCoy’s 5A medium,
respectively, supplemented with 10% fetal bovine serum (FBS).
NBS-T cells were grown in DMEM with 10% FBS. Synchroni-
zation of RPE-hTERT cells was performed as described
previously [28]. To detect Nbs1 in unfractionated extracts, cells
were sonicated on ice with 265 s pulses of a 550 Sonic
Dismembrator (Fisher Scientific). Chromatin fractionation was
performed as described [42]. Immunoblots and immunoprecipi-
tations were carried out as described [33] with the following
antibodies: anti-Cdk2 (M2) and anti-cyclin A (H432) from Santa-
Cruz Biotechnology; anti-Cdk1 (POH1), anti-P-T160-Cdk2, anti-
P-T161-Cdk1 and anti-P-T68-Chk2 from Cell Signaling Tech-
nologies; anti-Chk2 (clone 7) from Upstate; and anti-Mre11, anti-
Rad50, and anti-Nbs1 from Novus Biologicals. For detection of
phosphorylated Nbs1 we used anti-P-S432-Nbs1 from Abcam:
either total Nbs1 was immunoprecipitated from extracts and
Cdk2 Phosphorylates Nbs1 to Promote IR Resistance
PLOS Genetics | 9 August 2012 | Volume 8 | Issue 8 | e1002935
probed for phospho-Ser432, or extracts were electrophoresed in
3–8% tris-acetate gradient gels (NuPAGE Novex, Invitrogen).
Kinase assays
The indicated CDK/cyclin complexes were reconstituted in
vitro from purified subunits expressed in baculovirus-infected
insect cells (Cdk1, Cdk2
, Cdk2
, cyclin A, cyclin B) and
activated by CAK; or co-expressed and purified as complexes
(Cdk7/cyclin H to which we added Mat1 purified separately, and
Cdk9/cyclin T1). GST-Nbs1(397-742) was expressed in bacteria.
Kinase reactions were carried out on histone H1, GST-Nbs1(397-
742) or Cdk2 substrates. Substrates (1.0–8.5
mg) were incubated
for 10–15 min at 22uC with CDK complexes in 20
ml kinase
buffer (10 mM HEPES, pH 7.4, 150 mM NaCl, 10 mM MgCl
mM ATP) with or without 10 mCi [c-
P]ATP. Reaction
products were analyzed by 10% SDS–PAGE followed by
autoradiography or immunoblot analysis.
Chemical-genetic methods
Treatment of cells with 3-MB-PP1 or 6-BAP was performed as
previously described [28]. To label substrates of Cdk2, 100
whole-cell extract protein was incubated for 15 min at room
temperature with 140 ng Cdk2
-His/cyclin A complex in a 60-ml
reaction containing an ATP regenerating system (25 mM Hepes,
pH 7.4, 10 mM NaCl, 2 mM MgCl
, 1 mM ATP, 40 mM creatine
phosphate, 0.2 mg/ml creatine phosphokinase) and 5
(benzyl)-ATP, as described previously [26,32,33]. Label-
ing was stopped by addition of 26 sample buffer. Phosphorylated
proteins were separated by sodium dodecylsulfate-polyacrylamide
gel electrophoresis (SDS-PAGE) and detected by autoradiography.
Labeling reactions were scaled up to 400
mg extract protein for
immunoprecipitation. To express Nbs1 in vitro, we used TNTH
Coupled Reticulocyte Lysate kit from Promega, and performed
analog-selective labeling, as previously described for mapping of
sites phosphorylated by Cdk7
[32]. 3-MB-PP1 was dissolved in
dimethylsulfoxide (DMSO) and used at 0–10
Transfection and retroviral infection of NBS-T cells
Small interfering RNA (siRNA) homologous to human Nbs1,59-
CCAACAAGGUUAUAUGAAU-39, and a negative control du-
plex of random sequence, 59-GGUGGACGGCAAGUUUGCU-
39, were synthesized by Dharmacon Research. For complementa-
tion of NBS-T cells, we used expression plasmids pcDNA3-
Nbs1myc and pCLN-Nbs1. Phosphorylation-site mutations
(S432A or S432D) were introduced by site-directed mutagenesis.
Logarithmically growing cells were transfected with siRNA and/or
expression plasmids using Lipofectamine 2000 (Invitrogen) follow-
ing manufacturer’s instructions. For retrovirus production we used a
Phoenix Retroviral Expression System.
Colony formation assay
To determine sensitivity of cells to different agents, 500–40,000
cells (depending on the treatment) were plated in 10-cm
Colonies were counted 14 d later after staining with crystal violet.
Additional methods used to generate data in Supporting Figures
are described in Text S1.
Figure 6. Specialized roles of Cdk2 in DNA damage response: a function of activation pathway insulation? We propose that a specific
requirement for Cdk2 activity to protect cells from IR reflects its distinct mode of activation. Cdk2 is phosphorylated as a monomer by CAK, and then
binds cyclin A to become active. Cdk1, in contrast, can only be phosphorylated by mammalian CAK in the presence of a cyclin (A or B), and only forms
stable complexes with cyclins upon T-loop phosphorylation. This effectively couples Cdk1-activating phosphorylation to inhibitory phosphorylation
by kinases such as Wee1 that also require a CDK/cyclin complex substrate [27,66], and makes Cdk1 intrinsically more sensitive to restraint by DNA
damage checkpoints. By evading that restraint, Cdk2 might take the lead role in phosphorylating Nbs1-Ser432 (and possibly other targets such as
CtIP) early in S phase.
Cdk2 Phosphorylates Nbs1 to Promote IR Resistance
PLOS Genetics | 10 August 2012 | Volume 8 | Issue 8 | e1002935
Supporting Information
Figure S1 Nbs1 protein levels and labeling by Cdk2
in extracts
of different human cell lines. Whole-cell extracts of indicated cell
lines (‘‘LCL2985wt’’ is a lymphoblastoid cell line; ‘‘NBS-T +Nbs1’’
is an NBS-T cell line stably complemented with wild-type Nbs1 )
were labeled with recombinant Cdk2
/cyclin A and [c-
(benzyl)-ATP, followed by anti-Nbs1 immunoprecipitation and
immunoblot analysis of Nbs1 (top) or autoradiography (second
from top). The same extracts were also probed directly by
immunoblot (without immunoprecipitation) for Nbs1 (third from
top) or the loading control glyceraldehyde 3-phosphate dehydro-
genase (GAPDH; bottom).
Figure S2 Ser432 is not required for Nbs1 localization to
chromatin. Fractionation of NBS-T cells stably expressing wild-
type, S432A, or S432D Nbs1 was performed as in Figure 4B.
Isolated nuclei were extracted with different concentrations of
NaCl, as indicated, and Nbs1 protein levels were measured in
soluble fractions derived from equal numbers of cell-equivalents.
Figure S3 Phenotypic characterization of Nbs1-Ser432 phos-
phorylation. (A) Total RNA of NBS-T cells treated either with
control siRNA (si-control) or siRNA targeting endogenous Nbs1
mRNA (si-Nbs1) was used as template in reverse transcriptase
polymerase chain reaction (RT-PCR) and subsequent PCR with
Nbs1-orCdk2-specific primers to estimate transcript abundance.
Treatment with siRNA specific to Nbs1 led to a reduction in Nbs1
mRNA levels whereas Cdk2 mRNA levels were not affected. (B)
G2/M checkpoint assay of Nbs1-deficient NBS-T cells or NBS-T
cells stably complemented with indicated alleles of Nbs1. Mitotic
fraction was quantified by phosphorylated histone H3 staining
1 hr after X-irradiation with 10 Gy. Error bars denote +/2 SD
from duplicate measurements of two independent clones of each
genotype. (C) Nbs1-Ser432 and Chk2-Thr68 phosphorylation of
cells in (B) after X-irradiation. Note that Chk2 Thr-68 phosphor-
ylation occurs at low levels in cells expressing Nbs1
even in
the absence of X-rays. (D) NBS-T/DR-GFP cells were treated
with siRNA targeting Nbs1 or control siRNA and transiently
complemented with empty vector or wild-type, S432A or S432D
alleles of Nbs1, as indicated, and tested for gene-conversion
frequency after I-SceI expression. Numbers of GFP-positive cells
are expressed as percentages of the value in cells. complemented
with wild-type Nbs1 (defined as 100%); error bars denote +/2 SD
of three independent experiments.
Figure S4 Nbs1-Ser432 is not required for DNA damage focus
formation. RPA focus formation was measured in parental NBS-T
cells or NBS-T cells stably expressing wild-type, S432A or S432D
alleles of Nbs1 . Cells were c-irradiated with 4 Gy and collected for
RPA immunostaining at 1 and 6 hr post-irradiation. RPA focus-
positive cells were counted from more than 130 randomly chosen
cells for each cell line.
Figure S5 CtIP levels are unaffected by Nbs1 Ser432 mutation
or Cdk2 inhibition. (A) CtIP protein levels were measured by
immunoblotting of NBS-T cells stably expressing wild-type,
S432A, or S432D alleles of Nbs1. (B) Wild-type or Cdk2
RPE-hTERT were treated with DMSO, 0.5 mM 6-BAP or 10 mM
3-MB-PP1 for 20 hr and tested for CtIP expression by
Figure S6 Determination of HU sensitivity. NBS-T cells
expressing either wild-type, S432A or S432D alleles of Nbs1 were
treated with the indicated doses of HU. Medium was changed
24 hr after start of treatment and cells were tested for colony
formation after 14 d. Values represent the means of duplicates +/
2 SD.
Text S1 Methods used to generate data in Figures S1, S2, S3,
S4, S5, S6 are described in Text S1.
We thank members of the Fisher lab for helpful discussions and critical
review of the manuscript, D. Morgan for providing the baculovirus
encoding Cdk2
, and M. Jasin for providing gene-conversion assay
Author Contributions
Conceived and designed the experiments: LW KAM SD RA JHK SL.
Performed the experiments: LW KAM RA JHK SD SL. Analyzed the
data: LW KAM SD RA JHK SL JHJP RPF. Contributed reagents/
materials/analysis tools: JJA CZ KMS. Wrote the paper: LW KAM JHJP
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Cdk2 Phosphorylates Nbs1 to Promote IR Resistance
PLOS Genetics | 12 August 2012 | Volume 8 | Issue 8 | e1002935
    • "In animals and yeast, it has been found that CDKs phosphorylate proteins in the MRN complex that process 3 0 ends, for example, Nbs1, and proteins that work in concert with this complex, such as the nuclease Sae2/CtIP/Com1 (Huertas et al, 2008; Huertas & Jackson, 2009; Wohlbold et al, 2012; Simoneau et al, 2014 ). Conversely, mutants of Nbs1 in which the phosphorylation site of Cdk2 was eliminated were hypersensitive to radiation in a similar manner as cells in which Cdk2 was chemically inhibited (Wohlbold et al, 2012). Here, we identified RAD51 as a possible target of DNA damage-induced CDK activity. "
    [Show abstract] [Hide abstract] ABSTRACT: Upon DNA damage, cyclin-dependent kinases (CDKs) are typically inhibited to block cell division. In many organisms, however, it has been found that CDK activity is required for DNA repair, especially for homology-dependent repair (HR), resulting in the conundrum how mitotic arrest and repair can be reconciled. Here, we show that Arabidopsis thaliana solves this dilemma by a division of labor strategy. We identify the plant-specific B1-type CDKs (CDKB1s) and the class of B1-type cyclins (CYCB1s) as major regulators of HR in plants. We find that RADIATION SENSITIVE 51 (RAD51), a core medi- ator of HR, is a substrate of CDKB1-CYCB1 complexes. Conversely, mutants in CDKB1 and CYCB1 fail to recruit RAD51 to damaged DNA. CYCB1;1 is specifically activated after DNA damage and we show that this activation is directly controlled by SUPPRESSOR OF GAMMA RESPONSE 1 (SOG1), a transcription factor that acts simi- larly to p53 in animals. Thus, while the major mitotic cell-cycle activity is blocked after DNA damage, CDKB1-CYCB1 complexes are specifically activated to mediate HR.
    Full-text · Article · Aug 2016
    • "This increased resection results from the high level of CDK activity, which promotes the functions of the core resection factors including MRN, CtIP, Exo1, and Dna2 [26][27][28][29][149][150][151][152][153][154][155][156] . The NBS1 subunit of the MRN complex is phosphorylated by CDKs at S432 in S, G2, and M phases (but not in G1), which is important for DNA end resection [149,150]. Mre11 interacts directly with CDK2 and promotes phosphorylation of CtIP/Sae2 by CDK2, which is also crucial for resection in S and G2 phases [147,[151][152][153]. In budding yeast, Sae2 is phosphorylated at S267 by Cdc28 (CDK1) and mutation of this residue to alanine inhibits DNA resection in vivo [152] . "
    [Show abstract] [Hide abstract] ABSTRACT: DNA end resection is a key process in the cellular response to DNA double-strand break damage that is essential for genome maintenance and cell survival. Resection involves selective processing of 5′ ends of broken DNA to generate ssDNA overhangs, which in turn control both DNA repair and checkpoint signaling. DNA resection is the first step in homologous recombination-mediated repair and a prerequisite for the activation of the ataxia telangiectasia mutated and Rad3-related (ATR)-dependent checkpoint that coordinates repair with cell cycle progression and other cellular processes. Resection occurs in a cell cycle-dependent manner and is regulated by multiple factors to ensure an optimal amount of ssDNA required for proper repair and genome stability. Here, we review the latest findings on the molecular mechanisms and regulation of the DNA end resection process and their implications for cancer formation and treatment.
    Full-text · Article · May 2016
    • "It was found that mutation of this site and 6 other S/T-P sites on Xrs2 specifically stimulates NHEJ (Simoneau et al. 2014). Recent studies have shown that Nbs1 is also phosphorylated by Cdk1/2 to promote DSB repair in human cells (Wohlbold et al. 2012). The phosphorylation on Nbs1 occurs at Ser432, which is not far away from Ser349 on Xrs2, based on protein/protein alignments ( "
    [Show abstract] [Hide abstract] ABSTRACT: In response to replication stress, a phospho-signaling cascade is activated and required for coordination of DNA repair and replication of damaged templates (intra-S phase checkpoint). How phospho-signaling coordinates the DNA replication stress response is largely unknown. We employed state-of-the-art liquid chromatography tandem mass spectrometry (LC-MS/MS) approaches to generate high-coverage and quantitative proteomic and phospho-proteomic profiles during replication stress in yeast, induced by continuous exposure to the DNA alkylating agent methyl methanesulfonate (MMS). We identified 32,057 unique peptides representing the products of 4,296 genes, and 22,061 unique phosphopeptides representing the products of 3,183 genes. 542 phosphopeptides (mapping to 339 genes) demonstrated an abundance change of ≥ 2-fold in response to MMS. The screen enabled detection of nearly all of the proteins known to be involved in the DNA damage response, as well as many novel MMS-induced phosphorylations. We assessed the functional importance of a subset of key phosphosites by engineering a panel of phosphosite mutants, in which an amino acid substitution prevents phosphorylation. In total, we successfully mutated 15 MMS-responsive phosphorylation sites in 7 representative genes including:APN1(base excision repair);CTF4andTOF1(checkpoint and sister chromatid cohesion);MPH1(resolution of HR intermediates);RAD50andXRS2(MRX complex); andRAD18(PRR). All of these phosphorylation site mutants exhibited MMS-sensitivity, indicating an important role in protecting cells from DNA damage. In particular, we identified MMS-induced phosphorylation sites on Xrs2 that are required for MMS resistance in the absence of the MRX activator, Sae2, and that affect telomere maintenance.
    Full-text · Article · Mar 2016
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