hSSB1 rapidly binds at the sites of DNA double-strand breaks and is required for the efficient recruitment of the MRN complex.
ABSTRACT hSSB1 is a newly discovered single-stranded DNA (ssDNA)-binding protein that is essential for efficient DNA double-strand break signalling through ATM. However, the mechanism by which hSSB1 functions to allow efficient signalling is unknown. Here, we show that hSSB1 is recruited rapidly to sites of double-strand DNA breaks (DSBs) in all interphase cells (G1, S and G2) independently of, CtIP, MDC1 and the MRN complex (Rad50, Mre11, NBS1). However expansion of hSSB1 from the DSB site requires the function of MRN. Strikingly, silencing of hSSB1 prevents foci formation as well as recruitment of MRN to sites of DSBs and leads to a subsequent defect in resection of DSBs as evident by defective RPA and ssDNA generation. Our data suggests that hSSB1 functions upstream of MRN to promote its recruitment at DSBs and is required for efficient resection of DSBs. These findings, together with previous work establish essential roles of hSSB1 in controlling ATM activation and activity, and subsequent DSB resection and homologous recombination (HR).
- SourceAvailable from: Derek J Richard[Show abstract] [Hide abstract]
ABSTRACT: Aberrant DNA replication is a primary cause of mutations that are associated with pathological disorders including cancer. During DNA metabolism, the primary causes of replication fork stalling include secondary DNA structures, highly transcribed regions and damaged DNA. The restart of stalled replication forks is critical for the timely progression of the cell cycle and ultimately for the maintenance of genomic stability. Our previous work has implicated the single-stranded DNA binding protein, hSSB1/NAPB2, in the repair of DNA double-strand breaks via homologous recombination. Here, we demonstrate that hSSB1 relocates to hydroxyurea (HU)-damaged replication forks where it is required for ATR and Chk1 activation and recruitment of Mre11 and Rad51. Consequently, hSSB1-depleted cells fail to repair and restart stalled replication forks. hSSB1 deficiency causes accumulation of DNA strand breaks and results in chromosome aberrations observed in mitosis, ultimately resulting in hSSB1 being required for survival to HU and camptothecin. Overall, our findings demonstrate the importance of hSSB1 in maintaining and repairing DNA replication forks and for overall genomic stability.Nucleic Acids Research 04/2014; · 8.81 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: The repair of DNA double-strand breaks (DSBs) is a critical cellular mechanism that exists to ensure genomic stability. DNA DSBs are the most deleterious type of insult to a cell's genetic material and can lead to genomic instability, apoptosis, or senescence. Incorrectly repaired DNA DSBs have the potential to produce chromosomal translocations and genomic instability, potentially leading to cancer. The prevalence of DNA DSBs in cancer due to unregulated growth and errors in repair opens up a potential therapeutic window in the treatment of cancers. The cellular response to DNA DSBs is comprised of two pathways to ensure DNA breaks are repaired: homologous recombination and non-homologous end joining. Identifying chemotherapeutic compounds targeting proteins involved in these DNA repair pathways has shown promise as a cancer therapy for patients, either as a monotherapy or in combination with genotoxic drugs. From the beginning, there have been a number of chemotherapeutic compounds that have yielded successful responses in the clinic, a number that have failed (CGK-733 and iniparib), and a number of promising targets for future studies identified. This review looks in detail at how the cell responds to these DNA DSBs and investigates the chemotherapeutic avenues that have been and are currently being explored to target this repair process.Frontiers in Oncology 01/2014; 4:86.
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ABSTRACT: Oligonucleotide/oligosaccharide-binding (OB) fold is a ssDNA or RNA binding motif in prokaryotes and eukaryotes. Unexpectedly, we found that the OB fold of human ssDNA-binding protein 1 (hSSB1) is a poly(ADP ribose) (PAR) binding domain. hSSB1 exhibits high-affinity binding to PAR and recognizes iso-ADP ribose (ADPR), the linkage between two ADPR units. This interaction between PAR and hSSB1 mediates the early recruitment of hSSB1 to the sites of DNA damage. Mutations in the OB fold of hSSB1 that disrupt PAR binding abolish the relocation of hSSB1 to the sites of DNA damage. Moreover, PAR-mediated recruitment of hSSB1 is important for early DNA damage repair. We have screened other OB folds and found that several other OB folds also recognize PAR. Taken together, our study reveals a PAR-binding domain that mediates DNA damage repair.Proceedings of the National Academy of Sciences 05/2014; · 9.81 Impact Factor
hSSB1 rapidly binds at the sites of DNA
double-strand breaks and is required for the
efficient recruitment of the MRN complex
Derek J. Richard1,*, Kienan Savage2, Emma Bolderson1, Liza Cubeddu3, Sairei So4,
Mihaela Ghita2, David J. Chen4, Malcolm F. White5, Kerry Richard6, Kevin M. Prise2,
Giuseppe Schettino2and Kum Kum Khanna1,*
1Signal Transduction Laboratory, Queensland Institute of Medical Research, Brisbane, Queensland 4029,
Australia,2Centre for Cancer Research and Cell Biology, Queen’s University Belfast, Belfast, BT9 7BL, UK,
3School of Molecular and Microbial Biosciences, University of Sydney, Sydney, NSW 2006, Australia,
4Radiation Oncology, Southwestern Medical School, Dallas, TX 75390, USA,5Centre for Biomolecular Sciences,
University of St Andrews, North Haugh, St Andrews, Fife KY16 9ST, UK and6Conjoint Endocrine Laboratory,
Clinical Research Centre, Royal Brisbane and Women’s Hospital and Pathology Queensland, Herston,
Queensland 4029, Australia
Received September 1, 2010; Revised and Accepted October 15, 2010
DNA (ssDNA)-binding protein that is essential for ef-
ficient DNA double-strand break signalling through
ATM. However, the mechanism by which hSSB1
functions to allow efficient signalling is unknown.
Here, we show that hSSB1 is recruited rapidly to
sites of double-strand DNA breaks (DSBs) in all
interphase cells (G1, S and G2) independently of,
CtIP, MDC1 and the MRN complex (Rad50, Mre11,
NBS1). However expansion of hSSB1 from the DSB
site requires the function of MRN. Strikingly,
silencing of hSSB1 prevents foci formation as well
as recruitment of MRN to sites of DSBs and leads to
a subsequent defect in resection of DSBs as evident
by defective RPA and ssDNA generation. Our data
suggests that hSSB1 functions upstream of MRN to
promote its recruitment at DSBs and is required for
efficient resection of DSBs. These findings, together
with previous work establish essential roles of
hSSB1 in controlling ATM activation and activity,
and subsequent DSB resection and homologous
It is essential that human cells detect, signal and repair
DNA damage in order to prevent chromosomal instability
or malignant transformation. DNA double-strand breaks
can be induced by a number of agents including ionizing
radiation (IR), reactive chemical species and during en-
dogenous DNA processing events such as DNA replica-
tion. These breaks must be repaired in order to maintain
cellular viability and genomic stability. Once a break has
occurred, cells respond by recruiting DNA repair proteins
to the DSB sites and initiating a complex DSB response
pathway, which includes altered transcriptional and trans-
lational regulation, activation of DSB repair and cell-cycle
checkpoint arrest. DSBs that occur in the S or G2 phases
of the cell cycle can be repaired by the homologous recom-
bination machinery (1–3). The process of HR is initiated
by the recruitment of the MRN complex to the site of the
DSB. MRN has a number of functions, including
tethering of the DNA ends and the activation of the
ATM kinase, resulting in the initiation and maintenance
of signalling pathways and the resection of DSBs to
provide a single-stranded DNA (ssDNA) substrate
for Rad51 mediated strand exchange (4,5). Recent work
has also revealed a role for MRN in both classical and
The most extensively studied human single-stranded
DNA-binding protein (SSB) is replication protein A
(RPA). RPA is widely believed to be a central component
of both DNA replication and DNA repair pathways
(8–10). It does not however, have any similarities in
oligomeric structure to the bacterial SSBs. Recently, we
identified two other chromosomally-encoded members of
the SSB family in humans, named hSSB1 and hSSB2 (11).
*To whom correspondence should be addressed. Tel: +61 7 33620339; Fax: +61 7 33620105; Email: email@example.com
Correspondence may also be addressed to Kum Kum Khanna. Tel: +61 7 33620338; Fax: +61 7 33620105; Email: firstname.lastname@example.org
Nucleic Acids Research, 2011, Vol. 39, No. 5Published online 3 November 2010
? The 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.
hSSB1 and hSSB2 are structurally much more closely
related to the bacterial and archaeal SSBs than to RPA
(12). Both hSSBs are composed of a single polypeptide
containing a ssDNA-binding OB fold, followed by a di-
vergent spacer domain and a conserved C-terminal tail
predicted to be required for protein:protein interactions
(11). The crenarchaeal SSB, from Sulfolobus solfataricus,
also has a flexible spacer followed by basic and acidic
regions near the C-terminus which plays no part in
DNA binding but is known to modulate protein:protein
Our studies on the functional characterization of
hSSB1 have revealed that hSSB1 is stabilized following
exposure of cells to IR and forms distinct foci in inter-
phase cells (G1, S, G2 cells), which colocalize with the
known DSB marker gH2AX within 30min of exposure
(11). In addition, hSSB1 interacts with the ATM kin-
ase in vivo and is phosphorylated by the ATM kinase on
Threonine 117. This phosphorylation event is required
lacking hSSB1 are radiosensitive
functional HR pathway (11). We have also shown
that hSSB1 is a component of a complex containing
IntS3 (14,15). IntS3 is required for the normal tran-
scription of hSSB1 and depletion of IntS3 as expected
gives a similar phenotype to hSSB1 depletion. Consistent
with this, ectopic expression of hSSB1 from a CMV
promoter is able to reverse the IntS3 depletion phenotype
Although we have shown hSSB1 is an ATM target, our
data also demonstrates that hSSB1 is required for efficient
ATM activation and downstream signalling following
DNA damage (11). This is seen by the defective ability
of hSSB1-deficient cells to initialize G1/S and G2/M
checkpoints following IR induced DSBs and significantly
reduced phosphorylation of various ATM targets in
hSSB1-deficient cells (11). However, the mechanism by
which hSSB1 functions to allow efficient activation of
ATM and DSB signalling as yet remains unclear.
In this study, we demonstrate that hSSB1 forms distinct
foci at sites of DSBs generated by IR, a-particles, soft
X-rays and laser tracks. We show that hSSB1 plays an
essential role in the recruitment and function of MRN
and downstream repair proteins at DSBs. The MRN
complex is believed to be the primary sensor of DSBs
and is required for the optimal activation of ATM and
the subsequent downstream DSB signalling. MRN also
functions in the resection of the DSB, a process required
for ATR signalling and Rad51 mediated strand invasion
(4,16,17). Our data now demonstrates that the recruitment
of hSSB1 to DSBs is rapid and is independent of the MRN
complex. We further demonstrate that hSSB1 is essential
for the recruitment of other known HR repair factors.
Further as expected, the lack of recruitment of MRN
also prevents the normal downstream processing events.
Our data suggests that hSSB1 may be required for the
recognition of the initial DSB and may function in the
stability of the DSB and the recruitment of other repair
MATERIALS AND METHODS
Cell lines, plasmids and siRNA
HeLa, HEK293T, MCF7, U2OS and NFF cells were
maintained in DMEM supplemented with 10% foetal
bovine serum (Gibco). Transfection of plasmids and
(Invitrogen) asper manufacturer’s
Full-length hSSB1 and truncations were cloned into bac-
terial expression vectors encoding a His-tag (pET28c).
GFP-hSSB1 was expressed from pEGFP-C1. Small
Invitrogen. The target sequences for siRNA were hSSB1:
GACAAAGGACGGGCATGAG; hSSB1 (2): GCTCAC
CAAAGGGTACGCTTCAGTT; Mre11: GATGCCATT
GAGGAATTAG; Rad50 CTTTGAAGATGTTAACTG
GGCTTCC; CtIP: TAATGATCTTGTTCACTTCAGA
CCC; MDC1 TCGGTCCTATAAGCCTCAGAGAGTT.
Calbiochem (Rad50, Rad51), Sigma (Mre11, actin),
EMD Chemicals (NBS1), Upstate (gH2AX, CtIP),
Roche [BrdUrd (BRDU)], and Invitrogen [Alexa second-
ary antibodies, (raised in Donkey)]. Sheep antiserum to
hSSB1 has been described previously (11). Sheep anti-
serum to MDC1 was a kind gift from Prof. Martin Lavin.
usedin this studywere suppliedby
Immunofluorescence was performed as described previ-
ously (11). Cells treated with IR were grown on Ibidi
8-wellm-slides. Prior to exposure to desired antibodies
cells were pre-permeabilized in the following buffer
(NP40 buffer) for 30min at 5?C: 20mM Tris pH8,
50mM NaCl, 5mM MgCl2, 0.2% NP40, 0.5mM DTT,
1mM Na3VO4, 1mM NaF. Cells were then washed in
ice-cold PBS prior to fixation in 4% paraformaldehyde
(PBS). Images were taken on a Deltavision PDV micro-
scope for IR-induced foci, while X-ray microbeam and
a-particle images were taken on a Zeiss Apotome and
Axiovert 200M microscope. Images captured on an
In-cell-2000 microscope (GE Helathcare) are indicated in
were taken on an
Purification of recombinant protein
hSSB1 was purified as described earlier (11).
DNA pull-down assays
Annealed double-stranded oligos with 6-bp overhangs
were generated from the following two sequences; oligo
GGAT, oligo 2 50TCCATGATCCGGGCAATGTCCG
GCGTTAACCCAGTGGATC or double-strand oligo 3
C. Oligo 1 was modified with a 50-biotin. Oligos were
annealed and bound to streptavidin agarose prior to
assay. Each assay consisted of 10ng of oligo bound to
beads with 2mM hSSB1. Binding was performed for
Nucleic Acids Research,2011, Vol.39, No. 51693
15min at room temperature in DNA pull-down buffer:
20mM HEPES pH 8, 150mM KCl, 5mM MgCl2, 5%
glycerol, 0.05% NP40, prior to being loaded onto a
immunoblotted and stained with anti-hSSB1 antibody.
Sub-cellular protein fractionation
Cellular fractionation was performed using a Thermo
Scientific, Pierce Subcellular Protein Fractionation Kit,
as manufacturer’s instructions.
In-nuclear-western (chromatin loading)
These assays were performed utilizing the GE Healthcare
In Cell Analyzer 2000 and data analysed using In Cell
Investigator software. A macro was written to measure
the total fluorescence of the nuclear compartment. Cells
were plated out on a 96-well GE Healthcare Matriplate.
Prior to exposure to desired antibodies, cells were
pre-permeabilized in the following buffer for 30min at
5?C: 20mM Tris pH8, 50mM NaCl, 5mM MgCl2,
0.2% NP40, 0.5mM DTT, 1mM Na3VO4, 1mM NaF.
Cells were then washed in ice-cold PBS prior to fixation
in 4% paraformaldehyde (PBS). All other stages were per-
formed as for Immunofluorescence microscopy. The In
Cell Analyser automatically counted 800 cells from each
well prior to measuring the mean nuclear intensity for
each signal (Alexa 488 or 594). U2OS cells were used
during this study. Cells were treated at 2Gy IR and
analysed at the time-points indicated in the study. We
used 2Gy of IR as this is a non-lethal does in U2OS cells.
Slot blot analysis was performed on chromosomal DNA
isolated from BrdUrd labelled cells using Invitrogen’s
Pure link Genomic DNA extraction kit. The nitrocellulose
membrane was dried for 24h at room temperature prior to
blocking with SSC buffer (saline sodium citrate). As a
control DNA heated for 15min at 90?C was also loaded.
Introduction of DSBs by microirradiation with a pulsed
365-nm nitrogen laser was performed as described previ-
Microbeam irradiation was performed using the Queen’s
University Belfast X-ray microbeam using a 2mm
diameter characteristic carbon KaX-ray beam (278eV)
at a dose rate of 0.1Gysec?1.
Particle irradiation was performed using a small (7-mm
diameter) a source (activity 1mCi). at a dose rate of
?1Gymin?1corresponding to ?4 a-particle traversals
per cell nucleus per minute.
hSSB1 localizes rapidly to sites of DSBs
hSSB1 has recently been shown to localize at sites of DSBs
and form discrete foci that localize with the DSB marker
gH2AX (11). hSSB1 is required for Rad51 foci formation
and facilitates Rad51 mediated strand exchange. To inves-
tigate the role of hSSB1 further we initially compared the
kinetics of hSSB1 and gH2AX foci formation following
induction of DSBs by IR. Both hSSB1 and gH2AX
accumulated at foci within 15min of induction of DSBs
following IR; however, unlike hSSB1 foci, which persist
for up to 8h, gH2AX foci had largely disappeared by this
time point (Figure 1a). This was further confirmed by
measuring chromatin loading of hSSB1, Mre11 and the
phosphorylation of H2AX following IR treatment.
Chromatin loading was measured using an ‘In-Cell-2000
microscope’ and ‘In-cell-analyser’ software. The average
nuclear fluorescence intensity of the subject antigen (sec-
ondary antibody labelled with Alexa 488 or 594) was
measured from at least 800 cells following extraction of
non-chromatin bound proteins, as described in experimen-
tal procedures (Figure 1b). This demonstrated that hSSB1,
like gH2AX and Mre11, localizes rapidly to chromatin.
hSSB1 is however, retained on chromatin for a longer
period of time than both Mre11 and gH2AX, confirming
the immunofluorescence data. The persistence of hSSB1
foci is consistent with its role in the later stages of HR
(11); importantly however, the immediate accumulation of
hSSB1 implies that hSSB1 may also act at early stages of
repair. We further confirmed the chromatin loading of
hSSB1 by sub-cellular fractionation and immunoblotting
and compared it with loading of the Nbs1 component of
the MRN complex (Supplementary Figure S1). hSSB1 was
also observed to localize to DSBs generated by laser and
a-particles (Figure 1c). The MRN complex is also re-
cruited rapidly to sites of DSBs and is believed to be the
initiating factor in DSB signalling and repair (4,16,17).
We next used a focused soft X-ray microbeam, laser
micro-irradiation and a-particle radiation to study the re-
cruitment of hSSB1 to DSBs at very early time points
a-particle irradiation showed that like Mre11 and
gH2AX, hSSB1 localized to DSBs rapidly (within
<1min), supporting a role for hSSB1 at the earliest
stages of DSB repair (Figure 2a–c). This recruitment is
significantly faster than that observed for RPA suggesting
a differential function of these two proteins.
Recently, hSSB1 has been reported to form a complex
with IntS3 and the newly named hSSBIP1 (hSSB1 inter-
acting protein1) formerly
(14,20,21). IntS3 is a subunit of the integrator complex,
which interacts with RNA Pol II and promotes transcrip-
tion of snRNAs (small nuclear RNAs) (22). Although
IntS3 is required for hSSB1 foci formation at DSBs, this
requirement can be circumvented by ectopic expression of
hSSB1 from a constitutive promoter, in IntS3 depleted
cells (14,20). Furthermore, IntS3 is required for efficient
hSSB1 mRNA expression, suggesting that IntS3 functions
as a transcription factor, regulating hSSB1 levels and may
not play a direct role in hSSB1 recruitment and function at
laser irradiation and
1694Nucleic Acids Research, 2011,Vol.39, No. 5
Figure 1. hSSB1 localizes to sites of DSBs. (a) Foci formation kinetics of hSSB1 and gH2AX. Neonatal foreskin fibroblasts (NFF) were irradiated at
6Gy and immunostained at indicated time points. Cells were pre-extracted with NP40 buffer (as described in experimental procedures) before
fixation and immunostaining as described previously (9) (b) In-nuclear-western analysis of chromatin bound hSSB1, gH2AX and Mre11. U2OS cells
were treated with 6Gy IR, at indicated time points and non-chromatin bound proteins extracted. Mean fluorescence signal from the nuclear
compartment was then calculated from at least 800 cells. (c) Localization of hSSB1 and Mre11 at laser micro-irradiated (U2OS cells) or
a-particle-induced DNA damage (MCF7 cells) as indicated. Fixation and staining was performed 30min after treatment.
Figure 2. hSSB1 locates rapidly to sites of DSBs. (a) Immunostaining showing rapid (within 1min) localization of hSSB1 and gH2AX to DSBs
generated by a soft X-ray microbeam (MCF7 cells). (b) Immunostaining showing rapid (within 3s) localization of hSSB1 and gH2AX to DSBs
generated by a a-particle irradiation (MCF7 cells). (c) Rapid localization (within 10s) of GFP-hSSB1 to DSBs generated by laser micro-irradiation
Nucleic Acids Research,2011, Vol.39, No. 51695
DSBs (14). In support of this, while we observed rapid
recruitment of hSSB1 to sites of DSBs generated by
laser, a-particles or X-rays, we were unable to observe
recruitment of IntS3 at these DSBs. In two of our
previous studies we were unable to observe IntS3 foci at
IR induced DSBs (14,21), however another study has
demonstrated IntS3 foci, which colocalize with g-H2AX
6h after treatment with 10Gy of IR (20). We were unable
to observe any significant colocalization between these
two proteins with the pan nuclear staining of IntS3 ap-
pearing not to change following IR, laser, a-particle or
soft X-ray treatments. We were however, consistently
able to observe hSSB1 colocalization with components
of MRN within 30min of DSB induction by IR (Figure 3).
The MRN complex is not required for IR-induced hSSB1
Generally, DNA damage induced foci formation repre-
sents a hierarchical accumulation of repair proteins in
the vicinity of the DSB site (23). Since MDC1 is
required for the normal maintenance and amplification
of the ATM signal we next decided to determine if deple-
tion of MDC1 effected hSSB1 recruitment. We were
however, unable to observe any defect in hSSB1 localiza-
tion to repair foci or a change in chromatin loading in
MDC1-depleted cells (siRNA) (Supplementary Figure
S2a–c). We also utilized fibroblasts defective for MDC1
and again were unable to observe a defect in hSSB1 foci
formation (Supplementary Figure S2d). CtIP is required
for the efficient resection of DSBs by MRN and is
required for RPA loading following DSB induction
(24,25). Since hSSB1 is a ssDNA-binding protein, it is
possible that it coats ssDNA generated by CtIP. Again
we were unable to observe any defect in hSSB1 foci for-
mation or chromatin loading in CtIP-deficient cells
(Supplementary Figure S3a–c).
The MRN complex is thought to be the sensor and
initiator of the DSB response pathway, and activates
ATM and downstream DSB signalling, by tethering
broken DNA ends together and recruiting ATM which
facilitates ATM activation (4,16,17). Given the rapid re-
cruitment of hSSB1 to DSBs we explored the possibility
that hSSB1 foci formation is dependent on the classical
DNA damage sensor MRN. Surprisingly, hSSB1 foci
formed within 30min of exposure to IR, in U2OS cells
(Supplementary Figure S4a–c). We also observed only a
slight defect in hSSB1 chromatin loading in these cells
(Figure 4a and b). Fibroblasts defective for Mre11
(AT-LD) or NBS1 (ILB1) (26,27) also failed to show a
dramatic effect on hSSB1 foci formation or chromatin
loading (Figure 4c, Supplementary Figure S5a and b).
To further confirm these observations we exposed Mre11
or Rad50 depleted MCF7 cells to a-particle and soft X-ray
microbeam irradiation to analyse the effect on hSSB1 foci
formation. Again like IR, hSSB1 recruited with normal
kinetics to the DSBs generated
(Figure 4d, e and Supplementary Figure S6). However,
analysis of hSSB1 foci in MRN depleted cells indicated
these foci were ?40% smaller than foci formed in
wild-type cells (Figure 4f). This suggests that although
hSSB1 can recognize and bind a DSB independently
of MRN, expansion of hSSB1 at the site may require
MRN activity. This would likely be due to a lack of
MRN dependent resection of DSBs leading to reduced,
ssDNA at DSB ends. The slight defect in chromatin
(Figure 4). The presence of hSSB1 at the DSB prior to
MRN was surprising as little ssDNA would be present at
this site; however, other SSBs including Sulfolobus
solfataricus SSB are capable of melting duplex DNA
(28). DSBs represent areas of destabilized duplex with
increased rates of DNA breathing which exposes ssDNA
to which hSSB1 can bind. Also the majority of DSBs
generated within a cell represent two proximal ssDNA
breaks that melt to form a DSB. This could be
bound by hSSB1. Indeed we were able to observe
hSSB1 binding to both duplex DNA and duplex
DNA with a short 6bp ssDNA overhang in this assay
(Supplementary Figure S7).
hSSB1 is required for DSB resection
As the recruitment of hSSB1 to DSBs is independent of
MRN, we next looked to see if depletion of hSSB1 from
cells by siRNA (sihSSB1) impaired the early stages of DSB
processing. Following resection of the DSB by MRN, the
generated ssDNA becomes coated with RPA (29).
Interestingly, RPA foci formation was impaired in
hSSB1 depleted cells, following IR (Figure 5a and b).
Since depletion of hSSB1 had no effect on replication
Figure 3. hSSB1 localizes with components of the MRN complex
within 15min of IR. Immunostaining showing co-localization of
hSSB1 with Rad50 and Mre11 in MCF7 cells treated with 6Gy IR.
After treatment cells were allowed to recover for 30min and
pre-extracted NP40 buffer before fixation and staining as described
previously (9). Images were captured on a Deltavision PDV microscope
using a 100? objective. Images were deconvolved using softWoRx
1696Nucleic Acids Research, 2011,Vol.39, No. 5
Figure 4. Depletion of components of the MRN complex does not affect hSSB1 chromatin loading or foci formation. (a and b) Depletion of Mre11
or Rad50 does not impair hSSB1 chromatin loading. MCF7 cells transfected with control, Mre11, or Rad50 siRNA’s were irradiated (2Gy, IR) and
immunostained with hSSB1 30min after irradiation. Chromatin loading was measured using an In-cell-2000 and In-cell-analyser software.
(c) Deficiency of NBS1 and Mre11 does not impair hSSB1 foci formation. NBS1-deficient fibroblasts (ILB1) transfected with retroviral vector
alone or full-length NBS1 cDNA and Mre11-deficient (ATLD) and control fibroblasts, were pre-extracted with NP40 buffer, fixed and
immunostained with anti-hSSB1 antibody 30min after irradiation (2Gy) or mock treated. Chromatin loading was measured using an In-cell-2000
and In-cell-analyser software. (d) Depletion of Mre11 or Rad50 does not impair hSSB1 foci formation at soft X-ray microbeam induced DSBs.
Immunostaining of hSSB1 in MCF7 cells transfected with control, Mre11 or Rad50 siRNA’s (48h) after soft X-ray microbeam irradiation.
(e) MCF7 cells were treated with control, Mre11 or Rad50 siRNA as indicated. Forty-eight hours after treatment cells were exposed to focused
a-particle radiation and immunostained with antibodies as indicated. (f) Mean foci size of hSSB1 foci from at least 50 cells treated as above.
Nucleic Acids Research,2011, Vol.39, No. 51697
associated RPA foci in S-phase cells prior to DSB induc-
tion, it is likely the defect observed is primarily in
repair-associated foci. Supporting this, depletion of
hSSB1 from normally cycling cells has no effect on
S-phase progression (11). We next reasoned whether the
defect in RPA foci formation is due to defective gener-
ation of ssDNA formed after resection of DSBs. CtIP is
known to be required for the generation of extended
Figure 5. hSSB1 is required for efficient DSB resection. (a) Defective RPA34 loading represented by impaired foci formation in NFF cells trans-
fected with control or hSSB1 siRNAs and immunostained for RPA34 1h after IR (6Gy). (b) RPA34 positive cells from above were counted and the
percentage RPA34 positive cells calculated from at least 100 cells from replicate experiments. (c) Defective CtIP foci formation in NFF cells
transfected with control or hSSB1 siRNAs and Immunostained for CtIP 1h after IR (6Gy). (d) CtIP positive cells from above were counted and
the percentage CtIP positive cells calculated from at least 100 cells from replicate experiments. (e) Defective ssDNA formation after IR in control and
hSSB1-depleted NFF cells. Cells labelled with BrdUrd were fixed 1h after IR (6Gy). Cells were immunostained with BrdUrd antibody, which under
native conditions (non-denaturing) is only able to detect BrdUrd in exposed ssDNA. (f) DNA slot blot analysis of cells from above. Exposed BrdUrd
in membrane bound ssDNA was detected with anti-BrdUrd antibody. (g) Relative BrdUrd intensity from DNA slot blot above. Error bars were
calculated from standard deviations. Asterisk indicates significant differences P<0.005.
1698 Nucleic Acids Research, 2011,Vol.39, No. 5
regions of ssDNA required for RPA loading (30). In
hSSB1-defficient cells we observed a defect in CtIP foci
formation suggesting there may be a defect in ssDNA
generation (Figure 5c and d). We next studied the appear-
ance of ssDNA using a BrdUrd incorporation assay, a
non-denaturing staining assay, which detects BrdUrd,
only in cells with exposed ssDNA (31). In response to
IR, 33% of control siRNA treated cells showed BrdUrd
foci formation whereas most of the hSSB1-depleted cells
did not exhibit ssDNA foci formation (Figure 5e). We also
observed some cytoplasmic BrdUrd staining in both
mitochondria staining, which are known to exhibit long
stretches of ssDNA (32). To confirm that the staining
was specific we also analysed BrdUrd exposed ssDNA
by Slot Blot of isolated genomic DNA (Figure 5f and g).
This data suggests either that MRN dependent processing
of DSBs to ssDNA extensions is defective or that MRN
generated ssDNA is no longer stable in hSSB1-depleted
hSSB1 is required for MRN recruitment to DSBs
In light of the above, we next looked for the presence of
the MRN complex at DSBs in hSSB1-deficient cells fol-
lowing treatment with IR. Indeed, Rad50, NBS1 and
Mre11 foci were easily detectable in >90% of control
siRNA transfected cells, whereas NBS1, Rad50 and
Mre11 foci formation was clearly defective in cells
depleted of hSSB1 by
Supplementary Figure S8a and b). To further confirm
these observations, chromatin loading of Mre11 was
analysed in both control and hSSB1 depleted cells.
Mre11 retention to chromatin was severely impacted in
hSSB1-depleted cells (Figure 6c). Laser tracks also con-
firmed that hSSB1 depleted cells failed to efficiently recruit
Mre11 (Figure 6d). An immunoblot of sub-cellular frac-
tions also confirmed that the MRN complex failed to load
onto chromatin following IR treatment in hSSB1 depleted
cells. It also confirmed that RPA chromatin loading was
also defective in hSSB1-depleted cells. MDC1 does not
load onto chromatin following IR treatment but is
cells we observe MDC1 post-translational modification
following IR, however, these modifications are absent
in hSSB1-depleted cells, consistent with the observed gen-
eral chromatin loading and signalling defect (Figure 6e
The MRN complex, like hSSB1, is required for normal
ATM signalling following DSB induction (11,16,33). Our
data now demonstrates that the signalling defect in
hSSB1-deficient cells is likely due to a deficiency in
MRN recruitment/stability at DSBs, which is subsequent-
ly compounded by the loss of RPA loading required for
ATR signalling (29).
modified. In control
In summary, we have shown that hSSB1 is recruited
rapidly to sites of DSBs in all interphase cells (G1, S
and G2). This recruitment is not dependent on the DSB
repair proteins CtIP or MDC1 as depletion of these
proteins has little effect on the recruitment of hSSB1.
Interestingly CtIP is required for the recruitment of
RPA indicating that hSSB1 functions upstream of RPA.
Indeed we have confirmed that RPA, which is loaded onto
ssDNA generated by CtIP and MRN, does not load onto
chromatin following DSB induction and that it does not
form repair foci. This is also consistent with our observa-
tion that IR induced ssDNA cannot be detected in
Since these results now confirm distinct roles for hSSB1
and RPA in the repair of DSBs we then further
determined where in this pathway hSSB1 functions. The
MRN complex is thought to be the initiating factor in the
HR repair process. However, this study now demonstrates
that depletion of hSSB1 by two distinct siRNAs results in
a loss of IR induced MRN chromatin loading and a severe
defect in foci formation. This is again consistent with the
previous published work indicating hSSB1 is required for
ATM mediated signalling. Loss of the MRN components
results in a very similar signalling defect as seen in hSSB1
depleted cells. However, a study by Huang et al. (20), has
conflicting data to our observation. Their work suggested
that IntS3 formed nuclear IR induced foci, which
colocalized with g-H2AX. We were unable to repeat
these observations as we observe pan nuclear staining of
IntS3, which does not change following IR, soft X-ray or
a-particle treatment. Huang et al. (20), also observed that
depletion of MRN causes abrogation of IntS3 and hSSB1
foci formation at DSBs after long repair times (6h) fol-
lowing high doses of irradiation (10Gy). In contrast, we
find that at early time points (after g-irradiation, soft
X-ray, microbeam and a-particle irradiation), depletion
of MRN does not abrogate hSSB1 foci formation but
rather results in reduced foci size, which is likely due to
reduced ssDNA generated at DSBs. We were also unable
to study hSSB1 chromatin loading or foci formation at
10Gy IR in MRN depleted cells (6h post-IR) as these
depleted cells are highly sensitive and become pro-
apoptotic during the course of the experiment.
Usinga numberof techniques
demonstrated that DNA damage-induced hSSB1 foci
occur independently of MRN, but that MRN foci and
chromatin association depend on hSSB1, which potential-
ly explains defects in homologous recombination and
ATM signalling conferred by hSSB1 silencing (Figure 7).
Interestingly hSSB1 also
phosphorylated by the ATM kinase (11). This may
indicate that hSSB1 has two distinct functions at the
early stages of the DSB response and processing
pathway. The initial function, required for the recruitment
of MRN, is ATM independent; a secondary function may
then require the modulation of hSSB1 activity by the
ATM kinase. The findings presented here are of interest
to the development of new anti-cancer drugs, as there is an
increasing focus on the inhibition of DNA repair
processes in the treatment of cancer. Therefore, further
studies of hSSB1, particularly as it acts at the earliest
stages of the DNA damage response, will provide
valuable information to aid drug development.
we have now
Nucleic Acids Research,2011, Vol.39, No. 5 1699
Figure 6. hSSB1 is required for MRN foci formation at DSBs. (a) Depletion of hSSB1 impairs NBS1, Mre11, and Rad50 foci formation in response
to IR. Cells were transfected with one of two different hSSB1 siRNA’s [sihSSB1 (1) or (2)] and treated with 6Gy IR 72h after transfection. Cells
were extracted with NP40 buffer, fixed and immunostained with the indicated antibodies 30min after IR. (b) Quantification of foci positive cells for
NBS1, Mre11 and Rad50 from experiments as above. Mean percentage of positive cells were calculated from at least 100 cells from replicate
experiments. (c) In-nuclear-western analysis of chromatin bound Mre11 in control and hSSB1 siRNA transfected cells. U2OS cells were treated with
6Gy IR, at indicated time points and non-chromatin bound proteins extracted. Mean fluorescence signal was then calculated from at least 800 cells.
(d) In hSSB1-depleted U2OS cells, Mre11 fails to efficiently recruit to Laser micro-irradiation induced DSBs. (e) IR induced chromatin loading of
Mre11, NBS1, RPA and post-translational modification of MDC1 is impaired in hSSB1-depleted U2OS cells. (f) Quantification of Sub-cellular
fractionation western blot from above. Error bars where present, were calculated from standard deviations.
1700 Nucleic Acids Research, 2011,Vol.39, No. 5
Supplementary Data are available at NAR Online.
The authors would like to thank all colleagues in the
Khanna laboratory for discussion and Stephen Miles for
Cancer Council Queensland Project Grant (to D.J.R.);
Program Grant from National Health and Medical
Research Council of Australia (to K.K.K); National
Institutes of Health grants (CA050519, CA134991 and
CA92584 to D.J.C.). Funding for open access charge:
Queensland Institute of Medical Research.
Conflict of interest statement. None declared.
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