Differential effects of poly(ADP-ribose) polymerase inhibition on DNA break repair in human cells are revealed with Epstein-Barr virus.
ABSTRACT Poly(ADP-ribose) polymerase (PARP) inhibitors can generate synthetic lethality in cancer cells defective in homologous recombination. However, the mechanism(s) by which they affect DNA repair has not been established. Here we directly determined the effects of PARP inhibition and PARP1 depletion on the repair of ionizing radiation-induced single- and double-strand breaks (SSBs and DSBs) in human lymphoid cell lines. To do this, we developed an in vivo repair assay based on large endogenous Epstein-Barr virus (EBV) circular episomes. The EBV break assay provides the opportunity to assess quantitatively and simultaneously the induction and repair of SSBs and DSBs in human cells. Repair was efficient in G1 and G2 cells and was not dependent on functional p53. shRNA-mediated knockdown of PARP1 demonstrated that the PARP1 protein was not essential for SSB repair. Among 10 widely used PARP inhibitors, none affected DSB repair, although an inhibitor of DNA-dependent protein kinase was highly effective at reducing DSB repair. Only Olaparib and Iniparib, which are in clinical cancer therapy trials, as well as 4-AN inhibited SSB repair. However, a decrease in PARP1 expression reversed the ability of Iniparib to reduce SSB repair. Because Iniparib disrupts PARP1-DNA binding, the mechanism of inhibition does not appear to involve trapping PARP at SSBs.
- SourceAvailable from: Geun-Hyoung HA[Show abstract] [Hide abstract]
ABSTRACT: Deregulation of the transforming acidic coiled-coil protein 3 (TACC3), an important factor in the centrosome-microtubule system, has been linked to a variety of human cancer types. We have recently reported on the oncogenic potential of TACC3; however, the molecular mechanisms by which TACC3 mediates oncogenic function remain to be elucidated. In this study, we show that high levels of TACC3 lead to the accumulation of DNA double-strand breaks (DSBs) and disrupt the normal cellular response to DNA damage, at least in part, by negatively regulating the expression of ataxia telangiectasia mutated (ATM) and the subsequent DNA damage response (DDR) signaling cascade. Cells expressing high levels of TACC3 display defective checkpoints and DSB-mediated homologous recombination (HR) and non-homologous end joining (NHEJ) repair systems, leading to genomic instability. Importantly, high levels of TACC3 confer cellular sensitization to radiation and poly(ADP-ribose) polymerase (PARP) inhibition. Overall, our findings provide critical information regarding the mechanisms by which TACC3 contributes to genomic instability, potentially leading to cancer development, and suggest a novel prognostic, diagnostic and therapeutic strategy for the treatment of cancer types expressing high levels of TACC3.Oncogene advance online publication, 28 April 2014; doi:10.1038/onc.2014.105.Oncogene 04/2014; · 8.56 Impact Factor
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ABSTRACT: DNA is subject to many endogenous and exogenous insults that impair DNA replication and proper chromosome segregation. DNA double-strand breaks (DSBs) are one of the most toxic of these lesions and must be repaired to preserve chromosomal integrity. Eukaryotes are equipped with several different, but related, repair mechanisms involving homologous recombination, including single-strand annealing, gene conversion, and break-induced replication. In this review, we highlight the chief sources of DSBs and crucial requirements for each of these repair processes, as well as the methods to identify and study intermediate steps in DSB repair by homologous recombination.Cold Spring Harbor perspectives in biology 08/2014; · 8.23 Impact Factor
Article: DNA single-strand break repairExperimental Cell Research 11/2014; · 3.37 Impact Factor
Differential effects of poly(ADP-ribose) polymerase
inhibition on DNA break repair in human cells are
revealed with Epstein–Barr virus
Wenjian Ma1, Christopher J. Halweg1,2, Daniel Menendez, and Michael A. Resnick3
Chromosome Stability Section, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709
Edited by Matthew Meselson, Harvard University, Cambridge, MA, and approved March 15, 2012 (received for review November 3, 2011)
Poly(ADP-ribose) polymerase (PARP) inhibitors can generate syn-
thetic lethality in cancer cells defective in homologous recombina-
tion. However, the mechanism(s) by which they affect DNA repair
PARP inhibition and PARP1 depletion on the repair of ionizing
radiation-induced single- and double-strand breaks (SSBs andDSBs)
in human lymphoid cell lines. To do this, we developed an in vivo
repair assay based on large endogenous Epstein–Barr virus (EBV)
circular episomes. The EBV break assay provides the opportunity
of SSBs and DSBs in human cells. Repair was efficient in G1 and G2
cells and was not dependent on functional p53. shRNA-mediated
knockdown of PARP1 demonstrated that the PARP1 protein was
not essential for SSB repair. Among 10 widely used PARP inhibitors,
none affected DSB repair, although an inhibitor of DNA-dependent
protein kinase was highly effective at reducing DSB repair. Only
Olaparib and Iniparib, which are in clinical cancer therapy trials, as
well as 4-AN inhibited SSB repair. However, a decrease in PARP1
expression reversed the ability of Iniparib to reduce SSB repair. Be-
cause Iniparib disrupts PARP1–DNA binding, the mechanism of in-
hibition does not appear to involve trapping PARP at SSBs.
base excision repair|BRCA
(1). Interest in PARPs, especially PARP1, was intensified by the
discovery that PARP inhibition is toxic for cancer cells that are
defective in the homologous recombination (HR) genes BRCA1
and BRCA2 (2, 3). These mutations are found in breast and
ovarian cancer (4). Increased sensitivity to PARP inhibition has
also been observed with cells defective in other DNA double-
strand break (DSB) repair genes such as MRE11 (5) and ATM
(6, 7). Despite these findings and the promising clinical utility of
PARP inhibitors in treating HR-deficient cancers, the underlying
mechanism(s) remains elusive. PARP1 and PARP2 are generally
(8–10). Therefore, the synthetic lethality between PARP in-
hibition and HR defects has been attributed to accumulation of
SSBs, which are subsequently converted to DSBs (11). However,
unlike PARP inhibition, depletion of PARP1 resulted in only
modest toxicity in BRCA2-deficient cells (2). Given the wide-
ranging biological consequences expected from PARP inhibition,
the synthetic lethality in cells with an HR defect might go beyond
effects on just DNA repair. Elucidating the mechanisms of action
is critical to addressing the efficacy of PARP inhibitors, un-
derstanding mechanisms of drug resistance, as well as extending
PARP-related treatment to other types of cancers.
Because PARP participates in DNA repair processes, it is
important to examine the effects of inhibitors on DNA damage
and repair, especially SSBs and DSBs, which can lead to genome
instability. Despite many repair studies in human cells, there is
a lack of robust systems for accurate in vivo measurement of
randomly induced SSBs and DSBs, thereby limiting opportunities
to investigate underlying mechanisms of induction and repair, as
that catalyze ADP ribosylation of a variety of cellular factors
well as the role of PARP. Currently, the most commonly used
approach to detect random DSBs uses immunostaining of DSB-
related biomarkers such as γ-H2AX (12). This approach is sen-
sitive and has provided considerable understanding of proteins
recruited to damage sites. However, detection of DSBs is indirect,
and there are limitations with regard to knowing which proteins
are directly required for repair and which are related cofactors
that are part of downstream signaling or chromatin modification
events. The comet assay, another commonly used approach, vis-
ualizes damage within the mass of nuclear DNA when DNA is
subjected to an electric field. Comet assays are widely used to
detect SSBs/DSBs as well as other lesions that can be converted
into DNA strand breaks, such as alkali-labile sites (13, 14).
However, the actual incidence of breaks cannot be determined
directly, and there are limitations on assessing the specificity of
the damage response (15, 16).
We have developed an approach for detecting SSBs and DSBs
in human cells based on our previous findings with circular chro-
mosomesinbuddingyeast.A singleDSBdramatically changesthe
migration pattern of the chromosome during pulsed-field gel
electrophoresis (PFGE) (17) because a DSB converts a circular
chromosome into a unit-length linear form. We describe a human
cell system based on Epstein–Barr virus (EBV) episomes. These
minichromosomes are large circular molecules (165–180 kb) that
have many human chromosome features, including nucleosomes
with spacing typical of human chromatin (18, 19). Moreover,
replication, which occurs only once per cell cycle, is controlled by
host proteins (20), which makes this system ideal to study cell
cycle-dependent DNA repair events. Here we show that the EBV
break assay can assess directly and accurately the formation and
repair of both SSBs and DSBs.
To address the impact of PARP and PARP inhibitors on DNA
repair, we have used human lymphoblastoid cells containing the
circular EBV episomal genome to assess ionizing radiation-
induced SSB and DSB as well as repair. We evaluated the inhibi-
tory properties of various widely used PARP inhibitors, including
those showing clinical potential for cancer treatment such as Ola-
parib and Iniparib (21, 22). In addition, we confirmed the impor-
tant difference between PARP knockdown and chemical inhibition
of PARP on DNA strand break repair in vivo.
Author contributions: W.M., C.J.H., and M.A.R. designed research; W.M., C.J.H., and D.M.
performed research; W.M. contributed new reagents/analytic tools; W.M. and M.A.R.
analyzed data; and W.M. and M.A.R. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
1W.M. and C.J.H. contributed equally to this work.
2Present address: Department of Genetics, North Carolina State University, Raleigh,
3To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
| April 24, 2012
| vol. 109
| no. 17www.pnas.org/cgi/doi/10.1073/pnas.1118078109
Simultaneous, Quantitative Detection of SSBs and DSBs in Human
Cells. Yeast circular chromosomes have been used in combination
starting well during PFGE; however, a single random DSB gen-
erates a unit-length linear molecule detectable as a single band by
Southern blot (Fig. 1). Previously, Johnson and Beerman (26)
EBV-infected cells when the DNA was irradiated in plugs. We
extended our yeast approach to EBV to quantitate radiation
damage and repair within human cells. The EBV system provides
the opportunity to monitor simultaneously the induction and re-
pair in vivo of not only DSBs but also SSBs.
Without irradiation, two forms of EBV are detected in the
DNA from Raji cancer cells (Burkitt lymphoma) or LCL35
EBV-transformed lymphoblast cells, as described in Fig. 1A. The
majority of the molecules detected with the EBV-specific probe
remained in the well. Following irradiation, there is loss of the
upper band as well as the appearance of a much faster moving
band that corresponds to linear EBV (based on a comparison
with DNA markers) resulting from random single DSBs. Multi-
ple DSBs result in a broad smear of DNA (17).
The mobility of the upper band and the loss at small doses
suggested that this might be due to EBV supercoils, in which
case SSBs would relax the material, preventing entry into the gel.
If this were the case, then loss of the upper band would provide
a direct measurement of SSBs. To demonstrate that the upper
band is indeed supercoiled EBV, we examined the consequences
of radiation on this band using the approach described in Fig. 1B,
because SSBs would relax these molecules. Several plugs of
unirradiated DNA were subjected to PFGE (6 h) to create lanes
that had EBV DNA (presumably relaxed DNA) retained in the
plug and a band of fast-running DNA (proposed supercoiled
form). The gels were sliced so that each slice had two lanes; four
slices with two lanes each were created. The four slices were
irradiated with 0, 25, 50, or 100 Gy and subjected to further
PFGE. If the upper band of DNA was actually due to super-
coiled EBV, irradiation would lead to nicks and relaxation,
preventing further movement upon the second round of PFGE.
As shown in Fig. 1B, the EBV band in the unirradiated slab
moved much farther during the subsequent PFGE, and there was
no additional material contributed by the DNA in the well
(compare left and right lanes after 0 dose to slab). However,
irradiation with as little as 25 Gy essentially prevented any fur-
ther migration of the band. We conclude that this is due to re-
laxation of the supercoiled form, which prevents further PFGE
migration. In the right lane of each irradiated slab there was also
a single higher-mobility band that was due to the linearization of
molecules in the supercoiled band. In the left lane there were
two fast-moving bands corresponding to linearized EBV from
the plug and linearized DNA from the supercoiled band.
Thus, the mobility of supercoiled EBV provides a sensitive
assay for monitoring SSB formation and repair in vivo. Because
a single SSB would transform the EBV supercoil into a relaxed
circle that remains trapped in the well, the efficiency of SSB
induction can be determined by applying Poisson distributions to
model the loss of material from the supercoiled band (SI Mate-
rials and Methods). The method does not allow an estimate of
nicked and unnicked circles that remain in the well. For example,
the incidence of SSBs resulting from 100 Gy is 11.3 SSBs/Mb,
which would correspond to 680 SSBs/Gy in the human genome,
as described in Table 1 (assuming 6 × 109bp per diploid ge-
nome). Similar values have been obtained with less direct
methods (27). The induction of DSBs can also be quantitated
based on Poisson distribution by determining the appearance of
material with a single DSB (i.e., the fast-moving linear band in
Fig. 1; SI Materials and Methods). There was a near-linear in-
crease in material corresponding to ∼0.016 DSBs·Mb−1·Gy−1or
EBV molecules in vivo in a dose-dependent manner. Following PFGE of ir-
radiated cells, breaks in chromosomes were detected by ethidium bromide
(Eb) staining (Left). The lower compaction band corresponds to broken high-
molecular weight molecules. Southern blotting with an EBV-specific probe
(Right) revealed three forms of EBV: relaxed circles (top band) that are not
able to enter the gel, unit-size linear EBV molecules (bottom band) that were
due to single DSBs in the EBV molecules, and supercoiled EBV (middle band).
Note: The linear molecules arose primarily from DSBs in relaxed molecules
that would have appeared in the well before IR. (B) Demonstration that the
middle band is supercoiled EBV. Duplicate chromosomal DNA samples
without damage were run on PFGE for 6 h to let the proposed “supercoil”
DNA run out of the well. The well was then removed from one of the lanes
to leave only the supercoiled DNA in the lane. Each gel slab containing the
two lanes was irradiated, followed by PFGE for another 18 h. EBV was
detected by Southern blotting. Without irradiation, the initial supercoiled
band moves to a new position. However, because of nicking by the radiation
the supercoiled DNA is relaxed, preventing further movement of the band.
The radiation also generated broken molecules (i.e., induced DSBs) that
would give rise to two linear EBV bands for the left lane (derived from DNA
in the well and supercoil band) and only one for the right lane.
Detection of SSBs and DSBs in EBV. (A) IR changes the topology of
Measurement of in vivo SSBs and DSBs induced by
IR (Gy) SSBs/MbDSBs/MbSSBs/cell DSBs/cell SSB:DSB
9.77 (9.24–10.95) 1.15 (0.71–1.21)
8.32.33 (1.53–2.48) 115,800
Number of SSBs and DSBs was calculated as described in SI Materials and
Methods. Values presented are in the format of median and range (in paren-
theses) from five independent measurements. SSBs or DSBs per cell were cal-
culated assuming a genome size of 6 × 109bp in G2 phase (4N DNA content).
Ma et al.PNAS
| April 24, 2012
| vol. 109
| no. 17
∼100 DSBs/Gy in the genome of human diploid cells (Table 1).
This value corresponds to the range of 63–70 DSBs/Gy per cell
from previous estimates (27) and nearly twice that measured in
yeast with the same approach using changes in a circular chro-
mosome (24). Comparable values of SSBs and DSBs were found
for LCL35 cells, which have fivefold fewer copies of EBV than
the Raji cell line (∼10 vs. ∼50), consistent with previous studies
in which the initial number of DNA breaks is independent of cell
type (28, 29).
Using the EBV system, the ratio of SSB:DSB lesions induced
in vivo by radiation was ∼10 over a 10–100 Gy dose range (Table
reduction in the SSB:DSB ratiowith increasing doses also fits with
previous data and proposed models (31, 32). Whereas a DSB
could be generated if SSBs on opposite strands are closely spaced,
we previously showed that for random SSBs generated during
repair of methyl methanesulfonate (MMS) damage, only a few
DSBs were produced following the generation of thousands
of SSBs (17). The present ionizing radiation (IR) results are
consistent with multiple free radicals being produced by single
radiation events within a radius of only a few nanometers to
generate DSBs (33).
Repair of IR-Induced DNA Breaks.The EBV system provides a unique
opportunity to quantitatively address repair of SSBs and DSBs.
As shown in Fig. 2, repair of SSBs was first detected within several
minutes after a dose of 100 Gy to a population of growing Raji cells
(most of which are in G1). There was a small increase in super-
coiled DNA by 10 min, and by 30 min about half the SSBs were
repaired. Eventually, the amount of supercoiled DNA reached
a level comparable to that before IR. The chromatin structure of
the EBV minichromosomes is likely to be retained, including
within the vicinity of breaks (34), so that repair of an SSB will result
in the reappearance of supercoils when the DNA is displayed with
PFGE. These results suggest that whereas before IR there is more
relaxed EBV than supercoiled (compare DNA in the well vs. the
supercoiled band), the reappearance of supercoiled molecules is
largely due to repair of the IR-nicked supercoiled molecules. In-
cluded among the sources of preexisting relaxed DNA in the well
are molecules that are replicating, gapped, or damaged.
Similar to SSBs, ∼20% of the DSBs were repaired in the first 20
min; however, the subsequent reduction in broken molecules was
less, with nearly half remaining at 60–90 min. To address a possi-
ble role for nonhomologous end joining (NHEJ), a key pathway
for DSB repair in humans (35), we examined repair in cells ex-
posed to NU7026, an inhibitor of DNA-dependent protein kinase
(DNA-PK). Before irradiation, the population had been enriched
for G2 cells using nocodazole to enhance the capability for re-
combinational repair. Because there wasconsiderable reduction in
DSB repair, with only∼10% repair at 2 h compared with over50%
inmock-treated controlcells,mostoftheearly DSBrepairwasdue
to NHEJ (Fig. 3B). Repair of SSBs and DSBs appears to be in-
dependent of one another, because the NU7026 effect on DSB
repair did not extend to SSBs. The subsequent limited repair could
be due to homologous recombination. In a less quantitative anal-
ysis, ethidium bromide-stained gels also showed slow repair of the
fragmented human genome in NU7026-treated cells compared
with that of no inhibitor and the PARP inhibitor 4-amino-1,8-
naphthalimide (4-AN) (Fig. S1).
Inhibition of SSB Repair by PARP Inhibitors. PARP inhibition has
been used in cancer chemotherapy when combined with various
drugs or in particular genetic backgrounds (11) where the degree
of PARP inhibition appears related to clinical outcome (36, 37).
γ-irradiated at a dose of 100 Gy and then incubated in complete medium at
37 °C to allow repair. Cells were collected at the indicated times and pro-
cessed for PFGE and Southern blot analysis. The repair efficiencies of the
SSBs and DSBs are indicated at the bottom.
Simultaneous detection of repair of SSBs and DSBs. Raji cells were
duced breaks. (A) Repair of IR-induced breaks (100 Gy) in nocodazole-
enriched G2 Raji cells treated with PARP inhibitors: 20 μM DPQ; 50 μM
NU1025; 10 μM PJ34; 10 μM 4-AN; 20 μM Olaparib. Cells were collected at the
indicated times and processed for PFGE and Southern blot analysis. The re-
pair efficiencies of SSBs and DSBs are indicated at the bottom of each panel.
(B) Inhibition of DSB repair by the DNA-PK inhibitor NU7026 (10 μM). (C)
Repair of IR-induced breaks in p53-competent cells (LCL35) in the absence or
presence of 4-AN. LCL35 cells (p53+) enriched in G2 were exposed to 100 Gy
followed by incubation with or without the PARP inhibitor 4-AN (10 μM) and
then processed for PFGE and Southern blot analysis.
Effect of PARP and NHEJ inhibitors on the repair of radiation-in-
| www.pnas.org/cgi/doi/10.1073/pnas.1118078109Ma et al.
The EBV system provides the opportunity to determine directly
the effect of commonly used inhibitors on the ability of cells to
repair IR-induced SSBs and DSBs.
Previously, the potencies of PARP inhibitors were mainly de-
termined based on in vitro evaluation of NAD+turnover or syn-
thesis of poly(ADP ribose) chains in vivo (38). Although the
side comparison of potential inhibitors on DNA repair of radia-
tion damage has been lacking. Using the EBV system, we tested
repair: 4-AN, NU1025, DPQ, PJ34, Olaparib, Iniparib, IQD,
BYK204165, 3-AB, and DR2313 (See SI Materials and Methods
for full chemical names). Cells were incubated with nocodazole
before treatment with chemicals and IR to enrich for G2 cells and
increase opportunities for HR repair. Most inhibitors (7/10; see
DPQ, NU1025, and PJ34 in Fig. 3A) had little or no effect on SSB
repair, with over70% of SSBsrepaired within 2 h following a dose
of 100 Gy. However, there was inhibition of SSB repair by 4-AN
(Fig. 3A), Olaparib (Fig. 3A), and Iniparib (Fig. 4D). Only 7% of
SSBs were repaired in 4-AN–treated cells at 2 h, and Olaparib
treatment resulted in almost no repair (Fig. 3A). The effect of
Iniparib is weaker compared with 4-AN and Olaparib, resulting in
55% repair by 2 h (Fig. 4D). The efficiency of the inhibitors on
PARP catalytic activity was examined with an in vitro poly(ADP
ribosyl)ation (PARylation) assay. As shown in Fig. S2, all inhib-
itors used at the concentrations in the repair study, except Ini-
parib, efficiently inhibited PARylation. PJ32 and NU1025, which
did not inhibit SSB repair as efficiently as 4-AN, caused over 95%
inhibition of PARylation, which was greater than 4-AN. Thus,
inhibition of SSB repair may not always reflect direct effects on
PARP catalytic activity. The less effective inhibition of Iniparib
PARP1. There was little or no effect on DSB repair by any of the
PARP inhibitors (see examples in Fig. 3A), suggesting that under
these conditions, PARP plays at most a minor role, even though it
targets many genes involved in DSB repair.
PARP1 Is Not Required for Repair of IR-Induced SSBs or DSBs. To
address further the possible role of PARP1 in DNA repair of IR-
induced breaks, we created LCL35 cell lines expressing shRNA
complementary to PARP1 (PARP1KD). The PARP1 mRNA
levels were decreased by more than 80% compared with control
cells expressing scrambled shRNA, and there was no detectable
protein (Fig. 4A). As shown in Fig. 4B, PARP1 is not required to
repair IR-induced SSBs or DSBs. Repair of both types of breaks
following a dose of 100 Gy was comparable between the PARP1
knockdown and the PARP1-competent control cells.
The dramatic difference between PARP inhibition by some
PARP inhibitors and PARP1 knockdown suggests that PARP1
protein per se is not required for SSB repair. Possibly it forms
blocked repair intermediates or affects other repair-associated
targets. To address the mechanism of PARP inhibition of SSB
repair inhibition, we examined the effects of 4-AN, Olaparib,
and Iniparib on repair in PARP1 knockdown cells. Control
(scramble) and PARP1KDcells, enriched in G2 by nocodazole,
were treated with 100 Gy and repair was determined. The
knockdown of PARP1 did not prevent Olaparib inhibition, in-
dicating that Olaparib may target other proteins involved in SSB
repair, such as PARP2. Previous results with base-damaging
agents suggested that inhibitors might trap PARP1 at DNA
breaks, thereby uncoupling base excision repair (39). This hy-
pothesis was tested using Iniparib. Unlike 4-AN and Olaparib,
which mimic nicotinamide and compete for the catalytic domain
of PARP1 and PARP2 (and possibly others), Iniparib is pro-
posed to disrupt the interaction between PARP1 and DNA (40,
41) based on in vitro results. Iniparib inhibited SSB repair and
the inhibition could be reversed by knockdown of PARP1 (Fig.
4D), suggesting that inhibition requires interaction with PARP1.
Possibly Iniparib alters an interaction between PARP1 and re-
pair components or even traps PARP1 at damage sites within
cells, contrary to in vitro findings (Fig. 4E).
Repair of IR-Induced Breaks Is Not Affected by p53 or the Cell Cycle.
This study used two cell lines that contain EBV but differ in
functional status of the tumor suppressor p53: Raji cells are
mutated for p53 (Arg-213 and Thr-234), whereas LCL35 cells
retain wild-type p53 function. Besides modulation of cell-cycle
arrest in response to DNA damage, it has been suggested that
p53 may play a direct role in at least some repair processes, in-
cluding nucleotide excision repair (42, 43), base excision repair
(44), and recombination repair (45). Here we tested whether p53
status would affect repair of SSBs and DSBs induced by IR. The
Raji and LCL35 cells were treated with 100 Gy and subsequently
with PARP inhibitors. As shown in Fig. 3C, repair of SSBs and
DSBs was similar in both cell lines, suggesting that p53 was not
required for the efficient repair of IR-induced breaks. Further-
more, the strong inhibition of SSB repair by the PARP inhibitor
4-AN was not related to p53 status at early times (Fig. 3C);
however, there appeared to be reduced 4-AN inhibition in p53+
cells at later times.
We also compared repair of IR-induced DSBs and SSBs in G1
was no apparent difference in break repair in Raji cells (Fig. S3).
repair by the PARP inhibitor Iniparib. (A) The level of PARP1 expression in
control cells (scramble) or PARP1 knockdown LCL35 cells (PARP1KD) was
determined by RT-PCR and Western blot analysis, as described in Materials
and Methods. (B) Repair of SSBs and DSBs in control and PARP1KDcells fol-
lowing a dose of 100 Gy. The effect of PARP inhibitors, (C) Olaparib (20 μM)
or (D) Iniparib (100 μM), on repair of SSBs and DSBSs in control and PARP1KD
cells following a dose of 100 Gy. Cells were enriched for G2 by nocodazole.
Following incubation, cells were collected and processed for PFGE and
Southern blot analysis. (E) Description of how the different effects between
Olaparib and Iniparib might arise. Olaparib inhibits the catalytic domain of
PARPs and could trap a dysfunctional enzyme at the breaks, whereas Ini-
parib might prevent PARP1 binding but not other PARPs.
PARP1 depletion does not affect repair, but relieves the inhibition of
Ma et al.PNAS
| April 24, 2012
| vol. 109
| no. 17
The ability to detect three forms of EBV—linear, supercoiled,
and relaxed—has led to the development of a sensitive system
for direct and quantitative assessment of induction and repair of
DSBs and SSBs that are randomly induced by IR. Because EBV
has many chromosomal properties including chromatin organi-
zation, S phase-dependent replication, and much larger DNA
size compared with plasmid-based assays, we propose that the
EBV break assay is representative of damage and repair events
in chromosomes. The precise measurements of breaks and repair
are consistent with chromosomal results based on more indirect
methods with chromosomal material including comet and gross
PFGE measurements of fast-moving chromosomal materials
(FAR analysis) (46).
The ability to simultaneously monitor SSBs and DSBs makes
the EBV break assay a unique system for addressing in vivo repair.
Supercoiled DNA of SV40 virus (which is much smaller, ∼5 kb)
had previously been used to address SSBs and DSBs induced
in vivo by γ radiation (47, 48). Cells containing recently transfected
virus were irradiated with high doses (1,000 and 2,000 Gy); how-
ever, there was no repair. The levels of DSBs were similar to those
we obtained with EBV minichromosomes as described here, and
the ratio of 20 for SSBs:DSBs was within the range we describe for
EBV. Some reports had indicated a higher ratio, but that might be
due to methods that used high-temperature or alkaline conditions,
which could lead to SSBs at sites of base damage.
We suggest that the SSB:DSB ratio can be used as an indicator
of the incidence of clustered damage, where single events might
give rise to localized, closely opposed SSBs. Based on our pre-
vious findings with MMS showing an SSB:DSB ratio in the range
of ∼1,000 (17), the SSB:DSB ratio for IR-induced breaks cannot
be attributed to random SSBs. Clustered DNA lesions are con-
sidered to be more destabilizing to the genome and require more
complex repair systems than isolated single lesions (49–51).
The EBV break assay provides a direct measure of the ability of
drugs/chemicals/environmental exposures along with genetic fac-
tors to affect the generation and repair of SSBs and DSBs. This
could be extended to the impact of chromatin, compaction, and
radiation quality on SSB and DSB induction as well as repair.
Because the replication of individual EBV molecules is short
relative to the genome in S phase, it should also be possible to
address EBV changes during this phase. Based on results with
circular chromosomes in yeast (52, 53), genetic factors that in-
fluence conversion of SSBs to DSBs can also be assessed. We
of 100 Gy. The rate of DSB repair is roughly half that of SSB
repair, although during the first 30 min they appear comparable.
Using the EBV break system, we were able to assess directly
how p53 participates in repair of IR-induced breaks. Although
there have been numerous studies suggesting p53 involvement in
DNA repair, including interactions with RAD51 as well as the
Bloom’s (BLM) helicase at stalled replication forks (45, 54, 55),
the studies typically rely on indirect analyses such as fluorescence-
based detection of foci formation. Our direct measurements of
DNA break formation and repair in p53 wild-type and p53 mu-
either SSBs or DSBs in the time frame investigated, although we
do not exclude an effect on repair of residual breaks. This is not
surprising, because initiation of DNA strand break repair, espe-
by p53 peaks at around 10 h following DNA damage and the
stabilization of p53 itself is not seen until 30–60 min after treat-
ment. However,we do not excludethepossibilitythat themutated
form of p53 in Raji cells might interact with repair proteins.
The EBV break system has also provided the opportunity to
address the role of PARP and PARP inhibitors in repair of IR-
induced breaks, which is relevant to chemotherapy because radi-
ation is often included in treatment regimens. We did not detect
consistent with other studies of SSBs produced by other agents
(39,56).Inaddition, PARP1 is notrequiredforrecruitmentof the
major base excision repair proteins to sites of DNA damage (57).
Depletion of PARP1 in cell extracts can even result in more rapid
binding of repair proteins to DNA substrates (58). Therefore, if
PARPs play a role in break repair, possibly other PARPs can
substitute for PARP1, or the role of PARP1 might be greater
during replication, which is not addressed here.
There was clearly an SSB-specific impact of the PARP inhib-
itors 4-AN, Olaparib, and Iniparib. These results are consistent
with clinical trials for cancer treatment showing the effectiveness
of Olaparib and Iniparib (21, 22, 59). Surprisingly, many inhib-
itors that can efficiently inhibit the catalytic activity of PARP
(such as NU1025 and PJ32) failed to show an evident inhibition
of SSB repair, raising the question of what property of the PARP
inhibitors is relevant to in vivo inhibition of repair. Although
PARP inhibition is considered to lead to DSB accumulation,
which accounts for the lethal effect of PARP inhibitors in HR-
deficient cells (2, 3, 60), none of the inhibitors resulted in ele-
vated DSB levels or showed any apparent delay in the repair
kinetics of IR-induced DSBs in G2 cells, suggesting SSBs are not
converted into DSBs. However, SSBs might be converted to
DSBs during replication (61–63), which would explain a re-
quirement for BRCA1/2 and recombinational repair to prevent
lethality that arises from PARP inhibitors.
by some inhibitors and PARP1 knockdown suggests generation of
blocked intermediates or inhibition of repair components. Be-
repair proteins (64), chemical inhibitors bound at the catalytic
domain might trap the nonfunctional PARP1, preventing other
repair proteins from being recruited. However, trapping probably
does not account for PARP1-dependent inhibition of SSB repair
by Iniparib, because it prevents PARP1 from binding to DNA (40,
41). Possibly the inhibitor leads to adverse interactions of PARP1
with other repair components that do not depend upon binding of
PARP1 at SSBs.
In conclusion, the EBV break system provides a physical
method to simultaneously monitor directly the in vivo formation
and repair of both SSBs and DSBs in human cells. Using this
system, we confirmed the dramatic difference between PARP
inhibition by some PARP inhibitors and PARP1 knockdown as
well as the inhibitory capacity of various widely used PARP
inhibitors on DNA repair of radiation-induced breaks, providing
insights into the mechanism of PARP inhibition on DNA repair.
Beyond addressing many questions about break repair in human
cells, the system is well-suited for use in studies that address
broader issues of repair of a variety of DNA-damaging agents as
well as applications in drug development and analysis.
Materials and Methods
Cell Lines, Plasmids, and Chemicals. The EBV-containing Raji cell line derived
from Burkitt’s lymphoma was obtained from the American Type Culture
Collection (CCL-86); EBV-immortalized lymphoblastoid cell line LCL35 was
from Micah Luftig (Duke University, Durham, NC). Depletion of PARP1 ex-
pression (PARP1KD) in LCL35 cells was effected using stably integrated PARP1
shRNA with MISSION shRNA lentiviral plasmids from Sigma-Aldrich. More
information about cell culture, shRNA transfection, and chemicals is given in
SI Materials and Methods.
Gene Expression by RT-PCR and Western Blot Analysis. To determine PARP1
mRNA and protein levels in PARP1KDor control scramble-shRNA transfected
LCL35 cells, real-time PCR and Western blot analysis were conducted as de-
scribed in SI Materials and Methods.
Ionizing Radiation, Drug Treatment, and PFGE Analysis. Cells kept on ice were
irradiated in a137Cs irradiator (J. L. Shepherd model 431, at a dose rate of 2.3
krad/min), and then treated with or without PARP/DNA-PK inhibitors for
incubation repair. The collection of cells and preparation of agarose-DNA
plugs for PFGE analysis are described in SI Materials and Methods.
| www.pnas.org/cgi/doi/10.1073/pnas.1118078109 Ma et al.
ACKNOWLEDGMENTS. We thank Dr. Norman Sharpless, Dr. Julie Horton,
Dr. Michelle Heacock, Dr. Kin Chan, and Jim Westmoreland for critical reading
of the manuscript, and Dr. Charles Romeo and Dr. Negin Martin at the
National Institute of Environmental Health Sciences (NIEHS) Viral Vector
Core Laboratory for generating LCL35 shRNA cell lines. This work was
supported by the Intramural Research Program of the NIEHS (National
Institutes of Health, Department of Health and Human Services) under
Project 1 Z01 ES065073 (to M.A.R.).
1. Krishnakumar R, Kraus WL (2010) The PARP side of the nucleus: Molecular actions,
physiological outcomes, and clinical targets. Mol Cell 39(1):8–24.
2. Bryant HE, et al. (2005) Specific killing of BRCA2-deficient tumours with inhibitors of
poly(ADP-ribose) polymerase. Nature 434:913–917.
3. Farmer H, et al. (2005) Targeting the DNA repair defect in BRCA mutant cells as
a therapeutic strategy. Nature 434:917–921.
4. Narod SA, Foulkes WD (2004) BRCA1 and BRCA2: 1994 and beyond. Nat Rev Cancer 4:
5. Vilar E, et al. (2011) MRE11 deficiency increases sensitivity to poly(ADP-ribose) poly-
merase inhibition in microsatellite unstable colorectal cancers. Cancer Res 71:
6. Weston VJ, et al. (2010) The PARP inhibitor Olaparib induces significant killing of
ATM-deficient lymphoid tumor cells in vitro and in vivo. Blood 116:4578–4587.
7. Williamson CT, et al. (2010) ATM deficiency sensitizes mantle cell lymphoma cells to
poly(ADP-ribose) polymerase-1 inhibitors. Mol Cancer Ther 9:347–357.
8. de Murcia JM, et al. (1997) Requirement of poly(ADP-ribose) polymerase in recovery
from DNA damage in mice and in cells. Proc Natl Acad Sci USA 94:7303–7307.
9. Schreiber V, Dantzer F, Ame JC, de Murcia G (2006) Poly(ADP-ribose): Novel functions
for an old molecule. Nat Rev Mol Cell Biol 7:517–528.
10. Wang ZQ, et al. (1997) PARP is important for genomic stability but dispensable in
apoptosis. Genes Dev 11:2347–2358.
11. Lord CJ, Ashworth A (2008) Targeted therapy for cancer using PARP inhibitors. Curr
Opin Pharmacol 8:363–369.
12. Aten JA, et al. (2004) Dynamics of DNA double-strand breaks revealed by clustering of
damaged chromosome domains. Science 303(5654):92–95.
13. Collins AR (2004) The comet assay for DNA damage and repair: Principles, applica-
tions, and limitations. Mol Biotechnol 26:249–261.
14. Singh NP, McCoy MT, Tice RR, Schneider EL (1988) A simple technique for quantitation
of low levels of DNA damage in individual cells. Exp Cell Res 175(1):184–191.
15. Collins AR, et al. (2008) The comet assay: Topical issues. Mutagenesis 23(3):143–151.
16. McKenna DJ, McKeown SR, McKelvey-Martin VJ (2008) Potential use of the comet
assay in the clinical management of cancer. Mutagenesis 23(3):183–190.
17. Ma W, Resnick MA, Gordenin DA (2008) Apn1 and Apn2 endonucleases prevent ac-
cumulation of repair-associated DNA breaks in budding yeast as revealed by direct
chromosomal analysis. Nucleic Acids Res 36:1836–1846.
18. Dyson PJ, Farrell PJ (1985) Chromatin structure of Epstein-Barr virus. J Gen Virol 66:
19. Shaw JE, Levinger LF, Carter CW, Jr. (1979) Nucleosomal structure of Epstein-Barr virus
DNA in transformed cell lines. J Virol 29:657–665.
20. Yates JL, Guan N (1991) Epstein-Barr virus-derived plasmids replicate only once per
cell cycle and are not amplified after entry into cells. J Virol 65:483–488.
21. Fong PC, et al. (2009) Inhibition of poly(ADP-ribose) polymerase in tumors from BRCA
mutation carriers. N Engl J Med 361(2):123–134.
22. O’Shaughnessy J, et al. (2011) Iniparib plus chemotherapy in metastatic triple-nega-
tive breast cancer. N Engl J Med 364:205–214.
23. Game JC, Sitney KC, Cook VE, Mortimer RK (1989) Use of a ring chromosome and
pulsed-field gels to study interhomolog recombination, double-strand DNA breaks
and sister-chromatid exchange in yeast. Genetics 123:695–713.
24. Westmoreland J, et al. (2009) RAD50 is required for efficient initiation of resection
and recombinational repair at random, γ-induced double-strand break ends. PLoS
25. Ma W, Westmoreland J, Nakai W, Malkova A, Resnick MA (2011) Characterizing re-
section at random and unique chromosome double-strand breaks and telomere ends.
Methods Mol Biol 745(Pt 1):15–31.
26. Johnson PG, Beerman TA (1994) Damage induced in episomal EBV DNA in Raji cells by
antitumor drugs as measured by pulsed field gel electrophoresis. Anal Biochem 220
27. Olive PL (1998) The role of DNA single- and double-strand breaks in cell killing by
ionizing radiation. Radiat Res 150(Suppl 5):S42–S51.
28. Banáth JP, Macphail SH, Olive PL (2004) Radiation sensitivity, H2AX phosphorylation,
and kinetics of repair of DNA strand breaks in irradiated cervical cancer cell lines.
Cancer Res 64:7144–7149.
29. Purschke M, Kasten-Pisula U, Brammer I, Dikomey E (2004) Human and rodent cell
lines showing no differences in the induction but differing in the repair kinetics of
radiation-induced DNA base damage. Int J Radiat Biol 80(1):29–38.
30. Yokoya A, Cunniffe SM, O’Neill P (2002) Effect of hydration on the induction of strand
breaks and base lesions in plasmid DNA films by γ-radiation. J Am Chem Soc 124:
31. Shikazono N, Yokoya A, Urushibara A, Noguchi M, Fujii K (2011) A model for analysis
of the yield and the level of clustering of radiation-induced DNA-strand breaks in
hydrated plasmids. Radiat Prot Dosimetry 143(2–4):181–185.
32. Taucher-Scholz G, Kraft G (1999) Influence of radiation quality on the yield of DNA
strand breaks in SV40 DNA irradiated in solution. Radiat Res 151:595–604.
33. Blaisdell JO, Harrison L, Wallace SS (2001) Base excision repair processing of radiation-
induced clustered DNA lesions. Radiat Prot Dosimetry 97(1):25–31.
34. Caldecott KW (2007) Mammalian single-strand break repair: Mechanisms and links
with chromatin. DNA Repair (Amst) 6:443–453.
35. Lieber MR (2010) The mechanism of double-strand DNA break repair by the non-
homologous DNA end-joining pathway. Annu Rev Biochem 79:181–211.
36. Tutt A, et al. (2010) Oral poly(ADP-ribose) polymerase inhibitor Olaparib in patients
with BRCA1 or BRCA2 mutations and advanced breast cancer: A proof-of-concept
trial. Lancet 376:235–244.
37. Audeh MW, et al. (2010) Oral poly(ADP-ribose) polymerase inhibitor Olaparib in pa-
tients with BRCA1 or BRCA2 mutations and recurrent ovarian cancer: A proof-of-
concept trial. Lancet 376:245–251.
38. Ratnam K, Low JA (2007) Current development of clinical inhibitors of poly(ADP-ri-
bose) polymerase in oncology. Clin Cancer Res 13:1383–1388.
39. Ström CE, et al. (2011) Poly (ADP-ribose) polymerase (PARP) is not involved in base
excision repair but PARP inhibition traps a single-strand intermediate. Nucleic Acids
40. Ellisen LW (2011) PARP inhibitors in cancer therapy: Promise, progress, and puzzles.
Cancer Cell 19(2):165–167.
41. Ferraris DV (2010) Evolution of poly(ADP-ribose) polymerase-1 (PARP-1) inhibitors.
From concept to clinic. J Med Chem 53:4561–4584.
42. Ford JM, Hanawalt PC (1997) Expression of wild-type p53 is required for efficient
global genomic nucleotide excision repair in UV-irradiated human fibroblasts. J Biol
43. Smith ML, et al. (2000) p53-mediated DNA repair responses to UV radiation: Studies
of mouse cells lacking p53, p21, and/or gadd45 genes. Mol Cell Biol 20:3705–3714.
44. Offer H, et al. (1999) Direct involvement of p53 in the base excision repair pathway of
the DNA repair machinery. FEBS Lett 450(3):197–204.
45. Akyüz N, et al. (2002) DNA substrate dependence of p53-mediated regulation of
double-strand break repair. Mol Cell Biol 22:6306–6317.
46. Iliakis GE, Cicilioni O, Metzger L (1991) Measurement of DNA double-strand breaks in
CHO cells at various stages of the cell cycle using pulsed field gel electrophoresis:
Calibration by means of125I decay. Int J Radiat Biol 59:343–357.
47. Krisch RE, Flick MB (1988) Further studies of the induction and intracellular repair of
DNA strand breaks using intranuclear SV40 as a test system. Radiat Res 116:462–471.
48. Krisch RE, Flick MB, Trumbore CN (1991) Radiation chemical mechanisms of single-
and double-strand break formation in irradiated SV40 DNA. Radiat Res 126:251–259.
49. Tounekti O, Kenani A, Foray N, Orlowski S, Mir LM (2001) The ratio of single- to
double-strand DNA breaks and their absolute values determine cell death pathway.
Br J Cancer 84:1272–1279.
50. Shikazono N, O’Neill P (2009) Biological consequences of potential repair inter-
mediates of clustered base damage site in Escherichia coli. Mutat Res 669(1–2):
51. Sage E, Harrison L (2011) Clustered DNA lesion repair in eukaryotes: Relevance to
mutagenesis and cell survival. Mutat Res 711(1–2):123–133.
52. Ma W, et al. (2009) The transition of closely opposed lesions to double-strand breaks
during long-patch base excision repair is prevented by the coordinated action of DNA
polymerase δ and Rad27/Fen1. Mol Cell Biol 29:1212–1221.
53. Ma W, Westmoreland JW, Gordenin DA, Resnick MA (2011) Alkylation base damage is
converted into repairable double-strand breaks and complex intermediates in G2 cells
lacking AP endonuclease. PLoS Genet 7:e1002059.
54. Sengupta S, et al. (2003) BLM helicase-dependent transport of p53 to sites of stalled
DNA replication forks modulates homologous recombination. EMBO J 22:1210–1222.
55. Süsse S, Janz C, Janus F, Deppert W, Wiesmüller L (2000) Role of heteroduplex joints in
the functional interactions between human Rad51 and wild-type p53. Oncogene 19:
56. Godon C, et al. (2008) PARP inhibition versus PARP-1 silencing: Different outcomes in
terms of single-strand break repair and radiation susceptibility. Nucleic Acids Res 36:
57. Woodhouse BC, Dianova II, Parsons JL, Dianov GL (2008) Poly(ADP-ribose) poly-
merase-1 modulates DNA repair capacity and prevents formation of DNA double
strand breaks. DNA Repair (Amst) 7:932–940.
58. Parsons JL, Dianova II, Allinson SL, Dianov GL (2005) Poly(ADP-ribose) polymerase-1
protects excessive DNA strand breaks from deterioration during repair in human cell
extracts. FEBS J 272:2012–2021.
59. Rottenberg S, et al. (2008) High sensitivity of BRCA1-deficient mammary tumors to
the PARP inhibitor AZD2281 alone and in combination with platinum drugs. Proc Natl
Acad Sci USA 105:17079–17084.
60. Rouleau M, Patel A, Hendzel MJ, Kaufmann SH, Poirier GG (2010) PARP inhibition:
PARP1 and beyond. Nat Rev Cancer 10:293–301.
61. Bryant HE, et al. (2009) PARP is activated at stalled forks to mediate Mre11-de-
pendent replication restart and recombination. EMBO J 28:2601–2615.
62. Horton JK, Stefanick DF, Zeng JY, Carrozza MJ, Wilson SH (2011) Requirement for
NBS1 in the S phase checkpoint response to DNA methylation combined with PARP
inhibition. DNA Repair (Amst) 10:225–234.
63. Heacock ML, Stefanick DF, Horton JK, Wilson SH (2010) Alkylation DNA damage in
combination with PARP inhibition results in formation of S-phase-dependent double-
strand breaks. DNA Repair (Amst) 9:929–936.
64. Lindahl T, Satoh MS, Poirier GG, Klungland A (1995) Post-translational modification of
poly(ADP-ribose) polymerase induced by DNA strand breaks. Trends Biochem Sci 20:
Ma et al.PNAS
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| vol. 109
| no. 17