Article

Transcription–replication conflicts underlie

sensitivity to PARP inhibitors

https://doi.org/10.1038/s41586-024-07217-2 Michalis Petropoulos1, Angeliki Karamichali1,4, Giacomo G. Rossetti2,4, Alena Freudenmann2,3,

Luca G. Iacovino2, Vasilis S. Dionellis1, Sotirios K. Sotiriou2,3 & Thanos D. Halazonetis1 ✉
Received: 14 December 2022

Accepted: 20 February 2024

An important advance in cancer therapy has been the development of poly(ADP-ribose)
Published online: xx xx xxxx
polymerase (PARP) inhibitors for the treatment of homologous recombination
Open access
(HR)-deficient cancers1–6. PARP inhibitors trap PARPs on DNA. The trapped PARPs are
Check for updates thought to block replisome progression, leading to formation of DNA double-strand
breaks that require HR for repair7. Here we show that PARP1 functions together with
TIMELESS and TIPIN to protect the replisome in early S phase from transcription–
replication conflicts. Furthermore, the synthetic lethality of PARP inhibitors with HR
deficiency is due to an inability to repair DNA damage caused by transcription–
replication conflicts, rather than by trapped PARPs. Along these lines, inhibiting
transcription elongation in early S phase rendered HR-deficient cells resistant to PARP
inhibitors and depleting PARP1 by small-interfering RNA was synthetic lethal with HR
deficiency. Thus, inhibiting PARP1 enzymatic activity may suffice for treatment
efficacy in HR-deficient settings.

Homologous recombination (HR) is a main pathway for repair of DNA enhance retention of PARPs on DNA, or be neutral, or favour release of
double-strand breaks (DSBs); yet, a fraction of human cancers, often PARPs from DNA, depending on how they affect, by means of reverse
of ovarian, breast, prostate and pancreas origin, are HR-deficient due allostery, the conformation of the DNA binding domain24.
to biallelic mutations of genes involved in HR repair8,9. Cells defec- The mechanism by which PARP inhibitors induce lethality of
tive in HR are very sensitive to PARP inhibitors5,6 and, as a result, PARP HR-deficient cancer cells is at present attributed to trapping of PARPs
inhibitors have been developed as therapeutic agents1–4. The normal on DNA7,21–23. Specifically, it has been proposed that trapped PARPs block
cells of these patients remain HR-proficient and, hence, are resistant progression of the replisome, leading to formation of DNA DSBs that
to PARP inhibitors. require HR for repair. Here, we describe a new role of PARP1, together
The human PARP family comprises 17 members, of which PARP1 with TIMELESS and TIPIN, to prevent transcription–replication conflicts
and PARP2 are the only known members that function in DNA repair (TRCs) and propose that this function is more relevant for the synthetic
and are capable of poly(ADP-ribosyl)ation10. PARP1 and PARP2 bind to lethality of PARP inhibitors with HR deficiency.
various types of DNA damage lesions, including single-strand nicks,
single-strand gaps and DSBs11,12. Binding to these lesions involves
a conformational switch that traps the PARPs on DNA and enhances Role of TIMELESS and TIPIN in TRCs
their catalytic activity13–16. Once activated, PARP1 and PARP2 PARylate In budding yeast, the proteins Tof1 and Csm3 protect the replisome
various substrates, including histones and themselves11,12. AutoPARyla- from conflicts with transcription25–27. We examined whether TIME-
tion facilitates their release from DNA and switches their conformation LESS and TIPIN, the mammalian orthologues of Tof1 and Csm3,
back to the catalytically inactive state13,15,16. respectively28, have a similar function. HeLa human cervical carci-
According to the enzymatic cycle described above, inhibiting the noma cells, transfected with small-interfering RNAs (siRNAs), were
catalytic activity of PARPs will prevent autoPARylation and keep these synchronized with thymidine at the G1/S boundary and then released
enzymes trapped on DNA; thus, the trapping potential of PARP inhibi- into S phase, either in the presence or absence of 5,6-dichloro-1-beta-d-
tors should be proportional to their ability to inhibit PARP catalytic ribofuranosylbenzimidazole (DRB), an inhibitor of transcription
activity17–20. However, several studies have reported a poor correla- elongation29; DNA damage was assessed 100 or 200 min later (Fig. 1a
tion between the inhibitory and trapping potentials of PARP inhibi- and Extended Data Fig. 1a–c). Depletion of TIMELESS or TIPIN led to
tors21–23; this poor correlation has been attributed to differences in formation of γH2AX, 53BP1 and RAD51 foci, in a manner dependent
reverse allostery24. In the normal catalytic cycle, binding of PARPs to on transcription elongation (Fig. 1b,c). Similar results were obtained
DNA damage sites transmits a conformational switch from the DNA with synchronized U2OS osteosarcoma and hTERT-RPE1 immortalized
binding domain to the catalytic domain to regulate catalytic activity; retinal pigment epithelial cells (Extended Data Fig. 1d) and with other
in reverse allostery, binding of an inhibitor to the catalytic domain inhibitors of transcription (Extended Data Fig. 1e–h).
transmits a conformational change from the catalytic domain to the In the above experiments, most cells were in S phase, as they were
DNA binding domain, thereby affecting trapping. PARP inhibitors may released from a thymidine-induced G1/S arrest. To determine whether

1
Department of Molecular and Cellular Biology, University of Geneva, Geneva, Switzerland. 2FoRx Therapeutics AG, Basel, Switzerland. 3Present address: Roche Pharma Research and Early
Development, Roche Innovation Center Basel, Basel, Switzerland. 4These authors contributed equally: Angeliki Karamichali, Giacomo G. Rossetti. ✉e-mail: thanos.halazonetis@unige.ch

Nature | www.nature.com | 1
Article
a HeLa d HeLa g HeLa
siRNA siRNA siRNA
transf. Thy Release IF transf. Thy Release PLA transf. Thy Release EdU Collect

–48 h –18 h 0h 100/200 min –48 h –18 h 0h 200 min –48 h –18 h 0h 60/90 90/120
±DRB ±DRB min min
±CPT
b siCTRL siTIM siTIP e h Fork progression-
–DRB +DRB –DRB +DRB –DRB +DRB siCTRL siTIM siTIP CPT average signal
EdUseq(V)
50
γH2AX –DRB

90 min
53BP1 +DRB

siCTRL
0
10
PCNA-RNAPII PLA
RAD51
120 min

c γH2AX positive Cells with 53BP1 Cells with RAD51 f 0

cells (%) foci (%) foci (%) –DRB +DRB 40
–DRB –DRB –DRB 60
+DRB +DRB +DRB <0.0001 <0.0001
80 80 80
<0.0001 90 min
<0.0001 50
<0.0001

PLA foci per cell

<0.0001

PCNA-RNAPII
60 60 60 <0.0001 40 0

siTIM
0.0002 <0.0001 10
100 min

30 NS
40 40 40 120 min
20
NS
NS
20 20 20 10 0
NS
10
0
0 0 0 60 <0.0001 <0.0001 90 min
80 <0.0001 80 <0.0001 80 <0.0001
<0.0001 <0.0001 <0.0001 50 <0.0001
0
PCNA-RNAPII pS2

siTIP
PLA foci per cell

60 60 60 40 10

30 120 min
200 min

40 40 40
20 NS
NS 0
NS NS 10 Gene
20 20 20
annotation
0 EUseq(V)
siCTRL

siTIM

siTIP

CPT
(100 nM)

0 0 0
–500 0 +500
siCTRL

siTIM

siTIP

siCTRL

siTIM

siTIP

siCTRL

siTIM

siTIP

Forward kb
Reverse

Fig. 1 | TIMELESS and TIPIN suppress the occurrence of TRCs. a–c, DNA plots show medians and value ranges of 25–75 and 10–90%; filled circles
damage response in HeLa cells depleted for TIMELESS (TIM) or TIPIN (TIP). indicate the cells in the top and bottom deciles; n = 2 replicates; more than
a, Outline of the experiment. b, Representative immunofluorescence (IF) 51 cells per group (range 51–106); ANOVA. g,h, Increased fork progression
images; the nuclei were counterstained with DAPI. c, Means ± 1 s.d. of percentage following depletion of TIMELESS or TIPIN. g, Outline of the experiment.
of cells with more than 20 γH2AX, more than 20 53BP1 or more than 10 RAD51 h, Average EdUseq signal over large (more than 300 kb) transcribed genes
foci; n = 3 replicates; more than 259 cells per group (range 259–414); analysis of 90 and 120 min after release into S phase. The genes were aligned by their
variance (ANOVA). A residual DNA damage response in cells treated with DRB transcription start site with their 5′–3′ orientation from left to right. Lower
could reflect TRC-independent mechanisms or transcription elongation panels show the gene annotation and EUseq signal for each genomic locus
complexes that had already escaped the promoter when DRB was added. used to generate the EdUseq plot. Scale bars in microscopy images, 5 μm.
d–f, Detection of TRCs by PLA. d, Outline of the experiment with camptothecin Span of genomic regions, 1 Mb. CTRL, control; EU, 5-ethynyl uridine;
(CPT) as positive control. e, Immunofluorescence images of PCNA-RNAPII PLA NS, not significant; σ, sigma value; Thy, thymidine; transf., transfection.
foci; the nuclei were counterstained with DAPI. f, Number of PLA foci per cell;

DNA replication was required for induction of the DNA damage Data Fig. 2a–c). Third, overexpression of wild-type RNase H1, which
response, we repeated the experiment with unsynchronized cells helps resolve R-loops, attenuated the DNA damage response induced
that were treated with 5-ethynyl-2′-deoxyuridine (EdU) to distinguish by TIMELESS or TIPIN depletion (Extended Data Fig. 2d–f). We note
replicating from non-replicating cells. Depletion of TIMELESS or TIPIN that R-loops accumulate in response to TRCs and are associated with
induced a DNA damage response only in the cells that were in S phase formation of DNA breaks30–32.
and that were not treated with DRB, suggesting the involvement of Previous studies have shown that DNA damage induced by TRCs in
TRCs (Extended Data Fig. 1i–k). early S phase can persist until mitosis, where it is repaired by mitotic
Several more experiments linked TRCs to the observed DNA dam- DNA synthesis (MiDAS)33,34. We wondered whether some of the DNA
age response. First, a proximity ligation assay (PLA), which monitored damage induced by depletion of TIMELESS or TIPIN also remained
physical proximity of Proliferating Cell Nuclear Antigen (PCNA) to unrepaired until mitosis. HeLa cells transfected with the appropriate
RNA-polymerase II (RNAPII), showed an enhanced signal after TIME- siRNAs were released into S phase in the presence of RO-3306, a Cdk1
LESS or TIPIN depletion; moreover, this enhanced signal was strongly inhibitor that prevents mitotic entry; DRB was also optionally admin-
attenuated by DRB (Fig. 1d–f). Second, depletion of TIMELESS or TIPIN istered, but only during the first 200 min of S phase; 11 h after release
in cells expressing catalytically inactive RNase H1 led to the emergence into S phase, RO-3306 was withdrawn and EdU was added to monitor
of discreet RNase H1 foci indicating the presence of R-loops (Extended MiDAS (Extended Data Fig. 2g). Depletion of either TIMELESS or TIPIN

2 | Nature | www.nature.com
 a c e
DMSO Olap (10 μM) Tal (100 nM) 40 –DRB
HeLa <0.0001
+DRB
–DRB +DRB –DRB +DRB –DRB +DRB
Thy Release IF/PLA
53BP1 30 <0.0001

PLA foci per cell

PCNA-RNAPII
–18 h 0h 200 min
±PARPi 20
RAD51 NS
±DRB

10

b d
<0.0001
80 <0.0001 –DRB –DRB <0.0001
<0.0001 <0.0001 0
+DRB +DRB
150 80 <0.0001 <0.0001
<0.0001

<0.0001 <0.0001
<0.0001
γH2AX positive cells (%)

<0.0001

60 <0.0001 50

RAD51 foci per cell

NS

53BP1 foci per cell

120
<0.0001

60

PCNA-RNAPII pS2
PLA foci per cell
NS 0.0048 40 <0.0001
0.0034
40 90
0.0390

40 30

20 60
20 NS
NS
20
30 10
0
(5 μM)

(1 μM)
(5 μM)
(10 μM)
(100 nM)
(1 μM)

(10 μM)
(100 nM)
DMSO

0 0 0
DMSO

Tal
(100 nM)
Sar
(100 nM)

DMSO

Tal
(100 nM)
Sar
(100 nM)

DMSO
Olap
(10 μM)
Vel
(10 μM)

Olap
(10 μM)
Vel
(10 μM)

Olap
(10 μM)

Vel
(10 μM)
Tal Olap Sar Vel

Fig. 2 | PARP inhibitors induce TRCs. a–e, DNA damage response and TRCs in show medians and value ranges of 25–75 and 10–90%; filled circles indicate the
HeLa cells treated with PARP inhibitors (PARPi). a, Outline of the experiment. cells in the top decile; n = 3 replicates; more than 142 cells per group (range
b, Means ± 1 s.d. of percentage of cells with more than 20 γH2AX foci; n = 3 142–542); ANOVA. e, Number of PLA foci per cell plotted as in d; n = 2 replicates;
replicates; more than 232 cells per group (range 232–461); analysis of variance more than 51 cells per group (range 51–99); ANOVA. Scale bar in microscopy
(ANOVA). c, Representative immunofluorescence (IF) images; the nuclei were images, 5 μm. NS, not significant; Olap, olaparib; Sar, saruparib; Tal, talazoparib;
counterstained with DAPI. d, Number of RAD51 and 53BP1 foci per cell; plots Thy, thymidine; Vel, veliparib.

led to MiDAS, except for the cells that were treated with DRB during the optionally, with DRB during early S phase (0–3.5 h after release from
first 200 min of S phase (Extended Data Fig. 2h,i), consistent with DNA a thymidine block), mid-S phase (3.5–7 h) or late S phase (7–10.5 h);
damage being induced in early S phase and persisting until mitosis. γΗ2ΑΧ levels were monitored at the end of the PARP inhibitor treat-
In budding yeast, DNA replication forks pause at centromeres and ment (Extended Data Fig. 3c). A DNA damage response, suppressible
at highly transcribed transfer RNA loci in a Tof1-dependent manner27. by DRB, was observed in the cells treated with PARP inhibitors in early
To determine whether TIMELESS and TIPIN affect fork progression in S phase; the DNA damage response was weaker in the cells treated
human cells, we depleted TIMELESS or TIPIN by siRNA and examined with PARP inhibitors in mid-S phase and practically absent in the late S
the kinetics of DNA replication over large expressed genes. We note that phase-treated cells (Extended Data Fig. 3d). The magnitude of the DNA
expressed genes lack intragenic origins and are, therefore, replicated damage response correlated with the number of expressed genes that
by forks originating from upstream and/or downstream intergenic map to the early, mid- and late S replicating genomic domains, respec-
regions35. The cells were released into S phase for 90 or 120 min, EdU tively (Extended Data Fig. 3e). Similar results were obtained when we
was added during the last 30 min and DNA synthesis was monitored by monitored MiDAS; HeLa cells exposed to olaparib or talazoparib during
the EdUseq method35 (Fig. 1g). In the control cells, 90 min after release early, but not late, S phase showed MiDAS, which was suppressed by
from the thymidine block, the large, transcribed genes had not yet been concurrent administration of DRB (Extended Data Fig. 3f–h).
replicated, whereas at 120 min replication had advanced but was still The above experiments indicate that PARP inhibitor treatment, par-
incomplete. In the cells depleted of TIMELESS or TIPIN, both at 90 and ticularly in early S phase, leads to TRCs. In further support of this conclu-
120 min, replication had advanced further into the gene bodies than in sion, treatment of cells expressing catalytically inactive RNase H1 with
the control cells (Fig. 1h and Extended Data Fig. 2j,k). PARP inhibitors led to the emergence of discreet RNase H1 foci indicat-
ing formation of R-loops (Extended Data Fig. 3i,j) and overexpression
of wild-type RNase H1, which disrupts R-loops, attenuated the DNA
PARP inhibitors induce TRCs in early S phase damage response induced by PARP inhibitors (Extended Data Fig. 3k,l).
PARP1 and TIMELESS physically interact36,37. We, therefore, examined
whether PARP inhibitors phenocopy depletion of TIMELESS or TIPIN.
HeLa cells were arrested at the G1/S boundary and released into S phase PARP inhibition, not trapping, linked to TRCs
with or without PARP inhibitors and with or without DRB; 200 min The above experiments do not address whether the induction of TRCs
later, γΗ2ΑΧ levels were monitored (Fig. 2a). Four PARP inhibitors were by PARP inhibitors requires trapping of PARPs on DNA or whether it is
examined: olaparib, talazoparib, veliparib and saruparib (also known as sufficient to inhibit the enzymatic activity of PARPs. As a first step to
AZD5305). All induced a DNA damage response, which was suppressed address this question, we characterized the PARP inhibitors for inhi-
by DRB and other inhibitors of transcription elongation (Fig. 2b–d bition of PARP enzymatic activity, PARP trapping and induction of a
and Extended Data Fig. 3a,b). Olaparib and veliparib were also examined TRC-dependent DNA damage response.
for their ability to induce physical proximity of PCNA with RNAPII; both In vitro, the enzymatic activity of PARP1 was inhibited almost equipo-
PARP inhibitors led to a positive PLA signal, which was dependent on tently by all four PARP inhibitors, whereas PARP2 was inhibited potently
transcription elongation (Fig. 2e). by talazoparib, olaparib and veliparib, but less so by saruparib, which
Next, we monitored whether the timing of exposure of the cells to is a PARP1-selective inhibitor38 (Extended Data Fig. 4a). By contrast, in
PARP inhibitors during S phase was important for inducing a DNA dam- cells, the IC50 (half-maximum inhibitory concentration) values for inhi-
age response. Synchronized cells were treated with PARP inhibitors and, bition of PARP enzymatic activity varied by more than 1,000-fold, from

Nature | www.nature.com | 3
Article
a c e
HeLa/DLD1 BRCA2+/+/DLD1 BRCA2–/– HeLa/DLD1 BRCA2+/+ HeLa/DLD1 BRCA2+/+
PARPi H2O2 (2 mM) IF (PAR) PARPi IF (Trapped PARP1/2) Thy Release IF (γH2AX)

–8 h 0h 10 min 0h 24 h –18 h 0h 200 min

±PARPi ± DRB

b HeLa d HeLa f HeLa HeLa

Tal: 0.79 nM Tal: 233 nM Tal: 74.1 nM –DRB Tal: 1.39 μM +DRB
Sar: 2.03 nM 400 Sar: >10 μM 60 Sar: 221 nM 60 Sar: >10 μM
120 Olap: 384 nM Olap: >10 μM Olap: 1.74 μM Olap: >10 μM

γH2AX positive cells (%)

Vel: 1.03 μM Vel: >10 μM Vel: >10 μM Vel: >10 μM

PAR positive cells (%)

mean intensity (r.u.)

100 50 50

Trapped PARP1
80 300 40 40

60 30 30

40 200 20 20

20 10 10

0 100 0 0
–10 –9 –8 –7 –6 –5 –4 –11 –10 –9 –8 –7 –6 –5 –4 –11 –10 –9 –8 –7 –6 –5 –4 –11 –10 –9 –8 –7 –6 –5 –4

DLD1 BRCA2+/+ DLD1 BRCA2+/+ DLD1 BRCA2+/+ DLD1 BRCA2+/+

Tal: 0.71 nM Tal: 812 nM Tal: 18.5 nM –DRB Tal: 208 nM +DRB
Sar: 1.02 nM Sar: >10 μM Sar: 65.2 nM Sar: >10 μM
120 Olap: 67.3 nM 180 Olap: >10 μM 60 Olap: 4.96 μM 60 Olap: >10 μM

γH2AX positive cells (%)

Vel: 1.62 μM Vel: >10 μM Vel: >10 μM Vel: >10 μM
PAR positive cells (%)

mean intensity (r.u.)

100 50 50

Trapped PARP1
150
80 40 40

60 120 30 30

40 20 20
90
20 10 10

0 60 0 0
–10 –9 –8 –7 –6 –5 –4 –11 –10 –9 –8 –7 –6 –5 –4 –11 –10 –9 –8 –7 –6 –5 –4 –11 –10 –9 –8 –7 –6 –5 –4
PARPi [log (M)] PARPi [log (M)] PARPi [log (M)]
DLD1 BRCA2–/–
Tal: 0.76 nM
Sar: 1.08 nM g HeLa h –DRB –DRB
120 Olap: 76.6 nM 2× siRNA <0.0001
+DRB <0.0001 +DRB
Vel: 1.33 μM transf. Thy Release IF 40 40
PAR positive cells (%)

100 <0.0001 0.0001

53BP1 positive cells (%)

γH2AX positive cells (%)
80 –48 h –18 h 0h 200 min 30 30
±DRB NS NS
60 NS
20 20 NS
siPARP1

siPARP2
siCTRL

siCTRL

40
20 10 10
0 PARP1 100 PARP2 55
–10 –9 –8 –7 –6 –5 –4 PCNA 35 PCNA 35
0 0
siCTRL

siPARP1

siPARP2

siPARP1&2

siCTRL

siPARP1

siPARP2

siPARP1&2
PARPi [log (M)]

Fig. 3 | Inhibiting PARP enzymatic activity induces TRCs. a,b, PARP enzymatic n = 2 replicates; for HeLa, more than 7,597 (range 7,597–10,114) cells per data
activity in cells treated with PARP inhibitors (PARPi). a, Outline of the experiment. point; for DLD1, more than 5,512 (range 5,512–9,353) cells per data point.
b, Dose–response curves and calculated IC50 values for inhibition of PARP g,h, DNA damage response in cells transfected with siRNA targeting PARP1
enzymatic activity. One of n = 2 replicates is shown; 100 cells per data point. and/or PARP2. g, Outline of the experiment and immunoblotting to monitor
c,d, PARP1 trapping in cells treated with PARP inhibitors. c, Outline of the PARP1 and PARP2 depletion; PCNA is loading control. h, Means ± 1 s.d. of
experiment. d, Dose–response curves and calculated EC50 values for PARP1 percentage of cells with more than 20 γΗ2ΑΧ or more than 20 53BP1 foci; n = 3
trapping; means ± 1 s.d. shown; n = 2 replicates; for HeLa, more than 1,905 replicates; more than 148 cells per group (range 148–243); analysis of variance
(range 1,905–9,368) cells per data point; for DLD1, more than 1,789 (range (ANOVA). CTRL, control; IF, immunofluorescence; NS, not significant; Olap,
1,789–10,815) cells per data point. e,f, DNA damage response in cells treated olaparib; r.u., relative units; Sar, saruparib; Tal, talazoparib; Thy, thymidine;
with PARP inhibitors. e, Outline of the experiment. f, Dose–response curves transf., transfection; Vel, veliparib.
and calculated EC 50 values for induction of γH2AX; means ± 1 s.d. shown;

about 1 nM for talazoparib and saruparib to about 1 µM for veliparib and PARP2 by siRNA and monitored DNA damage markers in early
(Fig. 3a,b and Extended Data Fig. 4b). Trapping of PARP1 and PARP2 S phase HeLa cells (Fig. 3g). Depletion of PARP1 induced a DNA damage
on chromatin (Fig. 3c,d and Extended Data Fig. 4c,d) did not correlate response, in a manner dependent on transcription elongation, whereas
well with inhibition of PARP catalytic activity. For example, talazoparib depletion of PARP2 had no effect (Fig. 3h). These results support our
trapped PARP1 much more potently than saruparib, even though both conclusion that inhibiting PARP enzymatic activity is sufficient to
inhibited equally well the enzymatic activity of PARP1 in cells (Fig. 3b). induce TRCs, because depleted PARPs cannot be trapped. Moreover,
These results are consistent with previous studies showing that PARP it seems that only PARP1 prevents TRC-induced DNA damage, even
inhibitors have different capacities to trap PARPs on DNA due to dif- though during mouse embryonic development there is some partial
ferences in reverse allostery24. overlap of the functions of PARP1 and PARP2 (ref. 39).
The half-maximum effective concentration (EC50) values of the
four PARP inhibitors for inducing a TRC-dependent DNA damage
response in early S phase (Fig. 3e,f and Extended Data Fig. 4e), cor- TIMELESS and PARP1 act through the same pathway
related best with inhibition of PARP enzymatic activity, rather than PARP1 and TIMELESS interact with each other36,37, suggesting that
with trapping of PARP1 or PARP2 on chromatin (Fig. 3a–d and Extended they may act through the same molecular pathway to prevent TRCs.
Data Fig. 4d). Talazoparib and saruparib were equipotent in inducing As a first step to explore this hypothesis, we depleted TIMELESS or
a TRC-dependent DNA damage response, despite having very different TIPIN by siRNA in cells treated with PARP inhibitors. The depletion of
PARP trapping activities, and olaparib and veliparib, which were weak TIMELESS or TIPIN did not enhance further the DNA damage response
inducers of a DNA damage response, were also the weakest inhibitors induced by olaparib or talazoparib (Extended Data Fig. 5). Next, we
of PARP enzymatic activity in cells (Fig. 3b,f). examined if PARP1 would be hyper-activated in cells, in which TIMELESS
To further explore whether PARP trapping is required for induc- was depleted, the rationale being that if PARP1 signals the presence
tion of a TRC-dependent DNA damage response, we depleted PARP1 of TRCs to the replisome by means of TIMELESS, then in the absence

4 | Nature | www.nature.com
 a c d TIMWT TIMR1081G g

+0.3 μg

+0.3 μg
siCTRL

siTIMb

+1 μg

+1 μg
HeLa asynchronous HeLa HeLa
kDa
siRNA siRNA Plasmid Release IF siRNA
transf. PARGi IF transf. transf. Thy ±DRB PLA TIM transf. Thy Release EdU Collect
130
PCNA 35
–48 h 0h 16 h –48 h –24 h –18 h 0h 200 min –48 h –18 h 0h 90 120 min
+siTIMb

HeLa synchronized
siRNA e –DRB f h
80 50 <0.0001 Fork progression-
transf. Thy Release IF 0.0089 +DRB
average signal

γH2AX positive cells (%)

0.0097 40 0.0001 EdUseq(V) 120 min release

PCNA-RNAPII pS2
–48 h –18 h 0h 200 min 60

PLA foci per cell

PARGi 0.0250 10
30
NS
40 NS siCTRL
20
NS 0
20
10 10
b
Async 0
0 siPARP1
Sync <0.0001
40 80 –DRB 50 <0.0001
0.0004
+DRB 0
Cells with 53BP1 foci (%)
PAR positive cells (%)

0.0152 40
30 60 10

PLA foci per cell

<0.0001

PCNA-RNAPII
0.0257
30
<0.0001 NS siPARP2
20 40
NS
20 0
NS
10 20 Gene
10 annotation
–500 0 +500
0 0 0 Forward kb
siCTRL

siTIM

siCTRL

siTIM

siCTRL

+0.3 μg
TIMWT
+1 μg
TIMWT
+0.3 μg
TIMR1081G
+1 μg
TIMR1081G

siCTRL
–
+TIMWT
+TIMR1081G
Reverse

+siTIMb siTIMb

Fig. 4 | The TIMELESS–PARP1 interaction is required to avert TRCs. cell; plots show medians and value ranges of 25–75 and 10–90%; filled circles
a,b, PAR chain formation following depletion of TIMELESS (TIM) by siRNA. indicate the cells in the top and bottom deciles; n = 2 replicates; more than
a, Outline of the experiment. b, Means ± 1 s.d. of percentage of PAR positive 89 cells per group (range 89–138); ANOVA. g,h, Increased fork progression
cells; n = 3 replicates; more than 512 cells per group (range 512–921); analysis following depletion of PARP1, but not PARP2. g, Outline of the experiment.
of variance (ANOVA). c–f, The TIMELESS–PARP1 interface is important for h, Average EdUseq signal over large (more than 300 kb) transcribed genes
averting TRCs. c, Outline of the experiment. Transfected plasmids drove 120 min after release into S phase. Data from three independent replicates have
expression of exogenous wild-type (TIMWT) or mutant (TIMR1081G) TIMELESS, been merged; the individual replicates are shown in Extended Data Fig. 6. The
while endogenous TIMELESS was depleted by siRNA (TIMb). d, Immunoblot genes were aligned by their transcription start site with their 5′–3′ orientation
monitoring TIMELESS levels. Ectopic TIMELESS has a higher molecular weight from left to right. The lower panel shows gene annotation for each genomic
than endogenous TIMELESS. The TIMb siRNA targets only the endogenous locus used to generate the EdUseq plot. Span of genomic regions, 1 Mb.
TIMELESS gene. PCNA served as loading control. e, Means ± 1 s.d. of percentage Async, asynchronous; CTRL, control; IF, immunofluorescence; NS, not
of cells with more than 20 γΗ2ΑΧ or more than 20 53BP1 foci; n = 3 replicates; significant; PARGi, poly(ADP-ribose) glycohydrolase inhibitor; σ, sigma
more than 97 cells per group (range 97–445); ANOVA. f, Number of PLA foci per value; Sync, synchronized; Thy, thymidine; transf., transfection.

of TIMELESS, TRCs would not be averted and PARP1 activity would be TIMELESS or TIPIN. We monitored EdU incorporation over large, tran-
augmented. Indeed, depletion of TIMELESS enhanced poly(ADP-ribose) scribed genes 120 min after release from a thymidine block (Fig. 4g).
(PAR) chain formation in unsynchronized HeLa cells and, even more Depletion of PARP1 accelerated fork progression to a similar extent to
so, in HeLa cells synchronized in early S phase (Fig. 4a,b). the depletion of TIMELESS or TIPIN, whereas depletion of PARP2 had no
To examine more directly the importance of a physical interac- obvious effect (Fig. 4h and Extended Data Fig. 6). These results are con-
tion between TIMELESS and PARP1 in preventing TRCs, the endog- sistent with PARP1 signalling through TIMELESS the presence of TRCs
enous TIMELESS and/or PARP1 proteins were depleted by siRNA and to the replisome, whereas PARP2 does not interact with TIMELESS36,37.
exogenous wild-type or mutant versions of the above proteins were
expressed. The mutant versions had single amino acid substitutions
targeting the TIMELESS–PARP1 interface37. The transfected cells were TRCs mediate the synthetic lethality of PARP
synchronized in G1/S, released into S phase, and 200 min later, DNA inhibitors
damage response markers and TRCs were monitored by immunofluo- The TRC-dependent DNA damage response induced by PARP inhibitors
rescence and PLAs, respectively (Fig. 4c). Consistent with the binding of raised the question of whether TRCs drive the synthetic lethality of
PARP1 to TIMELESS being functionally important, ectopic expression PARP inhibitors with HR deficiency. To help answer this question, DLD1
of wild-type TIMELESS and PARP1 proteins prevented the induction BRCA2+/+ and BRCA2−/− cells were released from a thymidine block into
of TRCs and TRC-dependent DNA damage, whereas expression of the S phase and PARP inhibitors were administered with or without DRB
mutant TIMELESS or PARP1 proteins did not (Fig. 4d–f and Extended either during early or late S phase (0–3.5 and 7–10.5 h after release from
Data Fig. 5c,d). the thymidine block, respectively). Twenty-four hours after release
Finally, we examined whether depletion of PARP1 or PARP2 affected from the thymidine block, when the cells should have progressed into
fork progression, similar to what we observed in cells depleted of the next cell cycle, we scored for micronuclei, markers of aberrant

Nature | www.nature.com | 5
Article
a b +Olap c BRCA2+/+ BRCA2–/–
DLD1 BRCA2+/+/DLD1 BRCA2–/– BRCA2+/+ BRCA2–/– –DRB +DRB –DRB +DRB
Early S Late S

DAPI γH2AX
EF

Micronuclei
Thy Release Wash Wash IF

+Olap
–18 h 0h 3.5 h 7h 10.5 h 24 h
±PARPi ±PARPi
±DRB ±DRB

d PARPi 0–3.5 h e PARPi 0–3.5 h f PARPi 7–10.5 h

BRCA2+/+ BRCA2–/– BRCA2+/+ BRCA2–/– BRCA2+/+ BRCA2–/–
–DRB +DRB 0.0016 –DRB +DRB –DRB +DRB

0.0001
15 15 40 50 15 15

0.0130
<0.0001
Cells with micronuclei (%)

Cells with micronuclei (%)

0.0099

0.0083
γH2AX positive cells (%)

0.0339
0.0096
40
30
10 10 10 10
30

NS
20
NS

NS
NS

NS

NS
NS
NS

NS
20

NS
NS

NS
NS
NS
NS

5 5 5 5

NS
NS
NS

NS
NS

NS
10

NS
10

0 0 0 0 0 0

DMSO
Olap
Tal
Vel
Sar
DMSO
Olap
Tal
Vel
Sar

DMSO
Olap
Tal
Vel
Sar

DMSO
Olap
Tal
Vel
Sar

DMSO
Olap
Tal
Vel
Sar

DMSO
Olap
Tal
Vel
Sar
g DLD1 BRCA2+/+/DLD1 BRCA2–/– i DLD1 BRCA2–/– k HeLa HeLa/DLD1 BRCA2+/+/DLD1 BRCA2–/–
Early S
siRNA siRNA FACS siRNA
Thy Release Replate CFA transf. Thy Release Replate CFA transf. (γH2AX) transf. Replate CFA

–18 h 0h 3.5 h 14 days –48 h –18 h 0h 200 min 14 days 0h 72 h –72 h 0h 14 days
±PARPi
±DRB
±DRB

h PARPi 0–3.5 h j l m
BRCA2+/+ BRCA2–/– DLD1 BRCA2–/– HeLa HeLa DLD1
–DRB +DRB –DRB +DRB
Cells with pan-nuclear γH2AX (%)

–DRB +DRB <0.0001 BRCA2+/+

<0.0001
20 <0.0001 BRCA2–/–
NS <0.0001
0.0026
NS
NS

0.0051
NS
NS

0.0088

0.0008
NS

0.0488
Survival-CFA (%)

Survival-CFA (%)
NS

100 <0.0001

Survival-CFA (%)
15 <0.0001
100 100 100

10
50
50 50 50
5

0 0 0 0 0
DMSO
Olap
Tal
Vel
Sar

siCTRL

siPARP1

siCTRL
siTIM
siTIP

siTIM
siTIP
DMSO
Olap
Tal
Vel
Sar

siCTRL
siTIM
siTIP
siCTRL
siTIM
siTIP
–

siCTRL
siTIM
siTIP
–
siTIM
siTIP
siBRCA2 siBRCA2

Fig. 5 | Inhibition of transcription elongation confers resistance to PARP by colony formation assay (CFA); DMSO-treated cells serve as reference; n = 3
inhibitors. a–f, DNA damage by PARP inhibitors (PARPi) is suppressed by replicates; ANOVA. i,j, Synthetic lethality induced by PARP1 depletion is
inhibiting transcription elongation. a, Outline of the experiment. PARP alleviated by inhibiting transcription elongation. i, Outline of the experiment.
inhibitors and, optionally, DRB were administered during early or late S phase j, Means ± 1 s.d. of percentage of surviving cells, as assessed by CFA; non-
(0–3.5 or 7–10.5 h after release from a thymidine (Thy) block, respectively). transfected cells serve as reference; n = 3 replicates; ANOVA. k–m, Depletion
b,c, Representative images of cells treated with olaparib (Olap, 10 μΜ) during of TIMELESS (TIM) or TIPIN (TIP) are synthetic lethal with HR deficiency.
early S phase showing micronuclei (b) and γΗ2ΑΧ foci (c) 24 h after release from k, Outline of the experiments. l, Means ± 1 s.d. of percentage of cells with
the thymidine block. d–f, Means ± 1 s.d. of percentage of cells with micronuclei pan-nuclear γΗ2ΑΧ staining; n = 3 replicates; ANOVA. m, Means ± 1 s.d. of
(d,f) or more than 20 γΗ2ΑΧ foci (e) after treatment with PARP inhibitors in percentage of surviving cells, as assessed by CFA; control-transfected cells
early (d,e) or late (f) S phase; n = 3 replicates; more than 250 cells per group serve as reference; n = 3 replicates; ANOVA. Scale bars for microscopy images,
(range 250–400); analysis of variance (ANOVA). g,h, Synthetic lethality induced 10 μm. CTRL, control; EF, epifluorescence; FACS, fluorescence-activated cell
by PARP inhibitors is alleviated by inhibiting transcription elongation. g, Outline sorting; IF, immunofluorescence; NS, not significant; Sar, saruparib (1 μΜ);
of the experiment. h, Means ± 1 s.d. of percentage of surviving cells, as assessed Tal, talazoparib (100 nM); transf., transfection; Vel, veliparib (10 μΜ).

mitoses, and for γH2AX (Fig. 5a). A significant fraction of the BRCA2−/− inhibitors, paralleled the presence of micronuclei and γH2AX signal
cells treated with PARP inhibitors in early but not late S phase scored and was alleviated by DRB, whereas the BRCA2+/+ cells survived well
positive for both markers; moreover, the emergence of these markers the PARP inhibitor treatment (Fig. 5g,h and Extended Data Fig. 7c,d).
was suppressed by administering DRB together with the PARP inhibi- Cell lethality induced by PARP inhibitors in PEO1 HR-deficient ovarian
tors (Fig. 5b–f and Extended Data Fig. 7a,b). By contrast, the BRCA2+/+ cancer cells40 was also reversed by DRB, whereas HR-proficient PEO4
cells were generally devoid of micronuclei and γH2AX signal, indicat- ovarian cancer cells, derived from the same cancer as the PEO1 cells40,
ing that any TRC-related DNA damage induced by the PARP inhibitors were resistant to PARP inhibitors (Extended Data Fig. 7e,f). In further
(Fig. 2) had been repaired after removal of the PARP inhibitors from the support of the notion that TRCs are synthetic lethal with HR deficiency,
tissue culture media. Cell lethality of the BRCA2−/− cells, as determined HR-deficient cells were more sensitive to the topoisomerase I inhibi-
by a colony formation assay 14 days after treatment with the PARP tor camptothecin than HR-proficient cells (Extended Data Fig. 7g,h).

6 | Nature | www.nature.com
 a c e g DLD1 BRCA2+/+/DLD1 BRCA2–/–
DLD1 BRCA2+/+/DLD1 BRCA2–/– DLD1 BRCA2+/+ DLD1 BRCA2+/+/DLD1 BRCA2–/–
siRNA Replate
Plating ±PARPi CTG ±PARPi + 50 μM TMZ IF Plating ±PARPi ± TMZ CTG transf. ±PARPi ± TMZ Wash CTG

–24 h 0h 5 days 0h 24 h –24 h 0h 5 days –24 h 0h 2 days 5 days

b d f h
DLD1 BRCA2+/+ DLD1 BRCA2+/+ DLD1 BRCA2+/+ DLD1 BRCA2+/+ BRCA2+/+ + TMZ 20 μM BRCA2+/+ + TMZ 50 μM
Tal: 767 nM Tal + TMZ: 3.73 nM Tal: 807 nM siCTRL: 391 nM siCTRL: 8.87 nM siCTRL: 2.40 nM

Trapped PARP1 mean intensity (r.u.)

Sar: 120 μM Olap + TMZ: 5.44 nM Tal + 20 μM TMZ: 54 nM siPARP1: 23 μM siPARP1: 1.23 μM siPARP1: 67.6 nM
Olap: 59.0 μM Sar + TMZ: 6.51 nM Tal + 50 μM TMZ: 4.30 nM
120 Vel: >500 μM 350 120 140

100 100 120

Survival-CTG (%)

Survival-CTG (%)

Survival-CTG (%)
300
100 59×
80 80
80 140×
60 250 60 190×
60 28×
40 40
200 40
20 20 20
150
0 0 0
–10 –9 –8 –7 –6 –5 –4 –10 –9 –8 –7 –6 –5 –4 –10 –9 –8 –7 –6 –5 –4 –12–11–10 –9 –8 –7 –6 –5 –4 –12–11–10 –9 –8 –7 –6 –5 –4 –12–11–10 –9 –8 –7 –6 –5 –4

DLD1 BRCA2–/– DLD1 BRCA2+/+ DLD1 BRCA2–/– DLD1 BRCA2–/– BRCA2+/+ + TMZ 20 μM BRCA2+/+ + TMZ 50 μM
Trapped PARP2 mean intensity (r.u.)

Tal: 1.51 nM Tal + TMZ: 2.83 nM Tal: 1.16 nM siCTRL: 0.88 nM siCTRL: 0.30 nM siCTRL: 0.14 nM
Sar: 3.92 nM Olap + TMZ: 75.5 nM Tal + 20 μM TMZ: 0.40 nM siPARP1: ND siPARP1: ND siPARP1: ND
120 Olap: 356 nM Sar + TMZ: >10 μM 120 Tal + 50 μM TMZ: 0.16 nM
Vel: 4.59 μM 140
100 600 100 120
Survival-CTG (%)

Survival-CTG (%)

Survival-CTG (%)
80 80 100
500 80
60 60 7×
60
40 400 40
40
20 20 20
300
0 0 0
–10 –9 –8 –7 –6 –5 –4 –10 –9 –8 –7 –6 –5 –4 –12 –11 –10 –9 –8 –7 –6 –14–13–12 –11–10 –9 –8 –7 –6 –14–13–12 –11–10 –9 –8 –7 –6 –14–13–12 –11–10 –9 –8 –7 –6
PARPi [log (M)] PARPi [log (M)] Talazoparib [log (M)] Talazoparib [log (M)] Talazoparib [log (M)] Talazoparib [log (M)]

Fig. 6 | PARP trapping reduces the selectivity of PARP inhibitors for HR for talazoparib-mediated lethality of DLD1 BRCA2+/+ and DLD1 BRCA2−/− cells
deficiency. a,b, Sensitivity of DLD1 BRCA2+/+ and DLD1 BRCA2−/− cells to PARP with and without added TMZ. Horizontal arrows indicate the fold-change in
inhibitors (PARPi). a, Outline of the experiment. Survival was assessed using EC50 values as a result of administering 50 μM TMZ. Data from one of n = 2
the CellTiter-Glo (CTG) assay. b, Dose–response survival curves and calculated replicate experiments. g,h, Depletion of PARP1 renders HR-proficient cells
EC50 values for the various PARP inhibitors. Data from one of n = 2 replicates. resistant to talazoparib. g, Outline of the experiment. h, Dose–response
c,d, TMZ enhances trapping of PARP1 and PARP2. c, Outline of the experiment. survival curves and calculated EC50 values for induction of lethality of DLD1
d, Dose–response curves and calculated EC50 values for trapping of PARP1 and BRCA2 +/+ and DLD1 BRCA2−/− cells by talazoparib following depletion of PARP1
PARP2; data from one of n = 2 replicates; for PARP1 > 120 (range 120–4,848) in the absence and presence of TMZ. Horizontal arrows indicate the fold-
cells per data point; for PARP2 > 2,293 (range 2,293–5,377) cells per data point. change in EC50 values as a result of depleting PARP1; n = 2 replicates. CTRL,
e,f, TMZ reduces the selectivity of talazoparib (Tal) for HR deficiency. e, Outline control; IF, immunofluorescence; ND, EC50 values not determined; Olap,
of the experiment. f, Dose–response survival curves and calculated EC50 values olaparib; r.u., relative units; Sar, saruparib; transf., transfection; Vel, veliparib.

Depletion of PARP1 by siRNA induces a TRC-dependent DNA damage To address this further, we determined dose–response curves by
response similar to the one induced by PARP inhibitors (Fig. 3g,h). We, which the four PARP inhibitors induced lethality of DLD1 BRCA2−/−
therefore, examined whether depletion of PARPs by siRNA induced and BRCA2+/+ cells (Fig. 6a,b). For the HR-deficient BRCA2−/− cells, the
synthetic lethality with HR deficiency. We studied cancer cell lines that dose–response lethality curves (Fig. 6b) matched the curves for inhibi-
were naturally HR-deficient or in which HR deficiency was induced tion of PARP1 cellular enzymatic activity (Fig. 3b), but not the curves
by targeting the BRCA2 gene. Depletion of PARP1 or both PARP1 and for PARP1 or PARP2 trapping (Fig. 3d and Extended Data Fig. 4d). The
PARP2 compromised the survival of the HR-deficient cancer cells, mir- inverse was observed when we plotted the dose–response curves for
roring the effect of the PARP inhibitors, whereas depletion of PARP2 the HR-proficient BRCA2+/+ cells; here, the dose–response curves for
had no effect (Extended Data Fig. 8). The synthetic lethality induced induction of lethality (Fig. 6b) matched the curves for PARP1 and PARP2
by PARP1 depletion could be partially suppressed by treating the trapping (Fig. 3d and Extended Data Fig. 4d).
siRNA-transfected cells with DRB during early S phase (Fig. 5i,j). To explore further the role of PARP trapping on survival of
Finally, because PARP1, TIMELESS and TIPIN function in the same HR-deficient and HR-proficient cells, we co-administered PARP inhibi-
pathway to prevent TRCs, we examined whether depletion of TIME- tors with temozolomide (TMZ). TMZ, a DNA alkylating prodrug, induces
LESS or TIPIN, similar to PARP1 depletion, were synthetic lethal with HR DNA nicks in which PARPs are recruited and, potentially, trapped17,41,42.
deficiency (Fig. 5k). Codepletion of BRCA2 and TIMELESS or BRCA2 and We determined dose–response curves for trapping of PARP1 and PARP2
TIPIN led to a strong DNA damage response in HeLa cells, as revealed by on chromatin in cells treated with talazoparib, saruparib or olaparib in
pan-nuclear γH2AX staining, and to cell lethality; whereas, depletion the presence of 50 µM TMZ. Addition of TMZ enhanced significantly
of TIMELESS or TIPIN or BRCA2 on their own had a much smaller effect PARP trapping by all PARP inhibitors (Fig. 3d versus Fig. 6c,d).
(Fig. 5l). Similarly, depletion of TIMELESS or TIPIN compromised the Next we examined whether the increased PARP trapping affected
viability of DLD1 BRCA2−/− cells, while sparing the HR-proficient DLD1 the dose–response curves for induction of lethality in DLD1 BRCA2−/−
BRCA2+/+ cells (Fig. 5k,m). and DLD1 BRCA2+/+ cells treated with PARP inhibitors. TMZ, at 50 µM,
enhanced 190-fold the potency by which talazoparib induced
lethality of HR-proficient cells, whereas the corresponding effect
PARP trapping reduces the selectivity of PARP inhibitors for HR-deficient cells was only sevenfold, meaning that the selec-
The experiments presented so far indicate that the synthetic lethality tivity for HR deficiency was significantly reduced (Fig. 6e,f). Simi-
of PARP inhibitors with HR deficiency is due, at least in part, to TRCs lar effects were observed with saruparib and olaparib (Extended
and that TRCs can be induced without trapping PARPs on DNA. Data Fig. 9a,b).

Nature | www.nature.com | 7
Article
Following on from the above observations, we determined dose– PARP1 was depleted by siRNA5. Moreover, the studies demonstrating
response lethality curves for talazoparib-treated DLD1 BRCA2−/− and that PARP1 trapping is required for PARP inhibitors to be cytotoxic
DLD1 BRCA2+/+ cells, in which endogenous PARP1 was depleted by siRNA examined mostly HR-proficient cells and often stimulated PARP trap-
(Fig. 6g). Depletion of PARP1 by siRNA severely reduced the viabil- ping by combining PARP inhibitors with TMZ or other DNA damaging
ity of DLD1 BRCA2−/− cells (Fig. 6h), suggesting that the loss of PARP1 agents21–23,42,50–52.
enzymatic activity is sufficient to induce lethality in HR-deficient cells. Our findings raise the question whether modulating PARP trapping
By contrast, PARP1-depleted DLD1 BRCA2+/+ cells were highly resistant can enhance the therapeutic window of PARP inhibitors in the clinic3,17,18.
to talazoparib, in accordance with its on-target inhibition (Fig. 6h). PARP inhibitors differ in their trapping potential, so this is a parameter
Finally, consistent with lethality being linked to a persistent DNA that can be optimized independently of inhibitory activity. We propose
damage response, depletion of PARP1 suppressed the DNA damage that reducing trapping potential may decrease the toxicity of PARP
response in talazoparib-treated HR-proficient, but not HR-deficient, inhibitors without compromising efficacy.
cells (Extended Data Fig. 9c–e).

Online content
Discussion Any methods, additional references, Nature Portfolio reporting summa-
Several mechanisms have been proposed to explain why PARP inhibi- ries, source data, extended data, supplementary information, acknowl-
tors are synthetic lethal with HR deficiency. The now-favoured mecha- edgements, peer review information; details of author contributions
nism posits that PARP inhibitors trap PARPs on DNA; in turn, the trapped and competing interests; and statements of data and code availability
PARPs block fork progression, leading to DNA DSBs that require HR for are available at https://doi.org/10.1038/s41586-024-07217-2.
repair7. Here, we propose a TRC mechanism; specifically, that PARP1
signals the presence of impending TRCs to TIMELESS and TIPIN, paus- 1. Fong, P. C. et al. Inhibition of poly(ADP-ribose) polymerase in tumors from BRCA mutation
carriers. N. Engl. J. Med. 361, 123–134 (2009).
ing the replisome until the TRCs are resolved. If PARP1 or TIMELESS
2. Konstantinopoulos, P. A., Ceccaldi, R., Shapiro, G. I. & D’Andrea, A. D. Homologous
and TIPIN fail to perform their function, TRCs lead to DNA damage recombination deficiency: exploiting the fundamental vulnerability of ovarian cancer.
that requires HR for repair. The main difference between the two pro- Cancer Discov. 5, 1137–1154 (2015).
3. Pommier, Y., O’Connor, M. J. & de Bono, J. Laying a trap to kill cancer cells: PARP inhibitors
posed mechanisms is the nature of the object with which the replisomes
and their mechanisms of action. Sci. Transl. Med. 8, 362ps17 (2016).
collide: trapped PARPs versus transcription elongation complexes. 4. Lord, C. J. & Ashworth, A. PARP inhibitors: synthetic lethality in the clinic. Science 355,
Evidence supporting the revised mechanism is that the synthetic lethal- 1152–1158 (2017).
5. Bryant, H. E. et al. Specific killing of BRCA2-deficient tumours with inhibitors of
ity of PARP inhibitors with HR deficiency can be alleviated by inhibiting
poly(ADP-ribose) polymerase. Nature 434, 913–917 (2005).
transcription elongation and that depleting TIMELESS, TIPIN or PARP1 6. Farmer, H. et al. Targeting the DNA repair defect in BRCA mutant cells as a therapeutic
by siRNA is synthetic lethal with HR deficiency. strategy. Nature 434, 917–921 (2005).
7. Helleday, T. The underlying mechanism for the PARP and BRCA synthetic lethality:
Recent observations by others are consistent with the TRC mecha-
clearing up the misunderstandings. Mol. Oncol. 5, 387–393 (2011).
nism proposed here. Depletion of TIMELESS induces formation of 8. Moynahan, M. E. & Jasin, M. Mitotic homologous recombination maintains genomic
R-loops43, consistent with induction of TRCs; PARP1 binds to R-loops44; stability and suppresses tumorigenesis. Nat. Rev. Mol. Cell Biol. 11, 196–207 (2010).
9. Nielsen, F. C., van Overeem Hansen, T. & Sørensen, C. S. Hereditary breast and ovarian
PARP inhibitors enhance replication fork speed45 and HR repairs DNA
cancer: new genes in confined pathways. Nat. Rev. Cancer 16, 599–612 (2016).
damage induced by TRCs33,34. 10. Lüscher, B. et al. ADP-ribosyltransferases, an update on function and nomenclature. FEBS
Our study did not address the mechanism(s) by which PARP1 senses J. 289, 7399–7410 (2021).
11. Ray Chaudhuri, A. & Nussenzweig, A. The multifaceted roles of PARP1 in DNA repair and
TRCs. One possibility is that PARP1 is recruited by R-loops44 or other chromatin remodelling. Nat. Rev. Mol. Cell Biol. 18, 610–621 (2017).
DNA structures generated at sites of impending TRCs. A second, 12. Crawford, K., Bonfiglio, J. J., Mikoč, A., Matic, I. & Ahel, I. Specificity of reversible
non-mutually exclusive, possibility is that PARP1 is activated by topoi- ADP-ribosylation and regulation of cellular processes. Crit. Rev. Biochem. Mol. Biol. 53,
64–82 (2017).
somerase I, which is present at sites of TRCs to resolve DNA supercoil- 13. Satoh, M. S. & Lindahl, T. Role of poly(ADP-ribose) formation in DNA repair. Nature 356,
ing46,47. Supporting the latter hypothesis, PARP1 and topoisomerase I 356–358 (1992).
function together to maintain fork stability under conditions of DNA 14. Langelier, M. F., Planck, J. L., Roy, S. & Pascal, J. M. Structural basis for DNA damage-
dependent poly(ADP-ribosyl)ation by human PARP-1. Science 336, 728–732 (2012).
replication stress48 and, in budding yeast, Tof1, the orthologue of TIME- 15. Eustermann, S. et al. Structural basis of detection and signaling of DNA single-strand
LESS, functions together with topoisomerase I to pause the replisome breaks by human PARP-1. Mol. Cell 60, 742–754 (2015).
ahead of replication fork barriers49. Yet another possibility is that the 16. Bilokapic, S., Suskiewicz, M. J., Ahel, I. & Halic, M. Bridging of DNA breaks activates
PARP2-HPF1 to modify chromatin. Nature 585, 609–613 (2020).
TRCs are sensed by TIMELESS, which then recruits PARP1, along the 17. Hopkins, T. A. et al. Mechanistic dissection of PARP1 trapping and the impact on in vivo
lines proposed for a role of TIMELESS in sensing DSBs36,37. tolerability and efficacy of PARP inhibitors. Mol. Cancer Res. 13, 1465–1477 (2015).
An improved understanding of how PARP inhibitors target HR- 18. Hopkins, T. A. et al. PARP1 trapping by PARP inhibitors drives cytotoxicity in both cancer
cells and healthy bone marrow. Mol. Cancer Res. 17, 409–419 (2019).
deficient cells, could help guide their future clinical development, 19. Rudolph, J., Roberts, G. & Luger, K. Histone parylation factor 1 contributes to the inhibition
especially as it relates to isoform specificity and trapping potential. All of PARP1 by cancer drugs. Nat. Commun. 12, 736 (2021).
PARP inhibitors, used at present in the clinic, inhibit both PARP1 and 20. Rudolph, J., Jung, K. & Luger, K. Inhibitors of PARP: number crunching and structure
gazing. Proc. Natl Acad. Sci. USA 119, e2121979119 (2022).
PARP2. However, to protect replication forks from TRCs, the critical 21. Murai, J. et al. Trapping of PARP1 and PARP2 by clinical PARP inhibitors. Cancer Res. 72,
family member might be PARP1, because TIMELESS binds preferentially 5588–5599 (2012).
to this family member36,37. Thus, a PARP1-selective inhibitor, such as 22. Pettitt, S. J. et al. A genetic screen using the PiggyBac transposon in haploid cells
identifies Parp1 as a mediator of olaparib toxicity. PLoS ONE 8, e61520 (2013).
saruparib, might suffice for inducing synthetic lethality with HR defi- 23. Murai, J. et al. Stereospecific PARP trapping by BMN 673 and comparison with olaparib
ciency in the clinic, as demonstrated here with cell lines. and rucaparib. Mol. Cancer Ther. 13, 433–443 (2014).
The TRC mechanism may help inform whether PARP inhibitors should 24. Zandarashvili, L. et al. Structural basis for allosteric PARP-1 retention on DNA breaks.
Science 368, eaax6367 (2020).
have high or low trapping activity. The four PARP inhibitors we studied 25. Nedelcheva, M. N. et al. Uncoupling of unwinding from DNA synthesis implies regulation
here showed a very good correlation between induction of lethality of MCM helicase by Tof1/Mrc1/Csm3 checkpoint complex. J. Mol. Biol. 347, 509–521
of HR-deficient cells and inhibition of PARP enzymatic activity and (2005).
26. Tourrière, H., Versini, G., Cordón-Preciado, V., Alabert, C. & Pasero, P. Mrc1 and Tof1
no correlation with trapping potential. Moreover, depletion of PARP1 promote replication fork progression and recovery independently of Rad53. Mol. Cell 19,
by siRNA induced lethality of HR-deficient cells. Consistent with the 699–706 (2005).
model that trapping may not be important for therapeutic efficacy, 27. Hodgson, B., Calzada, A. & Labib, K. Mrc1 and Tof1 regulate DNA replication forks in
different ways during normal S phase. Mol. Biol. Cell 18, 3894–3902 (2007).
one of the original reports describing the synthetic lethality between 28. Leman, A. R. & Noguchi, E. Local and global functions of Timeless and Tipin in replication
PARP inhibitors and HR deficiency included experiments in which fork protection. Cell Cycle 11, 3945–3955 (2012).

8 | Nature | www.nature.com
29. Yankulov, K., Yamashita, K., Roy, R., Egly, J. M. & Bentley, D. L. The transcriptional elongation 44. Laspata, N. et al. PARP1 associates with R-loops to promote their resolution and genome
inhibitor 5,6-dichloro-1-beta-d-ribofuranosylbenzimidazole inhibits transcription factor stability. Nucleic Acids Res. 51, 2215–2237 (2023).
IIH-associated protein kinase. J. Biol. Chem. 270, 23922–23925 (1995). 45. Maya-Mendoza, A. et al. High speed of fork progression induces DNA replication stress
30. García-Muse, T. & Aguilera, A. R loops: from physiological to pathological roles. Cell 179, and genomic instability. Nature 559, 279–284 (2018).
604–618 (2019). 46. García-Muse, T. & Aguilera, A. Transcription-replication conflicts: how they occur and
31. Hamperl, S., Bocek, M. J., Saldivar, J. C., Swigut, T. & Cimprich, K. A. how they are resolved. Nat. Rev. Mol. Cell Biol. 17, 553–563 (2016).
Transcription-replication conflict orientation modulates R-loop levels and activates 47. Hamperl, S. & Cimprich, K. A. Conflict resolution in the genome: how transcription and
distinct DNA damage responses. Cell 170, 774–786 (2017). replication make it work. Cell 167, 1455–1467 (2016).
32. Stoy, H. et al. Direct visualization of transcription-replication conflicts reveals 48. Ray Chaudhuri, A. et al. Topoisomerase I poisoning results in PARP-mediated replication
post-replicative DNA:RNA hybrids. Nat. Struct. Mol. Biol. 30, 348–359 (2023). fork reversal. Nat. Struct. Mol. Biol. 19, 417–423 (2012).
33. Bhowmick, R. et al. RAD51 protects human cells from transcription-replication conflicts. 49. Shyian, M. et al. Fork pausing complex engages topoisomerases at the replisome. Genes
Mol. Cell 82, 3366–3381 (2022). Dev. 34, 87–98 (2019).
34. Groelly, F. J. et al. Mitotic DNA synthesis is caused by transcription-replication conflicts in 50. Kim, C., Wang, X. D. & Yu, Y. PARP1 inhibitors trigger innate immunity via PARP1 trapping-
BRCA2-deficient cells. Mol. Cell 82, 3382–3397 (2022). induced DNA damage response. eLife 9, e60637 (2020). (2020).
35. Macheret, M. & Halazonetis, T. D. Intragenic origins due to short G1 phases underlie 51. Verma, P. et al. ALC1 links chromatin accessibility to PARP inhibitor response in
oncogene-induced DNA replication stress. Nature 555, 112–116 (2018). homologous recombination-deficient cells. Nat. Cell Biol. 23, 160–171 (2021).
36. Young, L. M. et al. TIMELESS forms a complex with PARP1 distinct from its complex with 52. Higuchi, F. et al. Restoration of temozolomide sensitivity by PARP inhibitors in mismatch
TIPIN and plays a role in the DNA damage response. Cell Rep. 13, 451–459 (2015). repair deficient glioblastoma is independent of base excision repair. Clin. Cancer Res. 26,
37. Xie, S. et al. Timeless interacts with PARP-1 to promote homologous recombination repair. 1690–1699 (2020).
Mol. Cell 60, 163–176 (2015).
38. Illuzzi, G. et al. Preclinical characterization of AZD5305, a next-generation, highly Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in
selective PARP1 inhibitor and trapper. Clin. Cancer Res. 28, 4724–4736 (2022). published maps and institutional affiliations.
39. Ménissier de Murcia, J. et al. Functional interaction between PARP-1 and PARP-2 in
chromosome stability and embryonic development in mouse. EMBO J. 22, 2255–2263 Open Access This article is licensed under a Creative Commons Attribution
(2003). 4.0 International License, which permits use, sharing, adaptation, distribution
40. Sakai, W. et al. Functional restoration of BRCA2 protein by secondary BRCA2 mutations in and reproduction in any medium or format, as long as you give appropriate
BRCA2-mutated ovarian carcinoma. Cancer Res. 69, 6381–6386 (2009). credit to the original author(s) and the source, provide a link to the Creative Commons licence,
41. Miknyoczki, S. J. et al. Chemopotentiation of temozolomide, irinotecan, and cisplatin and indicate if changes were made. The images or other third party material in this article are
activity by CEP-6800, a poly(ADP-ribose) polymerase inhibitor. Mol. Cancer Ther. 2, included in the article’s Creative Commons licence, unless indicated otherwise in a credit line
371–382 (2003). to the material. If material is not included in the article’s Creative Commons licence and your
42. Murai, J. et al. Rationale for poly(ADP-ribose) polymerase (PARP) inhibitors in combination intended use is not permitted by statutory regulation or exceeds the permitted use, you will
therapy with camptothecins or temozolomide based on PARP trapping versus catalytic need to obtain permission directly from the copyright holder. To view a copy of this licence,
inhibition. J. Pharmacol. Exp. Ther. 349, 408–416 (2014). visit http://creativecommons.org/licenses/by/4.0/.
43. Andrs, M. et al. Excessive reactive oxygen species induce transcription-dependent
replication stress. Nat. Commun. 14, 1791 (2023). © The Author(s) 2024

Nature | www.nature.com | 9
Article
Methods (Gen5-Acoustic Transfer System; EDC Biosystems). The final concentra-
tion of the PARP inhibitors ranged from 0.2 to 100 nM using twofold
Cell culture and drug treatments dilution steps. The absorbance at 450 nm was measured using a Spark
The human cervical cell line HeLa and the human osteosarcoma cell 10M microplate reader (Tecan).
line U2OS were purchased from the American Type Culture Collec-
tion (ATCC) (CCL-2 and HTB-96 respectively) and were grown under siRNA and plasmid transfections
standard conditions (37 °C and 5% CO2) in Dulbecco’s modified Eagle’s Transfections of siRNAs (at a final concentration of 40 nM) were per-
medium (DMEM) (Invitrogen, catalogue no. 11960) supplemented formed with the cells at 60% confluency using INTERFERin (Polyplus,
with 10% fetal bovine serum (FBS) (Invitrogen, catalogue no. 10500) catalogue no. 409-01) or Lipofectamine RNAiMAX Transfection Rea-
and penicillin–streptomycin–glutamine (Invitrogen, catalogue no. gent (Thermo Scientific, catalogue no. 13778075) according to the
10378-016). The hTERT-RPE1 retinal pigment epithelial cells (CRL4000, manufacturer’s instructions. TIMELESS depletion was achieved by
ATCC), were cultured in DMEM and Ham’s F-12 (Invitrogen, catalogue transfection of 10 nM siRNA. Medium change was performed 24 h
no. 12634-010), supplemented with 10% FBS and penicillin–strepto- after siRNA transfection. The following siRNAs were used: negative
mycin–glutamine. DLD1 BRCA2+/+ (ATCC, CCL-221) and DLD1 BRCA2−/− control (AllStars Negative Control siRNA, Qiagen, catalogue no.
(Horizon, catalogue no. HD 105-007) were cultured in Roswell Park 1027281), siTIM (TIMELESS; Qiagen, catalogue no. SI04142194), siTIMb
Memorial Institute (RPMI) 1640 medium (Thermo Scientific, catalogue (Dharmacon, 5′-GUAGCUUAGUCCUUUCAAATT-3′), siTIP (TIPIN; Inv­
no. 11875093) supplemented with 10% FBS and penicillin–strepto- itrogen, catalogue no. S29864), siPARP1 (Qiagen, catalogue nos.
mycin–glutamine. HeLa cells expressing FLAG-tagged RNase H1 in a SI02662989 and SI02662996) and (Invitrogen, catalogue no. s1097),
doxycycline (DOX)-dependent manner53 were grown in DMEM sup- siPARP1b (Dharmacon, 5′-GGAAAGAUGUUAAGCAUUUTT-3′ and
plemented with 10% Tet system-approved FBS (Biowest, catalogue 5′-CAUGGGAGCUCUUGAAAUATT-3′ and 5′-AGAAAAGGCUGGAGAG
no. S181T) and antibiotics. Expression of FLAG-RNase H1 was induced AGATT-3′), siPARP2 (Invitrogen, catalogue no. S19504), siBRCA2 (Qia-
with addition of 2 μg ml−1 DOX (Sigma-Aldrich, catalogue no. D9891) in gen, catalogue no. SI02653434). Efficiency of siRNA-mediated deple-
the medium for 18 h. U2OS T-Rex cells expressing catalytically inactive tion was performed 72 h after transfection by western blotting. Empty
GFP-RNaseH1D201N in a DOX-dependent manner54 were grown in DMEM vector, full length GFP-TIMELESS WT, GFP-TIMELESS R1081G, full length
supplemented with 10% Tet system-approved FBS, 1 μg ml−1 puromycin FLAG-PARP1 WT and FLAG-PARP1 D993G plasmids37 were transfected
(Sigma, catalogue no. P8833) and 50 μg ml−1 hygromycin B (Thermo using the FuGENE HD transfection reagent (Promega, catalogue no.
Scientific, catalogue no. 10687010). Expression of catalytically inactive E2311) according to manufacturer’s instructions. Efficiency of plasmid
GFP-RNaseH1D201N was induced by addition of 1 ng ml−1 DOX for 18 h. transfection was performed by western blot for detection of TIMELESS
The human non-small cell lung carcinoma H1299 cells, expressing and PARP1 proteins.
a short-hairpin RNA against BRCA2 in a DOX-dependent manner55,
were grown in DMEM supplemented with 10% Tet system-approved Immunoblotting
FBS and antibiotics. Expression of shBRCA2 was induced by addition Protein cell extracts were resolved by SDS–PAGE in precast protein
of 2 μg ml−1 DOX in the medium. The human PEO1 and PEO4 ovarian gels (4–15% Mini-PROTEAN TGX, Bio-Rad, catalogue no. 4561083, or
cancer cell lines40 (provided by I. Labidi-Galy) were grown in RPMI 3–8% Criterion XT Tris-Acetate Protein Gel, Bio-Rad, catalogue no.
1640 supplemented with 2 mM sodium pyruvate and antibiotics; the 3450129) and transferred onto polyvinylidene fluoride membranes.
human colorectal carcinoma HCT116 cells (ATCC, catalogue no. CCL- Membranes were blocked with 5% milk powder diluted in TBS-Tween
247) were grown in McCoy’s 5A medium (Thermo Scientific, catalogue 20 (0.01%) for 1 h at room temperature. Incubation with primary anti-
no. 16600082) containing 10% FBS and antibiotics; the human OVSAHO bodies in blocking solution was applied for 1 h at room temperature.
ovarian cancer cell line was purchased from Sigma-Aldrich (catalogue The following primary antibodies were used for western blot analysis:
no. SCC294) and was grown in RPMI 1640 medium supplemented with PCNA mouse monoclonal (1:1,000, clone PC10, Millipore, catalogue no.
10% FBS and antibiotics. All cell lines were routinely tested for the MABE288); alpha-Tubulin mouse monoclonal (1:1,000, Calbiochem,
absence of mycoplasma contamination using the MycoGenie Rapid catalogue no. CP06); GAPDH mouse monoclonal (1:10,000, Abcam,
MycoPlasma Detection Kit (AssayGenie, catalogue no. MORV001) and catalogue no. ab8245); TIMELESS rabbit polyclonal (1:1,000, Abcam,
found negative. Drugs and chemical compounds used in this study catalogue no. ab109512); TIPIN rabbit polyclonal (1:250, Bethyl Labo-
were purchased from the following sources: thymidine (Sigma-Aldrich ratories, catalogue no. A301-474A); PARP1 rabbit polyclonal (1:1,000,
catalogue no. T1895), EdU (Thermo Fisher Scientific, catalogue no. Abcam, catalogue no. ab32138); PARP2 rabbit polyclonal (1:500, Active
A10044), 5-ethynyl uridine (EU) ( Jena Biosciences, catalogue no. Motif, catalogue no. 39743); BRCA2 mouse monoclonal (1:1,000, Cal-
CLK-N002-10), camptothecin (Sigma-Aldrich, catalogue no. C9911), biochem, catalogue no. OP95); RNase H1 rabbit polyclonal (1:500,
5,6-dichlorbenzimidazol 1-β-d-ribofuranosid (DRB; Sigma-Aldrich, ProteinTech, catalogue no. 15606-1-AP); FLAG mouse monoclonal
catalogue no. D1916), cordycepin (Tocris, catalogue no. 2294), trip- (1:1,000, Sigma-Aldrich, catalogue no. M2 F1804) and GFP rabbit poly-
tolide (Tocris, catalogue no. 3253), olaparib (Selleckchem, catalogue clonal (1:500, Abcam, catalogue no. ab290). Following incubation with
no. S1060), talazoparib (Selleckchem, catalogue no. S7048), veliparib primary antibodies, three washes with TBS-Tween 20 (0.01%) were
(Selleckchem, catalogue no. S1004), saruparib (Selleckchem, cata- performed. Membranes were incubated with secondary horseradish
logue no. S9875), hydrogen peroxide (Sigma-Aldrich, catalogue no. antimouse or antirabbit peroxidase-coupled antibodies IgG (1:2,500,
H3410), RO-3306 (Sigma-Aldrich, catalogue no. SML0569), TMZ Promega, catalogue nos. W401B and W402B, respectively) for 1 h at
(Sigma-Aldrich, catalogue no. T2577), nocodazole (Tocris, catalogue room temperature, before detection by ECL-based chemiluminescence.
no. 1228) and PARGi (Tocris, catalogue no. 7006). DRB, cordycepin and Uncropped western blot images are provided in Supplementary Fig. 1.
triptolide were used at concentrations of 75, 50 and 1 μΜ, respectively.
Flow cytometry
PARP1 and PARP2 biochemical assay Following siRNA transfection or drug treatment and, optionally, as
PARP1 and PARP2 activity in presence of increasing concentrations indicated, following pulse-labelling with 10 μΜ EdU for 30 min, cells
of PARP inhibitors was measured using PARP1 and PARP2 colorimet- were collected by trypsinization and fixed in 90% methanol overnight
ric assay kits (BPS Bioscience, catalogue no. 80580-1) according to at −20 °C. EdU detection was performed using the Click-it EdU Alexa
the manufacturer’s instructions. The assays were performed in tripli- Fluor 647 Flow Cytometry Assay Kit (Invitrogen catalogue no. C-10424)
cate. PARP inhibitors were dispensed with an acoustic liquid dispenser according to the manufacturer’s instructions. Detection of γH2AX
phosphorylation was performed using the Guava Histone H2AX Phos- Clonogenic assays were performed with a variety of cell lines fol-
phorylation Assay Kit (Luminex, catalogue no. FCCS100182) accord- lowing siRNA transfection or drug treatment. Briefly, following the
ing to the manufacturer’s instructions. The genomic DNA was stained indicated treatments, the cells were replated in triplicate in six- or
by incubating the cells in PBS containing RNase (Roche, catalogue 12-well plates (500–3,000 cells per well, depending on cell line) and
no. 11119915001) and propidium iodide (Sigma-Aldrich catalogue no. cultured for an additional 14 days (or more, depending on cell line) in
81845). EdU-DNA-γH2AX profiles were acquired by flow cytometry fresh medium. The cell culture medium was changed every two days.
(Gallios, Beckman Coulter); more than 20,000 cells were analysed per At the end of the experiment, medium was removed, and cells were
sample using Kaluza software (Beckman Coulter). The gating strategy rinsed with PBS and stained with 0.5% (w/v) crystal violet (Sigma-Aldrich)
is provided in Supplementary Fig. 2. in 20% (v/v) methanol for 30 min in the dark. The staining agent was
removed and the plates were rinsed three times in ddH20, air-dried and
EdUseq the cell colonies were counted.
The EdUseq protocol was performed as previously described35.
Briefly, HeLa cells were transfected with siRNA; 30 h later thymidine Immunofluorescence assays
(Sigma-Aldrich) at 2 mM final concentration was added for 18 h, at Cells were seeded onto autoclaved 12 mm glass coverslips or multiwell
which time the cells had reached 70–80% confluency. The cells were plates (μ-Plate 96 Wells, catalogue no. 89626) at 70–90% confluency.
washed four times with warm PBS and released in fresh medium for 90 Following any indicated treatment, the cells were pre-extracted for
or 120 min. EdU (25 μM) was added 30 min before the cells were col- 2 min with ice-cold 1× PBS containing 0.2% (v/v) Triton X-100 and then
lected, and the cells were then fixed with 90% ice-cold methanol over- fixed with 4% formaldehyde for 10 min at room temperature. After
night. Cells were stored until processed for isolation of EdU-labelled three washes with 1× PBS, the cells were permeabilized in 1× PBS con-
DNA. Following fixation, the cells were permeabilized with 0.2% triton taining 0.2% (v/v) Triton X-100 for 15 min at room temperature. For
X in PBS; then, the EdU incorporated into genomic DNA was coupled detection of trapped PARP1 and PARP2, the cells were pre-extracted
to a cleavable biotin-azide linker (Azide-PEG(3+3)-S-S-biotin; Jena with cold cytoskeleton buffer (0.5% Triton X-100, 10 mM PIPES pH 6.8,
Biosciences, catalogue no. CLK-A2112-10), using the reagents of the 3 mM MgCl2, 200 mM NaCl, 300 mM sucrose) for 10 min at 4 °C, fol-
Click-it Kit (Invitrogen, catalogue no. C-10424). Extraction of genomic lowed by fixation with ice-cold methanol for 15 min at −20 °C. Fol-
DNA was performed with phenol-chloroform ethanol precipitation, lowing three washes with PBS, the cells were blocked with 5% BSA/1×
followed by isolation of EdU-labelled DNA. Briefly, genomic DNA was PBS solution for 1 h at room temperature. Then, coverslips or multi-
sonicated to 100–500 bp nucleotide-long fragments using a bioruptor well plates were incubated for 2 h at room temperature with primary
sonicator (Diagenode). EdU-labelled DNA fragments were captured on antibodies diluted in 5% BSA/1× PBS. Following incubation with pri-
Dynabeads MyOne streptavidin C1 (Invitrogen, catalogue no. 65001). mary antibodies, coverslips or multiwell plates were washed three
The beads were washed three times with Binding and Washing Buffer times with 1× PBS and incubated for 1 h at room temperature with
1× (5 mM Tris-HCl pH 7.5, 0.5 mM EDTA, 1 M NaCL, 0.5% Tween 20) secondary antibodies diluted in 5% BSA/1× PBS. After three washes
and then were resuspended to twice the original volume with Bind- with 1× PBS, incubation with 1 μg ml−1 4,6-diamidino-2-phenylindole
ing and Washing Buffer 2×, mixed with an equal volume of sonicated (DAPI)/1× PBS for 15 min in dark at room temperature was performed.
EdU-labelled DNA incubated for 15 min on a rotating wheel at room Then, three washes with 1× PBS were performed and coverslips were
temperature. Following three washes of the beads with Binding and mounted on slides using the Fluoromount-G (Thermo Fisher Scientific,
Washing Buffer 1× and once with TE (10 mM Tris-HCl pH 8, 1 mM EDTA), catalogue no. 00-4958-02). For multiwell plates, following incubation
the EdU-labelled DNA was eluted by incubating the streptavidin beads with DAPI, 1× PBS was added in the wells. The primary antibodies used
with 2% β-mercaptoethanol (Sigma, catalogue no. M6250) for 1 h at for the immunofluorescence were: γH2AX (S139) mouse monoclonal
room temperature. The eluted DNA was used for library preparation (1:1,000, clone JBW301, Millipore, catalogue no. 05-636); RAD51 rab-
using the TruSeq ChIP Sample Prep Kit (Illumina, catalogue no. IP-202- bit polyclonal (1:1,000, Bioacademia, catalogue no. 70-002); 53BP1
1012). High-throughput 100-base-pair single-end sequencing was per- rabbit polyclonal (1:1,000, Novus Biologicals, catalogue no. NB100-
formed on an Illumina Hi-Seq 4000 sequencer. 304); poly (ADP-ribose) mouse monoclonal (1:500, Trevigen, cata-
logue no. 4335-MC-100 and 1-500, Enzo Life Sciences, catalogue no.
EdUseq data processing ALX-804-220-R100); PARP1 rabbit polyclonal (1:1,000, ProteinTech,
Sequencing reads were aligned on the non-masked human genome catalogue no. 13371–1-AP); PARP2 rabbit polyclonal (1:1,000, Active
assembly (GRCh37/hg19) using the Burrows–Wheeler Aligner software Motif, catalogue no. 39743). Secondary antibodies used: Alexa Fluor
as described previously35,56. Only the reads with the highest quality 488 goat-antirabbit IgG (1:500, Invitrogen, catalogue no. A110334);
score were retained. Previously described custom Perl scripts were Alexa Fluor 488 goat-antimouse IgG (1:500, Invitrogen, catalogue
used to assign the aligned reads to 10 kb genomic bins. Sigma (σ) values no. A11001); Alexa Fluor 594 goat-antirabbit IgG (1:500, Invitrogen,
were calculated as the normalized number of reads per bin divided catalogue no. A11037); Alexa Fluor 594 goat-antimouse IgG (1:500,
by its standard deviation. The data were visualized using previously Invitrogen, catalogue no. A11005); Alexa Fluor 647 goat-antirabbit
described scripts35. Assignment of replication timing was performed IgG (1:500, Invitrogen, catalogue no. A21244); Alexa Fluor 647 goat-
with REPLI-seq data generated previously35. antimouse IgG (1:500, Invitrogen, catalogue no. A21235). For micro-
nuclei detection, following any indicated treatment, cells were fixed
Cell viability and clonogenic assays with 4% paraformaldehyde for 15 min at room temperature and then
Viability assays were performed with DLD1 BRCA2+/+ and DLD1 BRCA2−/− permeabilized in PBS containing 0.2% Triton X-100 for 10 min at room
cells treated with various inhibitors and DNA damaging agents, and temperature. Nuclei were countestained with 1 μg ml−1 DAPI for 1 min at
following siRNA transfections, as indicated. In brief, 2,000 cells per well room temperature in dark, washed three times with PBS and mounted
were seeded in Advanced TC 96-well microplates; 24 h later, PARP inhibi- with Fluoromount-G.
tors were dispensed using a D300e digital dispenser (Tecan) at final
concentrations ranging from 0.6 nM to 10 µM using fourfold dilution In situ PLA
steps. The cells were incubated with the compounds for 5 days before Following the indicated treatments, cells grown on coverslips were
adding CellTiter-Glo 2.0 reagent (Promega, catalogue no. G9242) to washed twice with 1× PBS and pre-extracted for 10 min with ice-cold
each well, according to the manufacturer’s instructions. Luminescence 1× PBS containing 0.5% (v/v) Triton X-100 and protease inhibitor
was measured using a Spark 10M microplate reader (Tecan). cocktail (Complete, EDTA-free; Roche); they were then washed twice
Article
with 1× PBS and fixed with 4% (v/v) formaldehyde for 10 min at room at 20 foci per nucleus and for RAD51 at ten foci per nucleus. Micronu-
temperature, followed by two washes with 1× PBS. The cells were then clei, PLA foci and GFP-RNaseH1D210N foci were quantified manually.
incubated with 1× PBS containing 0.2% (v/v) Triton X-100 for 10 min at Automated, multi-channel image acquisition of multiwell plates was
room temperature, washed again twice with 1× PBS and blocked with performed in an unbiased fashion with an ImageXpress spinning disc
5% BSA/1× PBS solution for 1 h. The coverslips were then incubated confocal microscope (Molecular Devices), equipped with a sCMOS
O/N at 4 °C with primary antibodies diluted in 5% BSA/1× PBS. Follow- camera (Andor), and with a Nikon ×20 water 1.20 numerical aperture
ing incubation with primary antibodies, the coverslips were washed or ×60 water immersion 1.20 numerical aperture objective. The spin-
twice with 1× PBS and PLA was performed using Duolink PLA tech- ning disc confocal images were analysed using MetaXpress Custom
nology (Sigma-Aldrich, catalogue no. DUO92008) according to the Module Editor. The analysis pipeline started with the detection of the
manufacturer’s instructions. Briefly, coverslips were incubated with nuclei using the DAPI channel. This mask was then applied to quantify
antirabbit PLUS and antimouse MINUS PLA probes (Sigma-Aldrich, pixel intensities for γΗ2ΑΧ, PARP1/2 and EU incorporation for each
catalogue no. DUO92002 and DUO92004 respectively) for 1 h at 37 °C, individual cell. A light deconvolution was applied on the RAD51 and
followed by two washes in Wash Buffer A (0.01 M Tris, 0.15 M NaCl and 53BP1 channel to ease the detection of the small foci. The segmenta-
0.05% Tween 20, pH 7.4) for 5 min. Then, PLA probes were ligated for tion of foci was then performed using shape, size and intensity above
30 min at 37 °C, followed by two washes for 5 min in Wash Buffer A local background parameters. The masked foci were then attributed
and amplification using the Duolink In Situ Detection Reagents Red to their corresponding nuclei before the quantification of relevant
(Sigma-Aldrich, catalogue no. DUO92008), performed at 37 °C for parameters. Quantified values for each cell, were exported and
100 min. After amplification, the coverslips were washed twice in were subsequently used to generate graphs using GraphPad Prism 9
Wash Buffer B (0.2 M Tris and 0.1 M NaCl, pH 7.5) for 10 min and then software.
incubated with 1 mg ml−1 DAPI/1× PBS for 15 min in the dark at room
temperature. Finally, the coverslips were washed three times with 1× Statistical analysis
PBS and mounted on slides using Fluoromount-G. Primary antibodies Statistical analysis was performed using GraphPad Prism 9 software
used: RNAPII, H5 (1:500, BioLegend, catalogue no. 920204), RNAPII, (v.9.4.1). Detailed description of means or medians, error bars and
CTD4H8 (1:500, Millipore, catalogue no. 05-623) and PCNA (1:500, the number replicates and/or cells analysed is reported in the figure
Abcam, catalogue no. ab18197). legends. Statistical differences for grouped analyses were performed
using repeated-measures one-way analysis of variance (ANOVA) fol-
Quantification of nascent RNA production by EU labelling lowed by a Tukey’s multiple comparisons test. Statistical test results
Cells grown on multiwell plates were pulse-labelled with 1 mM EU for are provided as P values in the figures. Dose–response curves were
30 min, washed twice with 1× PBS and fixed in 4% formaldehyde for plotted using GraphPad Prism using as model the concentration of
10 min at room temperature. After three washes with 1× PBS, the cells the inhibitor versus response, variable slope (four parameters). No
were permeabilized with 1× PBS containing 0.2% (v/v) Triton X-100 statistical methods were used to determine the size sample size before
for 10 min at room temperature. EU incorporation was detected with conducting experiments. Experiments were not randomized and the
Click-iT EU Alexa fluor 488 Imaging Kit (Thermo Fisher Scientific, cata- investigators were not blinded to allocation. Data were assembled into
logue no. C10329). Multiwell plates were incubated with Hoechst 33342 figures using Adobe Illustrator CS6.
for 15 min in the dark at room temperature and subsequently washed
three times with 1× PBS. Reporting summary
Further information on research design is available in the Nature Port-
Detection of EdU incorporation in mitosis folio Reporting Summary linked to this article.
For detection of mitotic EdU foci, cells, cultured on coverslips,
were synchronized at the G1/S transition with 2 mM thymidine
(Sigma-Aldrich, catalogue no. T1895) for 18 h, washed three times Data availability
with 1× PBS and released in fresh medium containing 6 μM RO-3306 The fastq sequencing data and associated information described in this
(Sigma-Aldrich, catalogue no. SML0569) for 11 h. The cells were then study have been deposited in the Sequence Read Archive with Gene
washed three times with warm medium and released in medium con- Expression Omnibus accession number GSE220223. The EUseq data
taining 100 ng ml−1 nocodazole (Tocris, catalogue no. 1228) and 20 μM used in this study were previously published56. Unprocessed images of
EdU (Invitrogen, catalogue no. A10044) for 1 h. The cells were then western blots and the gating strategy for the flow cytometry experi-
fixed and permeabilized with 4% paraformaldehyde, 20 mM HEPES, ments are provided as Supplementary Information. All information
10 mM EGTA, 0.2% Triton X-100, 1 mM MgCl2 for 20 min at room supporting the conclusions are provided with the paper.
temperature and then washed three times with 1× PBS. EdU incorpora-
tion was performed using the Click-it EdU Alexa Fluor 647 Kit (Invitro-
gen, catalogue no. C10340), after which the cells were washed twice Code availability
with 1× PBS, incubated with DAPI (0.5 mg ml−1, Thermo Fisher, catalogue Computer codes and data files used to process and plot the data are
no. D1306) in 1× PBS for 15 min at room temperature, washed three available from our previous publications35,56.
times with PBS and rinsed in distilled water. The coverslips were then
mounted on slides using ProLong Gold Antifade Mountant (Thermo
Fisher, catalogue no. P10144). Quantification of EdU foci on metaphase 53. Sollier, J. et al. Transcription-coupled nucleotide excision repair factors promote
chromosome spreads was performed manually. R-loop-induced genome instability. Mol. Cell 56, 777–785 (2014).
54. Teloni, F. et al. Efficient pre-mRNA cleavage prevents replication-stress-associated
genome instability. Mol. Cell 73, 670–683 (2019).
Image acquisition and analysis 55. Zimmer, J. et al. Targeting BRCA1 and BRCA2 deficiencies with G-quadruplex-interacting
Images from coverslips were acquired with a Zeiss Imager M2 AX10 compounds. Mol. Cell 61, 449–460 (2016).
56. Macheret, M. et al. High-resolution mapping of mitotic DNA synthesis regions and
microscope equipped with ApoTome2 and a Plan-APOCHROMAT
common fragile sites in the human genome through direct sequencing. Cell Res. 11,
×100/1.4 oil immersion objective, using the ZEN3.4 (blue edition) 997–1008 (2020).
software. Images were analysed with ZEN3.4 (blue edition) or ImageJ/
FIJI software (National Healthcare Institute, USA). The threshold to Acknowledgements We thank the High-Throughput/Content Screening facility ACCESS and
determine whether a cell was positive for γΗ2ΑΧ or 53BP1 foci was set the Flow Cytometry and Genomics Platforms of the University of Geneva. This work was
supported by grants from the European Commission (REPLISTRESS) and the Swiss National Competing interests T.D.H. and S.K.S. are founders and stockholders of FoRx Therapeutics.
Science Foundation (grant nos. 182487 and 186230). S.K.S., G.G.R., A.F. and L.G.I. are employees of FoRx Therapeutics. The other authors declare no
competing interests.
Author contributions T.D.H. conceived the study and supervised the project. M.P., G.G.R.,
S.K.S. and T.D.H. designed the experiments. T.D.H. and M.P. wrote the manuscript. M.P. Additional information
performed the cell-based experiments with the contribution of A.K. and G.G.R. M.P. and A.K. Supplementary information The online version contains supplementary material available at
processed samples for sequencing. G.G.R., A.F. and L.G.I. performed the in vitro biochemical https://doi.org/10.1038/s41586-024-07217-2.
and biophysical experiments. M.P., G.G.R., V.S.D., S.K.S. and T.D.H. analysed the data. T.D.H. and Correspondence and requests for materials should be addressed to Thanos D. Halazonetis.
V.S.D. performed the bioinformatic analyses. All authors commented on the manuscript. Peer review information Nature thanks the anonymous reviewers for their contribution to the
peer review of this work. Peer reviewer reports are available.
Funding Open access funding provided by University of Geneva. Reprints and permissions information is available at http://www.nature.com/reprints.
Article
a b d
HeLa U2OS hTERT-RPE1

siCTRL

siTIM
±DRB EU Fix 80 -DRB +DRB 20
kDa <0.0001

γH2AX positive cells [%]

U2OS /
<0.0001
TIMELESS 0min 70min 100min hTERT-RPE1
100 60 15
a-tubulin 55 c siRNA <0.0001
Hoechst EU transf. Thy Release Collect <0.0001
40 10

siCTRL
-DRB -48h -18h 0h 200min

siTIP
±DRB 20 5
kDa NS NS

TIPIN 55
+DRB 0 0

siCTRL
siCTRL
a-tubulin

siTIM

siTIP
siTIM

siTIP
55

e g i j γH2AX+ γH2AX-
HeLa
siCTRL siTIM siTIP
HeLa <0.0001
siRNA <0.0001 HeLa / U2OS /
transf. Thy Release Collect 80 <0.0001 hTERT-RPE1 -DRB

EU mean intensity [r.u.]

siRNA
-48h -18h 0h 200min 60 transf. ±DRB EdU Collect
±CORD
±TLP +DRB
-48h 0h 170min 200min
f 40
DMSO DRB CORD TLP G1 G2 G1 G2 G1 G2
PI
20 k
EdU

HeLa U2OS hTERT-RPE1

50 50 10

γH2AX positive cells [%]

<0.0001 0.0018
0 <0.0001 <0.0001
G1 G2 G1 G2 G1 G2 G1 G2
TLP
DMSO

DRB

CORD
40 <0.0001
40 8
PI EdU+ 0.0251
EdU- NS
h 30 30 6
EdU+
HeLa HeLa 20 20 4
DMSO TLP DMSO TLP
NS
CORD <0.0001
CORD <0.0001
10 10 NS 2
<0.0001
γH2AX mean intensity [r. u].

400 50 <0.0001
<0.0001 <0.0001 <0.0001 0 0 0
γH2AX positive cells [%]

40 40 8
53BP1 foci per cell

NS
40 NS
<0.0001
-DRB
300 +DRB
30 30 30 6
NS
200 NS NS
EdU-

NS NS
20 NS 20 20 NS 4 NS
NS
100 10 NS
10 2
10 NS

0 0 0 0 0

siCTRL

siTIM

siTIP
siCTRL
siTIM

siTIP

siCTRL

siTIM

siTIP
siCTRL

siCTRL
siTIM

siTIP

siTIM

siTIP

Extended Data Fig. 1 | Depletion of TIMELESS or TIPIN by siRNAs induces plots show medians and value ranges of 25-75% and 10-90%, filled circles
a TRC-dependent DNA damage response in normal and cancer cells. indicate the individual cells in the top and bottom deciles; n = 2 replicates;
a, Efficiency of siRNA-mediated depletion of TIMELESS and TIPIN in HeLa >2624 cells per group (range: 2624-2899); ANOVA. h, Quantification of γH2AX
cells by immunoblotting. PCNA and α-tubulin served as loading controls. mean intensity and number of 53BP1 foci per cell; plots show medians and
b-c, Inhibition of transcription elongation by DRB. b, Outline of the experiment. value ranges of 25–75% and 10–90%, filled circles indicate the individual cells
c, Representative images of HeLa cells indicating inhibition of EU incorporation in the top and bottom deciles; n = 2 replicates; >66 cells per group (range:
by DRB; the nuclei were counterstained with Hoechst 33342. Scale bar: 10 μm. 66–194); ANOVA. i-k, Ongoing DNA replication is required for induction of a
d, Induction of a DNA damage response in U2OS and hTERT-RPE1 cells DNA damage response by depletion of TIMELESS or TIPIN. i, Outline of the
transfected with control siRNA or siRNAs targeting TIMELESS or TIPIN. γH2AX experiment. j, γH2AX levels and DNA content ascertained by flow cytometry.
levels were determined by flow cytometry; bars indicate means ± 1 s.d.; n = 3 k, Quantification of γH2AX positive cells determined separately for the
replicates; ANOVA. e-h, Transcription inhibitors cordycepin (CORD) and EdU-positive and EdU-negative cells; bars indicate means ± 1 s.d.; n = 3
triptolide (TLP) suppress the DNA damage response induced by depletion of replicates; ANOVA. CTRL, control; TIM, TIMELESS; TIP, TIPIN; transf.,
TIMELESS or TIPIN. e, Outline of the experiment. f, Flow cytometry profiles for transfection; Thy, thymidine; IF, immunofluorescence; PI, propidium iodide;
EdU incorporation and DNA content. g, Quantification of EU incorporation; r.u., relative units; NS, not significant.
 a d e HeLa DOX k Fork Progression-
U2OS T-REx GFP-RNaseH1D210N HeLa DOX FLAG-RNaseH1 FLAG-RNaseH1 average signal
siRNA ±DOX siRNA ±DOX DOX – + kDa 120min Release from G1/S
transf. Thy Release IF transf. Thy Release IF FLAG 35 EdUseq(σ)
RNH1 35 10
-48h -18h 0h 200min -48h -18h 0h 200min PCNA 35
siCTRL
b c f
<0.0001 <0.0001 -DOX 0
60 60 150 +DOX 10

γH2AX mean intensity [r.u.]

<0.0001
<0.0001
50 50
GFP-RNaseH1D210N
U2OS T-REx 125

53BP1 foci per cell

<0.0001
GFP-RNaseH1D210N <0.0001 siTIM
foci per cell 40 40 100
DOX - + kDa
GFP 55 30 30 75 NS

RNH1 0
55
20 20 50
PCNA 35 NS 10
10 10 25
siTIP
0 0 0

siCTRL

siTIM

siTIP
siCTRL
siTIM
siCTRL

siTIP

siTIP
siTIM
0
Gene
annotation
g h MiDAS
i -DRB +DRB
80 <0.0001 EUseq(σ)
HeLa siCTRL siTIM siTIP

with EdU foci >3 [%]

0.0001
siRNA Release in Release -500 0 +500
60 kb

Mitotic cells
transf. Thy S phase Wash in mitosis Fix Forward
-DRB
40 Reverse
-48h -18h 0h 200min 10.5h 12h
±DRB Noco/
+DRB
20 NS
RO-3306 EdU
0
EdU DAPI

siCTRL

siTIM

siTIP
j Fork Progression-average signal: 120min release from G1/S
RT RT:
Early S
Mid S
EdUseq
(σ) Gene annotation:
siCTRL Forward
siTIP Reverse
Ge
iG Overlap Bidirectional
Chr1 Chr2 Chr3 Chr3 Chr7
(Mb: 6.3-6.5) (Mb: 22.2-24.2) (Mb: 2.3-2.5) (Mb: 7.0-7.2) (Mb: 8.9-9.1)

Extended Data Fig. 2 | Induction of R-loops, MiDAS and increased mitosis; nocodazole (Noco) prevented exit from mitosis. h, Representative
replication fork speed following depletion of TIMELESS or TIPIN. images of prometaphase cells with MiDAS; the DNA was counterstained with
a-c, Induction of R-loops in cells after depletion of TIMELESS or TIPIN. DAPI. Scale bar: 5 μm. i, Quantification of the percentage of prometaphase cells
a, Outline of the experiment. The U2OS cells used in this experiment expressed with >3 EdU foci; bars indicate means ± 1 s.d.; n = 3 replicates; >294 prometaphase
GFP-RNaseH1D210N in a doxycycline (DOX)-dependent manner. b, Induction of cells per group (range: 294–315); ANOVA. j-k, Increased rates of fork progression
expression of GFP-RNaseH1D210N by DOX, as monitored by immunoblotting; over transcribed genes following depletion of TIMELESS or TIPIN in HeLa
PCNA served as loading control. c, Quantification of the number of GFP- cells. The outline of the experiment is shown in Fig. 1g. The experiment shown
RNaseH1D210N foci following depletion of TIMELESS or TIPIN; plots show medians here is a replicate of the experiment shown in Fig. 1h. j, EdUseq profiles at five
and value ranges of 25–75% and 10–90%, filled circles indicate the individual cells representative genomic regions. Replication timing (RT): blue, early S phase;
in the top decile; n = 2 replicates; >212 cells per group (range: 212–221); ANOVA. green, mid S phase. Genes (Ge): green, forward-transcribed genes; red, reverse-
d-f, The DNA damage response induced by depletion of TIMELESS or TIPIN is transcribed genes; yellow, overlap of forward and reverse-transcribed genes.
suppressed by expression of RNase H1. d, Outline of the experiment. The HeLa Intergenic regions (iG): gray. Bin resolution: 10 kb; ruler scale: 100 kb. k, Average
cells used in this experiment expressed FLAG-RNaseH1 in a DOX-dependent nascent DNA replication signal (EdUseq) at large (>300 kb) transcribed genes
manner. e, Induction of expression of FLAG-RNaseH1 by DOX, as monitored by 120 min after release in S phase. The genes are aligned by their transcription
immunoblotting. f, Quantification of the number of 53BP1 foci per cell and of start site and all genes are shown with their 5’-3’ orientation from left to right.
γH2AX mean intensity; plots show medians and value ranges of 25–75% and Lower panels: heatmaps showing gene annotation and EUseq signal for each
10–90%, filled circles indicate the individual cells in the top and bottom deciles; genomic locus used to generate the average EdUseq signal. Span of genomic
n = 2 replicates; >199 cells per group (range: 199–557); ANOVA. g-i, Induction regions: 1 Mb. transf., transfection; Thy, thymidine; IF, immunofluorescence;
of MiDAS in prometaphase cells following depletion of TIMELESS or TIPIN. RNH1, RNase H1; r.u., relative units; CTRL, control; TIM, TIMELESS; TIP, TIPIN;
g, Outline of the experiment; the Cdk1 inhibitor RO−3306 inhibited entry into σ, sigma value; NS, not significant.
Article
a c HeLa
HeLa
Collect Collect Collect
Thy Release IF Thy Release (Early S) (Mid S) (Late S)
e
-18h 0h 200min -18h 0h 3.5h 7h 10.5h
±PARPi ±PARPi ±PARPi ±PARPi High Tx
±CORD ±DRB ±DRB ±DRB 20 Mid Tx
±TLP Low Tx
b d No Tx

Percentage of genes [%]

DMSO TLP CORD Early S Mid S Late S
60 15
400 <0.0001 <0.0001 <0.0001 -DRB

γH2AX positive cells [%]

γH2AX mean intensity [r.u.]

<0.0001 <0.0001 50 +DRB

<0.0001
<0.0001
300 40 10
0.0016
30
200 0.9994
20 0.9972 5
NS
100 10 NS NS

0 0
Early Mid Late

DMSO
Olap

DMSO
Olap

DMSO
Olap
Tal

Tal

Tal
0
S phase
Olap
DMSO

Tal

h
f g Early S Late S

HeLa MiDAS 80 -DRB

Early S Late S Early S Late S <0.0001 +DRB

with EdU foci >3 [%]

Thy Release Wash Wash Collect DMSO Olap Tal Olap Tal 60 <0.0001

Mitotic cells
-DRB
-18h 0h 3.5h 7h 10.5h 12h 40
±PARPi ±PARPi NS NS
±DRB ±DRB Noco/ NS
+DRB 20 NS
RO-3306 EdU
EdUDAPI 0

DMSO

Olap

Tal

DMSO

Olap

Tal
i j k l
-DOX +DOX

γH2AX mean intensity [r.u.]

60 <0.0001
60 -DOX +DOX 100
U2OS T-REx HeLa DOX <0.0001 <0.0001
53BP1 foci per cell

GFP-RNaseH1D210N
GFP-RNaseH1D210N

50
<0.0001 FLAG-RNaseH1 50 <0.0001
80 <0.0001
±DOX ±DOX
foci per cell

Thy Release IF 40 Thy Release IF 40

60
30 30
-18h 0h 200min -18h 0h 200min 40
±PARPi 20 ±PARPi 20 NS NS

10 10 20

0 0 0

DMSO

Olap

Tal
DMSO

Olap

Tal
Olap
Tal
DMSO

Extended Data Fig. 3 | PARP inhibitors induce TRC-dependent DNA damage mitosis; nocodazole (Noco) prevented exit from mitosis. g, Representative
in early S phase and activate MiDAS. a-b, Transcription inhibitors cordycepin images of prometaphase cells with MiDAS; the DNA was counterstained with
(CORD) and triptolide (TLP) suppress the DNA damage response induced by DAPI. Scale bar: 5 μm. h, Quantification of the percentage of prometaphase
PARP inhibitors. a, Outline of the experiment. b, Quantification of γH2AX mean cells with >3 EdU foci; bars indicate means ± 1 s.d.; n = 3 replicates; >127
intensity; plots show medians and value ranges of 25–75% and 10–90%, filled prometaphase cells per group (range: 127–400); ANOVA. i-j, Induction of
circles indicate the individual cells in the top and bottom deciles; n = 2 R-loops in cells treated with PARP inhibitors. i, Outline of the experiment. The
replicates; >1668 cells per group (range: 1668–2282); ANOVA. c-d, The induction U2OS cells used in this experiment express GFP-RNaseH1D210N in a doxycycline
of a DNA damage response by PARP inhibitors depends on whether cells are (DOX)-dependent manner. j, Quantification of the number of GFP-RNaseH1D210N
exposed to these inhibitors in early, mid or late S phase. c, Outline of the foci following treatment with PARP inhibitors; plots show medians and value
experiment. Cells were exposed to PARP inhibitors 0−3.5, 3.5–7 or 7–10.5 h after ranges of 25–75% and 10–90%, filled circles indicate the individual cells in the top
release from a thymidine block, corresponding to early, mid or late S phase, and bottom deciles; n = 2 replicates; >192 cells per group; ANOVA. k-l, The DNA
respectively. d, Quantification of the percentage of γH2AX positive cells by flow damage response induced by PARP inhibitors is suppressed by expression of
cytometry; bars indicate means ± 1 s.d.; n = 3 replicates; ANOVA. e, Distribution RNase H1. k, Outline of the experiment. The HeLa cells used in this experiment
of human genes according to replication timing (early, mid or late S phase) and express FLAG-RNaseH1 in a DOX-dependent manner. l, Quantification of the
level of nascent transcription (High Tx, upper tertile of all expressed genes; Mid number of 53BP1 foci per cell and of γH2AX mean intensity; plots show medians
Tx, middle tertile; Low Tx, lower tertile; No Tx, non-expressed genes). Nascent and value ranges of 25–75% and 10–90%, filled circles indicate the individual
transcription was determined by EUseq analysis of HeLa cells. f-h, Induction of cells in the top and bottom deciles; n = 2 replicates; >141 cells per group (range:
MiDAS in prometaphase cells following treatment of cells with PARP inhibitors 141–206); ANOVA. Thy, thymidine; PARPi, PARP inhibitor; r.u., relative units;
in early or late S phase (0–3.5 or 7–10.5 h after release from a thymidine block). Olap, olaparib (10 μΜ); Tal, talazoparib (100 nM); NS, not significant.
f, Outline of the experiment; the Cdk1 inhibitor RO-3306 inhibited entry into
 a b d e
in vitro PARP1/2 activity HeLa HeLa
Inhibition of PAR chain formation
Tal: 2.59nM Olap Tal Sar Vel Tal: 454 nM γH2AX DAPI
Sar: 3.20nM Sar: >10 μM
120 Olap: 1.78nM DMSO (10 μM) (100 nM) (100 nM) (10 μM) 500 Olap: >10 μM
Vel: 5.17nM Vel: >10 μM

-DRB
100

mean intensity [r.u.]

PARP1 activity [%]

Trapped PARP2
PAR 400

DMSO
80
60 300

+DRB
40
DAPI 200
20
0 100

-DRB
-10 -9 -8 -7 -6 -11 -10 -9 -8 -7 -6 -5 -4

Tal (100 nM)

PARPi, [log(M)]
c DLD1 BRCA2+/+
PARP1/2 Trapping
Tal: 0.89nM Tal: 321 nM

+DRB
Sar: 35.6nM Tal Tal Sar: >10 μM
120 Olap: 1.03nM DMSO (2.5 μM) DMSO (2.5 μM) 400 Olap: >10 μM
Vel: 4.41nM Vel: >10 μM

mean intensity [r.u.]

100

Trapped PARP2
PARP2 activity [%]

80 PARP1 PARP2 300

60
40 200
DAPI DAPI
20
0 100
-10 -9 -8 -7 -6 -11 -10 -9 -8 -7 -6 -5 -4
PARPi, [log(M)] PARPi, (Log [M])

Extended Data Fig. 4 | Characterization of PARP inhibition and PARP c, Representative images of HeLa cells treated with different PARP inhibitors,
trapping activities of the four PARP inhibitors used in this study. pre-extracted and immunostained for PARP1 or PARP2; the nuclei were
a, Dose-response curves and calculated IC50 values for inhibition of PARP1 and counterstained with DAPI. Scale bar: 10 μm. d, Dose-response curves and
PARP2 enzymatic activities in vitro by the indicated PARP inhibitors. One of calculated EC50 values for PARP2 trapping in HeLa and DLD1 BRCA2+/+ cells;
n = 2 replicates is presented. The IC50 values determined by this assay might be means ± 1 s.d.; n = 2 replicates; for HeLa >2204 (range: 2204–10543), for
inaccurate, due to the assay not being sensitive enough for the most potent DLD1 > 1605 (range: 1605–11936) cells per data point. e, Representative
inhibitors; these inhibitors might appear less potent than they actually are20. images of HeLa cells treated with talazoparib and optionally with DRB and
b, Examples of images of HeLa cells treated with PARP inhibitors and H2O2 that immunostained for γH2AX; the nuclei were counterstained with DAPI.
were used to assess inhibition of PARP enzymatic activity in cells. The cells Scale bar: 10 μm. r.u., relative units; Tal, talazoparib; Sar, saruparib; Olap,
were treated as shown in Fig. 3a and were immunostained for poly(ADP-ribose) olaparib; Vel, veliparib.
(PAR) chains; the nuclei were counterstained with DAPI. Scale bar: 10 μm.
Article
a c siPARP1b

PARP1D993G
HeLa

siPARP1b
PARP1WT
siRNA

siCTRL
transf. Thy Release IF
kDa
-48h -18h 0h 200min PARP1
±PARPi 100
PCNA 35
b d
53BP1 γΗ2ΑΧ
70 70
30 <0.0001 <0.0001

γH2AX positive cells [%]

60 60

Positive cells [%]

50 50
20 0.0062
40 40 0.0308

30 30
20 20 10

10 10
0 0 0
siCTRL

siCTRL

siCTRL
-
+PARP1WT
+PARP1D993G
siCTRL
-
+PARP1WT
D993G
siTIM
siTIP

siTIM
siTIP
Olap
siTIM+ Tal

siTIP+ Olap
Tal

Olap
siTIM+ Tal

siTIP+ Olap
Tal
Olap
Tal

Olap
Tal

+PARP1
siPARP1b siPARP1b

Extended Data Fig. 5 | PARP inhibitors function in the same pathway as DNA damage responses. c, Levels of endogenous and ectopically-expressed
TIMELESS and TIPIN to prevent a TRC-induced DNA damage response. PARP1 proteins in cells transfected with siRNAs (CTRL or PARP1b) and plasmids
a, Outline of the experiment. b, Quantification of the percentage of cells with (PARP1WT or PARP1D993G). A representative immunoblot is shown; PCNA served
γH2AX and 53BP1 foci following depletion of TIMELESS or TIPIN or treatment as loading control. siPARP1b, siRNA targeting the endogenous PARP1 gene, but
with PARP inhibitors and/or combinations thereof; γΗ2ΑΧ and 53BP1-positive not the PARP1 genes expressed by the plasmids; PARP1WT, wild-type PARP1;
cells: >20 foci; bars indicate means ± 1 s.d.; n = 3 replicates; >207 cells per group PARP1D993G, D993G single amino acid substitution mutant. d, Quantification of
(range: 207–403). Olap, olaparib (10 μM); Tal, talazoparib (100 nM). CTRL, control; the percentage of cells with 53BP1 or γΗ2ΑΧ foci; bars indicate means ± 1 s.d.;
TIM, TIMELESS; TIP, TIPIN. c-d, Substitutions targeting the TIMELESS-PARP1 n = 3 replicates; for 53BP1 foci >246 (range: 246–657), for γΗ2ΑΧ foci >251
interface compromise the function of these proteins in averting TRC-dependent (range: 251–624) cells per group; ANOVA.
 HeLa: Fork Progression-average signal
120min Release from G1/S

EdUseq(σ) Replicate #1 Replicate #2 Replicate #3

10
10 10
10
siCTRL

0 0 0
10
10 10
10

siPARP1

0 0 0
10
10
10 10

siPARP2

0 0 0

Gene
annotation
-500 0 +500 -500 0 +500 -500 0 +500
kb kb kb
Forward Reverse

Extended Data Fig. 6 | Increased replication fork progression rates within averages of all three replicates are shown in Fig. 4h. The genes are aligned by
large, transcribed genes in HeLa cells depleted of PARP1. The outline of the their transcription start site and all genes are shown with their 5’−3’ orientation
experiment, performed in triplicate, is shown in Fig. 4g. Here, the average from left to right. Lower panels: heatmaps showing gene annotation for each
nascent DNA replication signals over large (>300 kb) transcribed genes 120 min genomic locus used to generate the average EdUseq profiles. Span of genomic
after release in S phase are shown separately for each replicate. The merged regions: 1 Mb; σ, sigma value; CTRL, control.
Article
a c
DLD1 BRCA2+/+/ DLD1 BRCA2-/- DLD1 BRCA2+/+/ DLD1 BRCA2-/-
Late S Late S
Thy Release Wash IF Thy Release Replate CFA

-18h 0h 7h 10.5h 24h -18h 0h 7h 10.5h 14days

±PARPi ±PARPi
±DRB ±DRB
b d
PARPi: 7-10.5h PARPi: 7-10.5h
BRCA2 +/+
BRCA2 -/- BRCA2 +/+ BRCA2 -/-
-DRB +DRB -DRB +DRB -DRB +DRB -DRB +DRB
40 40
γH2AX positive cells [%]

NS

NS

NS
Survival-CFA [%]

NS
NS
NS

NS
30 30 100 100

NS

NS

NS
NS
NS
20 NS
20
50 50
NS

NS
NS

NS
NS

NS
10 10
NS

0 0 0 0
DMSO

Tal
Vel
Sar
Olap

DMSO

Tal
Vel
Sar
Olap

DMSO

Tal
Vel
Sar
DMSO

Tal
Vel
Sar

Olap
e PEO4 / PEO1 Olap
Early S
g DLD1 BRCA2+/+/ DLD1 BRCA2-/-
Thy Release Replate CFA Plating ± CPT CTG

-18h 0h 3.5h 14days -24h 0h 5days

±PARPi
±DRB
f PEO4 PEO1 h DLD1
+/+
-DRB +DRB -DRB +DRB BRCA2-/- : 14.86 nM
BRCA2 : 2.95 nM
120
<0.0001

<0.0001
0.0019
NS
NS

100
NS

NS
Survival-CFA [%]

Survival-CTG [%]
NS
NS

100 100 80
0.0115

60
50 50 40
20
0 0 0
DMSO
Tal
Vel
Olap

DMSO
Tal
Vel
Olap
Sar

Sar

-10 -9 -8 -7 -6
CPT, (Log[M])

Extended Data Fig. 7 | The sensitivity of HR-deficient cells to PARP PEO1 cells treated with PARP inhibitors in early S phase is alleviated by inhibiting
inhibitors is affected by whether the cells are exposed to PARP inhibitors in transcription elongation. e, Outline of the experiment. PEO4 cells are HR-
early or late S phase. a-b, Exposure of HR-deficient cells to PARP inhibitors in proficient revertant cells derived from the same cancer as PEO1 cells.
late S phase does not lead to induction of a DNA damage response. a, Outline of f, Quantification of cell survival by CFA; bars indicate means ± 1 s.d.; n = 3
the experiment. b, Quantification of the percentage of cells with more than 20 replicates; ANOVA. g-h, HR-deficient cells have increased sensitivity to
γΗ2ΑΧ foci per cell. The bars indicate means ± 1 s.d.; n = 3 replicates; >283 cells camptothecin (CPT). g, Outline of the experiment. h, Dose-response survival
per group (range: 283–521); ANOVA. c-d, Exposure of cells to PARP inhibitors in curves and calculated EC50 values for DLD1 BRCA2+/+ and DLD1 BRCA2−/− cells
late S phase does not lead to synthetic lethality with HR deficiency. c, Outline of following treatment with CPT. Olap, olaparib (10 μΜ); Tal, talazoparib (100 nM);
the experiment. d, Quantification of cell survival by a colony formation assay Vel, veliparib (10 μM); Sar, saruparib (1 μΜ); CTG, CellTiter-Glo Cell Viability
(CFA) with the DMSO-treated cells serving as reference. The bars indicate Assay; NS, not significant.
means ± 1 s.d.; n = 3 replicates; ANOVA. e-f, The synthetic lethality of HR-deficient
 a b
HCT116 H1299-shBRCA2DOX
+siCTRL +siBRCA2 -DOX +DOX

siPARP1&2

siPARP1&2

siPARP1&2

siPARP1&2
siPARP1
siPARP2

siPARP1
siPARP2

siPARP1
siPARP2

siPARP1
siPARP2
siCTRL

siCTRL

siCTRL

siCTRL
Survival PARP1/2 siRNA
kDa kDa
2x siRNA Transf. Replate CFA
BRCA2 250 PARP1
100
-72h 0h 14days
PARP1 130 PARP2 * 55
PARP2 55 PCNA 35
PCNA 35

DLD1
BRCA2-/- PEO1 OVSAHO
Survival PARPi

siPARP1&2
siPARP1&2

siPARP1&2
+PARPi CFA

siPARP1
siPARP2
siPARP1
siPARP2

siPARP1
siPARP2

siCTRL
siCTRL

siCTRL
0 14days kDa kDa
kDa
PARP1 PARP1 130 PARP1 130
100
PARP2 PARP2 55 PARP2 55
55
PCNA 35 PCNA 35 GAPDH 35

c
HCT116 HeLa U2OS DLD1 H1299-shBRCA2DOX PEO1 PEO4 OVSAHO
siCTRL siCTRL siCTRL BRCA2+/+ -DOX <0.0001 0.0983 <0.0001
150 siBRCA2 siBRCA2 <0.0001 siBRCA2 BRCA2-/-<0.0001 +DOX 0.0001
<0.0001 0.0001 0.0005 NS NS
NS NS NS
0.0045 0.0064 NS <0.0001 0.0010
Survival-CFA [%]

<0.0001 <0.0001 <0.0001

0.0003 0.4303
100

50

siPARP1&2
siPARP1&2

siPARP1&2

siPARP1&2

siPARP1&2

siPARP1&2

siPARP1&2

siPARP1&2

siPARP1&2

siPARP1&2

siPARP1&2
siPARP1&2

siPARP1&2

siPARP1
siPARP2
siPARP1
siPARP2

siPARP1
siPARP2

siPARP1
siPARP2

siPARP1
siPARP2

siPARP1
siPARP2

siPARP1
siPARP2

siPARP1
siPARP2

siPARP1
siPARP2

siPARP1
siPARP2

siPARP1
siPARP2
siCTRL

siCTRL

siCTRL
siCTRL

siCTRL

siCTRL

siCTRL

siCTRL

siCTRL
siPARP1
siPARP2

siCTRL
siPARP1
siPARP2

siCTRL
siCTRL

siCTRL

siCTRL siCTRL siCTRL BRCA2+/+ -DOX

150 siBRCA2 siBRCA2 siBRCA2 BRCA2-/- +DOX <0.0001 <0.0001
NS
<0.0001 0.0001 <0.0001 <0.0001
<0.0001
0.0061 <0.0001 <0.0001 <0.0001
0.0007
Survival-CFA [%]

<0.0001 NS <0.0001

100

50

0
DMSO

DMSO

DMSO

DMSO

DMSO

DMSO

DMSO

DMSO

DMSO

DMSO

DMSO

DMSO

DMSO
Olap

Olap

Olap

Olap

Olap

Olap

Olap

Olap

Olap

Olap

Olap

Olap

Olap
Sar

Sar

Sar

Sar

Sar

Sar

Sar

Sar

Sar

Sar

Sar

Sar

Sar

Extended Data Fig. 8 | Depletion of PARP1 by siRNA is synthetic lethal with loading controls. The H1299-shBRCA2DOX cells induce expression of shRNA
HR-deficiency. a, Outline of the experiment. The cell lines were transfected targeting BRCA2 in a doxycycline (DOX)-dependent manner. c, Quantification
with siRNA or exposed to PARP inhibitors. Viability was assessed by a colony of cell survival; bars indicate means ± 1 s.d.; n = 3 replicates; ANOVA. PARPi,
formation assay (CFA). Note that in the siRNA-transfected cells, PARP1 and/or PARP inhibitor; transf., transfection; CTRL, control; Olap, olaparib (1 μΜ,
PARP2 were depleted for only a few days, whereas the PARP inhibitors were except for DLD1 and OVSAHO cells: 5 μΜ); Sar, saruparib (100 nΜ); NS, not
present over the entire 14 day-period. b, Assessment of the efficacy of depletion significant.
of BRCA2, PARP1 and PARP2 by immunoblotting. PCNA and GAPDH served as
Article
a +/+ -/- c +/+ -/-
DLD1 BRCA2 / DLD1 BRCA2 DLD1 BRCA2 / DLD1 BRCA2
2x siRNA ± Tal
Plating ±PARPi ±TMZ CTG transf. Replate ± TMZ IF

-24h 0h 5days -24h 0h 48h

b d
DLD1 BRCA2 +/+ DLD1 BRCA2 +/+ DLD1 BRCA2 +/+ DLD1 BRCA2 -/-
Olap: > 200 μM Sar: > 200 μM siCTRL: 1.45 μM siCTRL: 159 nM
Olap+20μΜ ΤΜΖ: 5.70 μM Sar+20μΜ ΤΜΖ: > 200 μM siPARP1: > 200 μM siPARP1: 316 nM
Olap+50μΜ ΤΜΖ: 2.95 μM Sar+50μΜ ΤΜΖ: 9.23 μM 150 100

γH2AX mean intensity [r.u.]

120 120 120 80
Survival-CTG [%]

100 100
90 60
80 80
>70x
60 60 >20x 60 40
40 40
30 20
20 20
0 0 0 0
-10 -9 -8 -7 -6 -5 -4 -10 -9 -8 -7 -6 -5 -4 -11 -10 -9 -8 -7 -6 -5 -4 -11 -10 -9 -8 -7 -6 -5 -4
Olaparib, (Log[M]) Saruparib, (Log[M]) Talazoparib, (Log[M]) Talazoparib, (Log[M])
e
DLD1 BRCA2 -/- DLD1 BRCA2 -/- DLD1 BRCA2 +/+ DLD1 BRCA2 -/-
Olap: 296 nM Sar: 2.21 nM siCTRL siPARP1 siCTRL siPARP1
Olap+20μΜ ΤΜΖ: 16.6 nM Sar+20μΜ ΤΜΖ: 0.74 nM <0.0001 <0.0001
Olap+50μΜ ΤΜΖ: 4.33 nM Sar+50μΜΤΜΖ: 0.32 nM <0.0001 NS
60 60

γH2AX mean intensity [r.u.]

120 120
50 50
Survival-CTG [%]

100 100
40 40
80 65x 80 7x
60 60 30 30

40 40 20 20

20 20 10 10

0 0 0 0
-10 -9 -8 -7 -6 -5 -4 -12 -11 -10 -9 -8 -7 -6
DMSO
Tal
TMZ
Tal+TMZ

DMSO
Tal
TMZ
Tal+TMZ

DMSO
Tal

Tal+TMZ

DMSO
Tal
TMZ

TMZ
Tal+TMZ
Olaparib, (Log[M]) Saruparib, (Log[M])

Extended Data Fig. 9 | PARP trapping is dispensable for the synthetic administered. d, Dose-response curves and calculated EC50 values for
lethality of PARP inhibitors with HR deficiency, yet toxic for HR-proficient induction of γΗ2ΑΧ. Data are from one of n = 2 replicates; for DLD1
cells. a-b, Temozolomide (TMZ) reduces the selectivity of olaparib and BRCA2 +/+ > 7732 (range: 7732-21772), for DLD1 BRCA2−/− > 3368 (range:
saruparib for HR-deficient cells. a, Outline of the experiment. b, Dose-response 3368–5803) cells per datapoint. e, Quantification of γΗ2ΑΧ intensity in DLD1
survival curves and calculated EC50 values of olaparib and saruparib-mediated BRCA2 +/+ and DLD1 BRCA2−/− cells transfected with control siRNA or siRNA
lethality of DLD1 BRCA2+/+ and DLD1 BRCA2−/− cells with and without added TMZ. targeting PARP1 and treated with talazoparib (1 μΜ) and optionally with TMZ
Horizontal arrows indicate the fold-change in EC50 values as a result of (50 μΜ); plots show medians and value ranges of 25–75% and 10–90%, filled
administering 50 μM TMZ. Data from one of n = 2 replicates. c-e, Depletion of circles indicate the individual cells in the top and bottom deciles; n = 2
PARP1 suppresses the induction of a DNA damage response in HR-proficient, replicates; >17091 cells per group (range: 17091−31634); ANOVA. PARPi, PARP
but not in HR-deficient cells, treated with talazoparib. c, Outline of the inhibitor; transf., transfection; CTRL, control; CTG, CellTiter-Glo Cell Viability
experiment. γΗ2ΑΧ intensity was monitored 48 h after talazoparib was Assay; NS, not significant.
σ
γ

γ
γ