Supplementary information
Increased replication initiation and conflicts with transcription underlie Cyclin Einduced replication stress
Rebecca M. Jones, Oliver Mortusewicz, Islam Afzal, Maëlle Lorvellec, Paloma García,
Thomas Helleday and Eva Petermann
Figure S1. Reduced G1 population and genomic instability in Cyclin Eoverexpressing cells.
(A) U2OS-Cyclin E cells were grown in presence (- Cyclin E) or absence (+ Cyclin
E) of tetracycline for 5 days. Samples were fixed each day, DNA was stained with
propidium iodide, and cell cycle profiles were analyzed using flow cytometry. (B)
Percentages of cells in different cell cycle stages after growing in presence (- Cyclin
E) or absence (+ Cyclin E) of tetracycline as above. The 3-day time point used
throughout this study is highlighted. (C) U2OS-Cyclin E cells were grown on
coverslips in presence (- Cyclin E) or absence (+ Cyclin E) of tetracycline for 72 h,
fixed, and DNA was stained with DAPI to identify micronuclei. Percentages of cells
with micronuclei are shown.
Figure S2. Cellular effects of Cyclin E overexpression are reversible.
U2OS-Cyclin E cells were grown for 72 h in presence or absence of tetracycline.
Cells were continued in presence (- Cyclin E) or absence (+ Cyclin E 6 days) of
tetracycline, or tetracycline was re-added (Cyclin E reversed) for another 72 h. (A)
Protein levels of Cyclin E and -Tubulin (loading control) in whole cell extracts. (B)
Cells were fixed and immunostained for γH2AX. Percentages of cells displaying
more than 10 γH2AX foci. (C) Cells were labeled with CldU and IdU for 20 min
1
each, DNA fiber spreads prepared and distributions of replication fork speeds
measured. Results from one experiment are shown. Average replication fork speeds: Cyclin E 0.51 kb/min; + Cyclin E 6 days 0.36 kb/min; Cyclin E reversed 0.54 kb/min.
Figure S3. Roscovitine prevents replication fork slowing by Cyclin E
overexpression.
U2OS-Cyclin E cells were grown for 72 h in presence (- Cyclin E) or absence (+
Cyclin E) of tetracycline, treated with 25μM roscovitine (rosc) or DMSO for 60 min
and labeled with CldU and IdU for 20 min each. (A) Protein levels of Cyclin E and Tubulin (loading control) in whole cell extracts from cells after treatment with rosc or
PHA-767491 (PHA). (B) New origin firing in U2OS-Cyclin E cells treated with rosc
for 100 min, shown as percentage of all red (CldU) labeled tracks. (C) Distribution of
replication fork speeds in Cyclin E cells treated with DMSO or rosc for 100 min. (D)
Cells were grown on coverslips, fixed and immunostained for γH2AX. Quantification
of U2OS-Cyclin E cells displaying more than 10 γH2AX foci. Means and SEM (bars)
of at least three independent experiments are shown. Values marked with asterisks are
significantly different (student’s t-test, * p < 0.05, ** p < 0.001, *** p < 0.001).
Figure S4. Slow replication fork progression in Cyclin E-overexpressing MRC5
cells depends on high levels of replication initiation and transcription
MRC5 human fibroblasts were transfected with pcDNA3-Cyclin E for 24 h, treated
with 10 μM PHA-767491 (PHA) or 50 μM cordycepin for 60 min and labeled with
CldU and IdU for 20 min each. (A) Protein levels of Cyclin E and -Tubulin (loading
control) in whole cell extracts from MRC5 cells after treatment with PHA and
cordycepin. (B) New origin firing in Cyclin E-overexpressing MRC5 cells treated
2
with PHA, shown as percentage of labeled tracks. (C) Distribution of replication fork
speeds in MRC5 cells treated with DMSO or PHA. (D) Distribution of replication
fork speeds in (-Cyclin E) MRC5 cells treated with DMSO or PHA. (E) Distribution
of replication fork speeds in MRC5 cells treated with DMSO or cordycepin. (F)
Distribution of replication fork speeds in (- Cyclin E) MRC5 cells treated with DMSO
or cordycepin. Means+/- SEM (bars) of three independent experiments are shown.
Figure S5. Slow replication fork progression in presence of overexpressed Cyclin
E is not caused by DNA resection.
(A) U2OS cells were treated with 1 M camptothecin in presence or absence of Mirin
as indicated for 60 min. Cells were fixed and immunostained for phospho-RPA32
(S4/S8). (B) U2OS cells were treated with camptothecin in presence or absence of
Mirin as indicated for 60 min. Levels of phospho-RPA32 (S4/S8) in whole cell
extracts were determined by Western blot. (C) U2OS-Cyclin E were grown for 72 h in
presence or absence of tetracycline and treated with 100 μM Mirin for 60 min before
labeling with CldU and IdU for 20 min each. Protein levels of Cyclin E and Tubulin (loading control) in whole cell extracts were determined. (D) Quantification
of new origin firing in U2OS-Cyclin E cells treated as indicated, shown as percentage
of all labeled tracks. (E) Distribution of replication fork speeds after treatment with
Mirin. Means +/- SEM (bars) of three independent experiments are shown.
3
Figure S6. Roscovitine and PHA-767491 do not prevent H2AX induction by
DNA damaging agents.
(A) U2OS cells were grown on coverslips and treated with 10 μM PHA-767491
(PHA) or 25 μM roscovitine (rosc) for 60 min before treatment with DNA damaging
agents hydroxyurea (HU, 2 mM for 2 h) or camptothecin (CPT, 1 µM for 1 h), or
exposure to gamma-irradiation (IR, 2 Gy). Cells were fixed and immunostained for
γH2AX (green). DNA was counterstained with DAPI (blue).
4
B
255
PI
- Cyclin E
1400
Events
1400
0
Events
255
PI
255
PI
255
C
%S
%G2
%G1
%S
%G2
1 day
52
31
17
49
36
15
2 days
51
34
15
47
34
19
3 days
44
33
23
35
45
20
4 days
45
33
22
42
33
25
5 days
54
26
20
48
32
20
255
PI
255
0
0
+ Cyclin E
%G1
PI
Events
Events
0
0
0
1400
PI
0
0
0
Used in this study
% cells with micronuclei
255
PI
255
Events
1400
Events
0
PI
1400
0
1400
255
PI
0
0
Events
1400
0
5 days
0
0
0
255
PI
4 days
Events
1400
Events
Events
0
+ Cyclin E
3 days
1400
2 days
1400
1 day
0
- Cyclin E
A
0
20
15
10
5
0
- Cyclin E + Cyclin E
Figure S1
5
B
6d 6d 3d tetracycline
+
- 3d +
kDa
46
¬ Cyclin E
58
¬ αTubulin
46
C
25
20
15
10
5
0
- Cyclin E + Cyclin E Cyclin E
reversed
60
+ Cyclin E 6 days
50
% of forks
% gH2AX positive cells
A
Cyclin E reversed
40
- Cyclin E
30
20
10
0
<0.2
<0.4
<0.6
<0.8
<1
<1.2
<1.4
fork speed (kb/min)
Figure S2
6
+
+
-
+
+
-
+
-
+
kDa
46
¬ Cyclin E
46
¬ aTubulin
C
+ Cyclin E + DMSO
+ Cyclin E + roscovitine
- Cyclin E
50
40
% of forks
tetracycline
roscovitine
PHA-767491
30
20
10
0
<0.2
<0.4
<0.6
<0.8
<1
fork speed (kb/min)
<1.2
<1.4
B
% new origins
+
-
+ Cyclin E + DMSO
+ Cyclin E + rosc
6
5
4
3
2
1
0
**
D
25
% gH2AX positive cells
A
- Cyclin E
***
20
+ Cyclin E
*
15
10
5
0
DMSO
Rosc
Figure S3
7
A
B
+
-
+
+
-
+
+
-
+
+
pcDNA3.1-Cyclin E
PHA-767491
cordycepin
46
¬ Cyclin E
58
¬ αTubulin
46
C
+ Cyclin E DMSO
+ Cyclin E PHA
- Cyclin E DMSO
D
20
10
0
<0.2
<0.6
<1
<1.4
<1.8
8
6
4
2
0
- Cyclin E DMSO
- Cyclin E PHA
20
10
0
<2.2
<0.2
<0.6
<1
<1.4
<1.8
<2.2
fork speed (kb/min)
fork speed (kb/min)
E
F
+ Cyclin E
+ Cyclin E cordycepin
- Cyclin E
30
20
10
0
<0.2
<0.6
<1
<1.4
fork speed (kb/min)
<1.8
<2.2
- Cyclin E
30
- Cyclin E cordycepin
% of forks
% of forks
+ Cyclin E
+ Cyclin E PHA
10
30
% of forks
30
% of forks
% new origins
kDa
-
20
10
0
<0.2
<0.6
<1
<1.4
<1.8
<2.2
fork speed (kb/min)
Figure S4
8
A
- Camptothecin
- Mirin
Blue: DNA
B
-
+
-
+
100
+ Camptothecin + Camptothecin
- Mirin
+ 10mM Mirin
+ Camptothecin
+ 50mM Mirin
+ Camptothecin
+ 100mM Mirin
Red: phospho-S4/8 RPA32
+
50
+
10
+ Camptothecin
1 Mirin (mM)
C
+
-
+
+
-
+
Tetracycline
Mirin
¬ Cyclin E
¬ phospho-RPA32
¬ aTubulin
¬ aTubulin
% new origins
+ Cyclin E DMSO
+ Cyclin E Mirin
6
5
4
3
2
1
0
E
50
+ Cyclin E + DMSO
+ Cyclin E + Mirin
- Cyclin E + DMSO
40
% of forks
D
30
20
10
0
<0.2 <0.4 <0.6 <0.8
<1
<1.2 <1.4 <1.6
fork speed (kb/min)
Figure S5
9
DMSO
PHA-767491
Roscovitine
gH2AX
2 mM HU
gH2AX
+ DNA
gH2AX
1 µM CPT
gH2AX
+ DNA
gH2AX
2 Gy IR
gH2AX
+ DNA
Figure S6
10
Table S1: Average fork speeds and statistical analysis for DNA fibre experiments
Experiment
Av. fork speed1
p2
p2
+ Cyclin E
0.32 +/- 0.02
0.032
+ Cyclin E + PHA-767491
0.47 +/- 0.02
0.006
0.347
- Cyclin E
0.46 +/- 0.01
0.032
- Cyclin E + PHA-767491
0.58 +/- 0.04
0.006
0.093
+ Cyclin E
0.33 +/- 0.03
0.025
+ Cyclin E + Mirin
0.31 +/- 0.02
0.098
0.015
- Cyclin E
0.49 +/- 0.02
0.025
- Cyclin E + Mirin
0.39 +/- 0.01
0.064
0.036
+ Cyclin E + con siRNA
0.34 +/- 0.03
0.002
+ Cyclin E + Cdc6 siRNA
0.71 +/- 0.10
0.007
0.021
- Cyclin E + con siRNA
0.54 +/- 0.06
0.002
- Cyclin E + Cdc6 siRNA
0.55 +/- 0.05
0.004
0.386
+ Cyclin E
0.34 +/- 0.02
0.018
+ Cyclin E + cordycepin
0.43 +/- 0.02
0.017
0.063
- Cyclin E
0.51 +/- 0.02
0.018
- Cyclin E + cordycepin
0.51 +/- 0.03
0.016
0.492
+ Cyclin E
0.40 +/- 0.04
0.076
+ Cyclin E + RNase H1
0.49 +/- 0.05
0.013
0.10
- Cyclin E
0.81 +/- 0.19
0.076
- Cyclin E + RNase H1
0.67 +/- 0.05
0.003
0.24
+ Cyclin E + con siRNA
0.30 +/- 0.02
0.061
4
+ Cyclin E + con + cordy
0.43 +/- 0.04
0.024
0.102
+ Cyclin E + Cdc6 siRNA
0.67 +/- 0.13
0.061
+ Cyclin E + Cdc6 + cordy
0.68 +/- 0.10
0.037
0.282
- Cyclin E + con siRNA
0.48 +/- 0.06
0.098
- Cyclin E + con + cordy
0.46 +/- 0.03
0.267
- Cyclin E + Cdc6 siRNA
0.54 +/- 0.07
0.098
0.116
- Cyclin E + Cdc6 + cordy
0.58 +/- 0.07
0.118
0.355
MRC5 + Cyclin E
0.54 +/- 0.04
0.010
5
MRC5 + Cyclin E + PHA
0.91 +/- 0.06
0.011
0.102
MRC5 + Cyclin E + cordy
0.79 +/- 0.06
0.006
0.015
MRC5
1.18 +/- 0.10
0.010
MRC5 + PHA
1.10 +/- 0.10
0.038
0.342
MRC5 + cordy
1.02 +/- 0.07
0.003
0.083
1
kb/min +/- SEM
2
1-tailed, paired student’s t-test compared to sample marked (-)
3
4
5
number of independent repeats
cordycepin
PHA-767491
n3
n tracks
3
3
3
3
3
3
3
3
5
5
5
5
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
1033
836
746
557
1198
1163
811
1149
2092
1040
1261
1134
1390
1065
925
783
1386
1527
746
747
1200
1105
609
589
848
858
579
525
983
661
674
330
389
378
11