Supplementary Information
Supplementary Figure 1:
Strong increase of replication extent after Rif1 depletion
.
Supplementary Figure 2: Non
-
homogenous organisation of replication tracks on combed fibers after
mock and Rif1 depletion.
Supplementary Figure 3:
Replication track patterns differ in the presence and absence of Rif1
.
Supplementary Figure 4:
Modeling experimental combing data with MM4
Supplementary Figure 5: Rif1 depletion increases replication foci number.
Supplementary Figure 6:
Rif1 depletion increases limiting replication factors and p
-
MTBP, p
-
Treslin on
chromatin
.
Supplementary Figure 7: MTBP protein levels decrease after MBT during early
Xenopus laevis
development.
Supplementary Figure 8:
Images of uncropped Western blots.
Supplementary
Table
1
:
DNA
combing
data
summary
after
Rif
1
depletion
.
Supplementary
Methods
1
:
list
of
antibodies
Supplementary Methods 2:
Definitions of replication parameters for DNA combing analysis
Rif1 restrains the rate of replication origin firing in
Xenopus laevis
Olivier Haccard
1
,
Diletta
Ciardo
2
,
Hemalatha
Narrissamprakash
1
, Odile Bronchain
3
, Akiko
Kumagai
4
, William G. Dunphy
4
, Arach Goldar
1
, Kathrin
Marheineke
*
1
Supplementary
Figure
2
:
Non
-
homogenous
organisation
of
replication
tracks
on
combed
fibers
after
mock
and
Rif
1
depletion
.
Using
the
dataset
from
Fig
.
2
d,
frequency
of
combed
fibers
versus
the
number
of
replication
tracks
per
fiber
(N)
for
early
S
phase
time
points
(exp)
compared
to
a
Poisson
distribution
(random)
with
identical
mean
(
theo
)
.
a
From
mock
depleted
extracts,
Chi
-
squared
test
(p
=
1
.
4
x
10
-
164
)
.
b
From
Rif
1
depleted
extracts,
Chi
-
squared
test,
p
=
0
.
a
b
0
1
2
3
4
5
6
7
8
9
1
0
1
1
0
.
0
0
.
2
0
.
4
0
.
6
0
.
8
N
t
r
a
c
k
s
/
f
i
b
e
r
s
f
r
a
c
t
i
o
n
o
f
f
i
b
e
r
s
M
o
c
k
e
x
p
M
o
c
k
t
h
e
o
0
1
2
3
4
5
6
7
8
9
1
0
1
1
1
2
1
3
1
4
1
5
1
6
1
7
1
8
1
9
2
0
0
.
0
0
.
2
0
.
4
0
.
6
0
.
8
N
t
r
a
c
k
s
/
f
i
b
e
r
s
f
r
a
c
t
i
o
n
o
f
f
i
b
e
r
s
R
i
f
1
e
x
p
R
i
f
1
t
h
e
o
N
tracks
/fiber
N
tracks
/fiber
Fraction of fibers
Fraction of fibers
∆
Rif1
exp
∆
Rif1
theo
∆
Mock
exp
∆
Mock
theo
Supplementary
Figure
1
:
Strong
increase
of
replication
extent
after
Rif
1
depletion
.
Rif
1
was
immunodepleted
from
egg
extracts,
DNA
was
isolated
at
the
indicated
times,
then
DNA
combing
was
performed
in
two
independent
experiments,
replication
extent
mean
with
SEM
(n=
2
)
,
and
ratios
∆Rif
1
/Mock
were
calculated
.
5
0
m
i
n
m
o
c
k
5
0
m
i
n
R
i
f
1
6
0
m
i
n
m
o
c
k
6
0
m
i
n
R
i
f
1
9
0
m
i
n
m
o
c
k
9
0
m
i
n
R
i
f
1
1
0
5
m
i
n
m
o
c
k
1
0
5
m
i
n
R
i
f
1
0
2
0
4
0
6
0
r
e
p
l
i
c
a
t
i
o
n
(
%
)
50
60
90
105
min
∆
Mock
∆Rif1
early
S
mid
S
8.0
7.0
2.7
2.0
∆Rif1
∆
Mock
Replication
(%)
Supplementary
Figure
3
:
Replication
track
patterns
differ
in
the
presence
and
absence
of
Rif
1
.
a
Example
of
combed
fibers
with
DNA
(green)
and
replication
eyes
(red)
from
Fig
.
2
d,
early
time
points
.
Positions
of
replication
tracks
indicated
below
each
fiber
in
bold
black
.
Third
fiber
in
Rif
1
depleted
conditions
is
a
composed
fiber
of
two
consecutive
microscope
fields
.
b
Mean
excluded
eye
-
to
-
eye
distances
(mean
excluded
ETED)
from
mock
and
Rif
1
depleted
extracts
of
experiment
1
;
corresponding
ratios
are
indicated
below
.
Mean
excluded
ETED
=
excluded
DNA/((N
of
forks/
2
)
-
N
of
ETED)
;
excluded
DNA
=
tot
DNA
-
(mean
ETED
x
N
of
ETED)
.
Mock
40kb
Rif1
260 kb
40kb
a
b
early
S
mid
S
5
0
6
0
9
0
1
0
5
0
2
0
0
4
0
0
6
0
0
8
0
0
m
i
n
m
e
a
n
e
x
c
l
u
d
e
d
E
T
E
D
(
k
b
)
M
o
c
k
R
i
f
1
min
Mean
excluded
ETED (kb)
5.3
4.6
2.2
1.2
Rif1
Mock
Supplementary
Figure
4
:
Modeling
experimental
combing
data
with
MM
4
41
.
The
potential
origins
are
heterogeneously
distributed
along
the
genome
.
Open
circles
represent
normalized
distribution
of
mean
experimental
values
for
indicated
replication
parameters
for
the
two
replicates
(
for
n
see
Suppl
.
Table
1
),
cross
represents
the
means
for
the
2
replicates
.
The
red
dashed
line
is
the
average
of
n
=
100
independent
fits
(grey
dashed
lines)
from
MM
4
41
.
The
red
error
bars
indicate
standard
deviations
.
a
Mock
depletion,
90
min
from
two
biological
replicates,
mean
16
%
replication
.
b
Rif
1
depletion,
90
min
from
two
biological
replicates,
mean
38
%
replication
.
c
Rif
depletion,
mean
14
%
replication,
from
two
biological
replicates
:
respectively
50
min
and
75
min
.
The
goodness
of
the
fit
(
GoF
)
is
calculated
as
퐺표퐹
=
1
−
푦
푓푖푡
−
푦
푒푥푝
2
푦
푒푥푝
−
푦
푒푥푝
2
and
can
vary
from
-
∞
(bad
fit)
to
1
(perfect
fit)
.
exp
. data
Simulations
Mean Simulation
Mean of
exp. data
a
b
c
G
oF
=0.7126
G
oF
=0.4254
G
oF
=0.8737
G
oF
=0.9989
G
oF
=0.9885
GoF
=
-
1.4387
G
oF
=0.9872
G
oF
=0.9452
G
oF
=0.9452
G
oF
=0.6401
G
oF
=0.9309
G
oF
=0.9309
G
oF
=0.61515
G
oF
=0.9147
G
oF
=0.4348
G
oF
=0.9436
G
oF
=0.9925
G
oF
=0.9871
∆Rif1 75+50 min (
mean
14 %
replication
)
∆Rif1 90 min (
mean
38 %
replication
)
∆
Mock
90 min (
mean
16 %
replication
)
Supplementary
Figure
5
:
Rif
1
depletion
increases
replication
foci
number
.
As
in
Figure
3
d,
quantification
of
second
biological
replicate,
mean
with
SD,
two
-
tailed
Mann
-
Whitney
test
.
m
o
c
k
c
o
r
r
R
i
f
1
c
o
r
r
0
2
0
4
0
6
0
1
9
.
1
1
.
2
1
f
o
c
i
c
o
u
n
t
m
o
c
k
F
o
c
i
n
u
m
b
e
r
/
n
u
c
l
e
u
s
Mock
(n=120)
Rif1
(n=73)
P< 0.0001
Foci
number
/ nucleus
Supplementary
Figure
6
:
Rif
1
depletion
increases
limiting
replication
factors
and
p
-
MTBP,
p
-
Treslin
on
chromatin
.
a
Chromatin
fractions
after
Rif
1
depletion
or
Mock
depletion
were
isolated
at
different
times
and
analyzed
by
western
blot
for
indicated
proteins,
second
biological
replicate
of
time
course
experiment
as
in
Fig
.
5
.
b
samples
from
Fig
.
5
b,
treated
with
l
-
phosphatase
.
b
a
Drf1
Cdc7
M
R
M
R
M
R
M
R
60
90
120
180
min
M
R
30
Orc2
MTBP
TopBP1
RecQ4
MCM4
Treslin
Rif1
Cdc45
R
M
M
R
MTBP
p
-
MTBP
-
+
-
+
Orc2
λ
Phosphatase
Treslin
p
-
Treslin
a
b
Supplementary
Figure
7
:
MTBP
protein
levels
decrease
after
MBT
during
early
Xenopus
laevis
development
.
a
Time
course
analysis
of
MTBP
expression
throughout
development,
whole
embryo
protein
extracts
were
analysed
by
western
blotting
against
indicated
proteins,
tubulin
was
used
as
a
loading
control
;
MBT
(mid
-
blastula
transition)
.
b
quantification
of
MTBP
abundance
;
three
biological
replicates
and
two
technical
replicates
of
western
blot
series
of
embryonic
whole
cell
extracts
were
quantified
and
Rif
1
abundance
was
plotted
as
mean
OD
normalized
to
Tubulin,
scaled
using
min
and
max
:
xscaled
=
(x
-
xmin
)
/
(
xmax
-
xmin
),
Mean
with
SED,
n
=
5
.
Tubulin
MTBP
2
5
6
7
8
8.5
9
10
11
19
23
1
28
34
Stages
MBT
Stages
MTBP (%)
1
2
5
6
7
8
8
.
5
9
1
0
1
1
1
9
2
3
2
8
3
4
0
5
0
1
0
0
S
t
a
g
e
s
M
T
B
P
(
%
)
Uncropped
immunoblots
used
in
Figure
1
c
MCM7
Rif1
Orc2
Orc1
Uncropped
immunoblots
used
in
Figure
1
a
Tubulin
Rif1
130
180
130
180
48
63
75
63
75
100
75
130
180
100
75
130
180
100
Supplementary Figure 8:
Images of uncropped Western blots.
48
Uncropped
immunoblots
used
in
Figure
5
b
Uncropped
immunoblots
used
in
Figure
5
d
Rif1
Orc2
MTBP
63
75
130
180
130
180
63
75
100
130
100
130
100
130
63
75
130
180
Orc2
TopBP1
RecQ4
Rif1
Drf1
Cdc7
MTBP
MCM4
Treslin
Cdc45
Rif1
130
180
100
130
48
63
75
48
Supplementary Figure 8 (continued)
Uncropped immunoblots used in
Supplementary Figure 6a
Orc2
TopBP1
RecQ4
Rif1
Drf1
Cdc7
MTBP
MCM4
Treslin
Rif1
Cdc45
Rif1
MTBP
Orc2
Treslin
Uncropped
immunoblots
used
in
Supplementary
Figure
6
b
100
180
130
63
75
130
130
180
180
130
180
63
75
63
75
48
100
130
100
130
130
100
130
63
75
Supplementary Figure 8 (continued)
130
180
Uncropped
immunoblots
used
in
Supplementary
Figure
7
a
Tubulin
MTBP
100
130
48
63
75
Supplementary
Table 1: DNA
combing
data
summary
after
Rif1
depletion
.
Replicate 1
early S phase
mid S phase
Min
50 min
60 min
75 min
90 min
105 min
sum total
condition
∆Mock
∆Rif1
∆Mock
∆Rif1
∆Mock
∆Rif1
∆Mock
∆Rif1
∆Mock
∆Rif1
Analysed DNA (kb)
57317.4
33913.6
50565.6
63104.9
34450.7
55022.6
32863.5
22546.7
24290.0
34122.3
408197.1
Replicated DNA(kb)
1376.3
6234.0
1778.9
14876.4
2705.7
18221.1
6299.0
9372.4
7151.3
17207.5
85222.5
Replication Extent
0.024
0.184
0.035
0.236
0.079
0.331
0.192
0.416
0.294
0.504
Number of analysed fibers
482
352
673
690
366
652
288
258
352
349
4462.0
Number of fully replicated fibers
2
15
6
29
33
9
11
14
34
15
Number of unreplicated Fibers
393
133
548
186
171
117
111
54
52
41
fraction of unreplicated fibers
0.82
0.38
0.81
0.27
0.47
0.18
0.39
0.21
0.15
0.12
mean size of all fibers (kb)
118.9
96.3
75.1
91.5
94.1
84.4
114.1
87.4
69.0
97.8
91.5
mean size of unreplicated fibers (kb)
117.3
91.8
71.0
85.9
106.1
72.3
129.0
83.7
85.6
95.7
Number of replication eyes
161
503
196
1112
226
1639
481
652
558
1247
6775.0
Mean Eye Length (kb)
6.3
8.5
6.7
9.6
6.8
9.1
10.7
10.4
8.7
10.1
Number of eye to eye distances (ETED)
82
341
101
737
125
1193
353
497
379
1000
4808.0
Mean ETED length (kb)
23.6
19.4
22.3
21.6
25.2
18.4
22.1
16.3
19.1
15.7
Mean excluded ETED length (kb)
586.1
109.6
381.9
83.8
182.5
53.3
130.8
60.7
56.9
47.3
Number of replication forks
353
1180
455
2600
593
3628
1089
1469
1357
2779
15503.0
Fork Density (forks/100 kb)
0.62
3.48
0.90
4.12
1.72
6.59
3.31
6.52
5.59
8.14
Rif/Mock FD
5.65
4.58
3.83
1.97
1.46
Number of gaps
192
677
259
1488
367
1989
608
817
799
1532
8728.0
Mean gap size (kb)
7.2
9.2
6.9
10.0
7.4
9.2
10.4
11.5
9.0
11.2
Replicate
2
early S phase
mid S phase
min
50 min
60 min
75 min
90 min
105 min
sum total
Condition
∆Mock
∆Rif1
∆Mock
∆Rif1
∆Mock
∆Rif1
∆Mock
∆Rif1
∆Mock
∆Rif1
Analysed DNA (kb)
29676.5
38244.1
47274.0
32949.2
24942.5
38001.2
40709.9
51519.9
47109.9
50042.1
400469.2
Replicated DNA(kb)
103.2
920.3
527.4
2552.9
964.2
3853.5
4824.0
17415.5
12500.3
26768.7
Replication Extent
0.003
0.024
0.011
0.077
0.039
0.101
0.118
0.338
0.265
0.535
Number of analysed fibers
312
457
371
402
352
525
675
917
780
1320
6111.0
Number of fully replicated fibers
0
3
1
23
4
5
56
78
152
367
Number of unreplicated Fibers
267
374
307
218
264
337
220
143
147
116
fraction of unreplicated fibers
0.86
0.82
0.83
0.54
0.75
0.64
0.33
0.16
0.19
0.09
Mean size of all fibers (kb)
95.1
83.7
127.4
82.0
70.9
72.4
60.3
56.2
60.4
37.9
65.5325086
Mean size of unreplicated fibers (kb)
88.2
79.4
127.3
89.8
70.0
69.6
73.2
61.0
75.7
54.7
Number of replication eyes
41
111
114
273
116
396
447
1296
1025
1400
5219.0
Mean Eye Length (kb)
1.8
5.8
4.0
6.5
4.9
7.3
5.7
8.7
7.5
10.0
Number of eye to eye distances (ETED)
5
53
55
161
64
251
202
839
701
953
3284.0
Mean ETED length (kb)
35.1
25.5
29.7
14.6
14.3
15.7
24.7
16.7
16.4
15.2
Number of replication forks
92
266
241
692
297
922
1215
3268
2627
4067
13687.0
Mean excluded ETED (kb)
nd
nd
696.8
165.4
284.3
162.1
88.1
47.1
58.2
32.92
Fork Density (forks/100 kb)
0.31
0.70
0.51
2.10
1.19
2.43
2.98
6.34
5.58
8.13
Rif/mock FD
2.24
4.12
2.04
2.13
1.46
Number of gaps
51
155
127
419
181
526
768
1972
1602
2667
8468.0
mean gap length (kb)
2.0
5.9
4.2
6.1
5.3
7.3
6.3
8.8
7.8
10.0
If, immunofluorescence; WB, western blot; comb, DNA combing
List of antibodies used in this study
Supplementary
Methods
1
Antigen
Host
Source
Reference
Application
RRID and/or
Doi
Primary antibodies
Anti
-
Xenopus
RIF1
Rabbit
Covalab,
Villeurbanne,
France
1:500 (IF)
1:1000 (WB)
10.4161/cc.11.6.19636
10.1093/n10.1093 /
nar
/gkab756
10.7554/eLife.75741
Anti
-
Xenopus
and Human
Treslin
Rabbit
A. Kumagai and
W. G. Dunphy,
Pasadena, USA
1:1000 (WB)
10.1016/j.cell.2009.12.049
Anti
-
Xenopus
MTBP
Rabbit
A. Kumagai and
W. G. Dunphy,
Pasadena, USA
1:1000 (WB)
10.1091/mbc.E17
-
07
-
0448
Anti
-
human
MCM4
Rabbit
Bethyl lab
A300
-
193A
1:2000 (WB)
AB_162720
Anti
-
Xenopus
Drf1
Rabbit
A. Kumagai and
W. G. Dunphy,
Pasadena, USA
1:1000 (WB)
10.1074/jbc.M307144200
Anti
-
Xenopus
Cdc7
Rabbit
J. Walter
1:1000 (WB)
10.1016/s1097
-
2765(00)80241
-
5
Anti
-
Xenopus
Cdc45
Rabbit
A. Kumagai and
W. G. Dunphy,
Pasadena, USA
1:500 (WB)
10.1091/mbc.e05
-
07
-
0671
Anti
-
Xenopus
RecQ4
Rabbit
A. Kumagai and
W. G. Dunphy,
Pasadena, USA
1:1000 (WB)
10.1016/j.cell.2005.05.015
Anti
-
Xenopus
TopBP1
Rabbit
A. Kumagai and
W. G. Dunphy,
Pasadena, USA
1:1000 (WB)
10.1016/j.cell.2005.12.041
Anti
-
Xenopus
ORC2
Rabbit
Gift from Dr. J. L.
Maller (EXRC),
Portsmouth, UK
1:1000 (WB)
10.1038/s41467
-
022
-
34779
-
4
10.15252/embj.201796585
Anti
-
Xenopus
ORC1
Rabbit
Gift from R. A.
Laskey,
Cambridge, UK
1:1000 (WB)
10.1016/S0960
-
9822(96)00746
-
4
Anti
-
Xenopus
MCM7
Rabbit
Gift from R. A.
Laskey
,
Cambridge, UK
1:1000 (WB)
10.1073/pnas. 93.19.10189
Anti
-
α Tubulin
Mouse
Sigma
T5168
1:10000 (WB)
AB_477579
Anti
-
human
ssDNA
Mouse
DSHB
autoanti
-
ssDNA
1:100 (comb)
AB_10805144
Secondary antibodies
AlexaFluor488
anti
-
mouse
Rabbit
Invitrogen
A11059
1:50 (comb)
AB_2534106
AlexaFluor488
anti
-
rabbit
Goat
Thermo Fisher
Scientific
A11008
1:200 (IF)
1:50 (comb)
AB_143165
Anti
-
streptavidin
biotinylated
Mouse
Abcyss
BA
-
0500
1:50 (comb)
AB_2336221
AlexaFluor594
conjugated
streptavidin
Mouse
Invitrogen
S11227
1:50 (comb)
HRP anti
-
mouse IgG
Goat
Sigma
-
Aldrich
A4416
1:10000 (WB)
AB_258167
HRP anti
-
rabbit IgG
Donkey
GE Healthcare
NA934
1:10000 (WB)
AB_772206
Supplementary
Methods
2
Replicated fraction of a fiber
f(t)
Sum of all red tracks (EL) on a fiber/length
of fiber (green track)
Total replicated fraction of a population
of fibers (
f(t))
Sum of all EL/ sum of fiber length
Percentage of
unreplicated
fibers
N of fibers with no red track/ N of all fibers
Fork density of a population of fibers
Number of forks/sum of fiber lengths
Active origin for
I(t)
calculation
EL <3 kb
Frequency of initiation
I(t)
Number of active origins /
((1
-
f(t))*
fiber length*180 sec)
Excluded DNA (kb)
Total DNA
-
(mean ETED x N of ETED)
Mean excluded
ETED (kb)
E
xcluded
DNA/((N
of
forks/
2
)
-
N
of
ETED)
Definitions
of
replication
parameters
for DNA
combing
analysis