Post on 20-Jan-2021
The human intra-S checkpoint response to UV-induced DNA
damage
William KaufmannDepartment of Pathology and Laboratory
MedicineUniversity of North Carolina at Chapel Hill
Intra-S Checkpoints
DNA damage Incomplete replication
IR UVC
Mechanisms and consequences of inhibition of DNA replication by UVC
TT TTATR
Chk1
DNA pol η/ζ
DNA dsb
ATM Intra-S & G2 checkpoint
Replicon initiation
Block pol α/δ
TT
NERp53 XPC/XPE
?
?
Velocity SedimentationSeparates DNA replication intermediates based on size and is used to quantify the inhibition of DNA synthesis in classes of
nascent DNA molecules.
5%
20%
LinearSucroseGradient
•Cells are irradiated, incubated for 30 minutes, and pulse-labeled with 3H-thymidine. Cells are harvested and lysed on top of a linear sucrose gradient. Nascent DNA molecules are separated by centrifugation.
•Bulk DNA is pre-labeled with 14C-thymidine for at least one population doubling.
DNA Profile
0.00
0.40
0.80
1.20
1.60
0 10 20 30
Fraction Number
Nor
mal
ized
3H
CPM
Sham Treated
DNA Profile
0.00%
2.50%
5.00%
7.50%
10.00%
0 10 20 30
Fraction Number
Perc
ent 1
4C
14C-thymidine
Dose-response and time-course for inhibition of DNA replication in UVC-treated
human fibroblasts
sham
0.8 J/m2
1.3 J/m2
5.2 J/m2
30’ post UV
60’
90’
120’
Dose: 30’ post UVC Time: 0.8 J/m2
Telomerized cell lines from patients with genetic instability syndromes.
ATM
ATR
Nbs1
Mre11
NHF1 ATM-/- Nbs1 -/- Mre11 -/-
ATM is not required for the UVC-induced inhibition of replicon initiation; caffeine reverses
the effectN
orm
aliz
ed 3 H
CPM
Fraction Number
NHF1
0.00
0.30
0.60
0.90
1.20
0 10 20 30
Sham 1J
NHF1
0.00
0.40
0.80
1.20
1.60
0 10 20 30
Sham 1J
ATM -/-
0.00
0.30
0.60
0.90
1.20
1.50
0 10 20 30
Sham 1J
ATM -/-
0.000
0.400
0.800
1.200
1.600
0 10 20 30
Sham 1J
Medium Caffeine
ATR (AT-and rad3-related)
• Member of the PI-3-kinase-related kinase family
• Essential gene product– ATR -/- mice die by E8.5– Cultured blastocysts display proliferation defects and mitotic
catastrophe
• Overexpression of kinase-inactive ATR (ATRKI) allele– Renders cells hypersensitive to DNA damaging agents– Abrogates the Decatenation, Replication, and G2 DNA Damage
Checkpoints.
Tet-on Inducible System
Tre
rTTA
ATRKITre
rTTA
ATRKI Tre ATRKITre ATRKI
Dox
rTTA
Dox
rTTArTTA
rTTADox
ATRWT ATRKD
Dox- + - +
rTTADox
rTTADox
rTTADox
ATRWT ATRKD
Dox- + - +
Overexpression of ATRKI abrogates the UVC-induced intra-S Checkpoint
U2OS Uninduced
0.00
0.05
0.10
0.15
0.20
0.25
0.30
0 10 20 30
Sham 1J
U2OS ATRKI
0.00
0.100.20
0.30
0.400.50
0.600.70
0 10 20 30
Sham Dox 1J Dox
U2OS Caffeine
0.00
0.05
0.10
0.15
0.20
0.25
0 10 20 30
Sham Caff 1J Caff
U2OS ATRWT
0.00
0.05
0.10
0.15
0 10 20 30
Sham 1J
Fraction Number
Nor
mal
ized
3 H C
PM
Chk1
• Essential gene product• Chk1 -/- mice die by E6.5• Cultured blastocysts display proliferation
defects
• Effector Kinase• Required for Replication, and G2 DNA Damage
checkpoints.• Sensitive to UCN-01 (7-hydroxystaurosporine)
Chk1 phosphorylation following UVC irradiation is dependent on ATR
Phospho-S317 Chk1
Phospho-S345 Chk1
Chk1
0 1 8 0 1 8 UVC J/m2
- - - + + + DoxU2OS U2OS ATRKI
ATR
No Virus Chk1wt Chk1ki GFP
UVC-induced inhibition of DNA synthesis
0
25
50
75
100
125
No Virus Chk1 wt Chk1 ki GFPDN
A S
ynth
esis
(Per
cent
of C
ontr
ol)
Sham 1 J/m2
Over-expression of Chk1ki overrides the UVC-induced intra-S checkpoint
* *P < 0.001
Cdc25A
Wee1
Myt 1
The Intra-S Checkpoint.
?
? ??
ATR
Chk1
Dbf4
Cdc7
Cyclin A/E
Cdk 1/2
ORI
ORC
Cdc6
6342
5 76342
5 7
Effective doses for UVC- and IR-induced inhibition of replicon initiation
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
0 10 20 30
Sham UV 1 J/m2
0.0
0.2
0.4
0.6
0.8
1.0
1.2
0 10 20 30
Sham IR 5 Gy
Fraction Number
Nor
mal
ized
3 H C
PM
UVC (1 J/m2) IR (5 Gy)
UVC IRSham 1 J/m2 Sham 5 Gy
Cdc25A
Chk1
P-Ser 317 Chk1
Chk2
P-Thr 68 Chk2
Cdc25A is not degraded under conditions that activate the UVC-induced intra-S checkpoint
Cyclin E/Cdk2 kinase is not inhibited in UVC-damaged fibroblasts
Chk1 phosphorylates Dbf4 in vitro and interacts with Dbf4 in vivo
Over-expression of Flag- or Myc-tagged Dbf4 attenuates intra-S checkpoint response to UVC but not IR
Over-expression of Flag-Dbf4 does not block Chk1 activation after UVC and Flag-Dbf4/Cdc7 interaction is
not affected by UVC
Add 10 μM IdU,10 min
(to label active replication units)
Add 10 μM IdU,10 min
(to label active replication units)
Add 10 ml of reserved medium containing 100μM CldU,
20 min(ongoing DNA synthesis, and the firing
of new replication origins)
Resuspend to 100 – 200
cells per μl.
Take 2 μl of cell solution and molecular comb the DNA
Cells released with trypsin,
washed with PBS
Resuspend to 100 – 200
cells per μl.
Take 2 μl of cell solution and molecular comb the DNA
Cells released with trypsin,
washed with PBS
Cells released with trypsin,
washed with PBS
Labelling patterns of DNA fibers reveal replicon dynamics
Add 10 mM IdU,10 min
+/- UVC (0, 1, 10 J/m2)
Add 10 ml of reserved medium containing 100 mM CldU, 20 min
Log Population of HeLa Cells
Count new origins (CldU-only tracks)and IdU-only tracks 0%
50%
100%
Sham 1 10Fluence (J/m2)
0%100%200%300%400%500%
Sham 1 10Fluence (J/m2)
Rel
ativ
e %
Red
-O
nly
trac
ksR
elat
ive
% G
reen
-O
nly
trac
ks
Add 10 mM IdU,10 min
+/- UVC(0, 1 J/m2)
Add 10 ml of reserved medium containing 100 mM CldU, 20 min
Log Population of HeLa Cells
Count new origins (CldU-only tracks)
+/- Caffeine(added 30 min prior to UV)
0%
50%
100%
150%
Sham 1 J/m2 CaffeineSham
Caffeine,1J/m2
Treatment
Rel
ativ
e %
of
New
Orig
in
1 J/m2 Caffeine,1 J/m2
Add 10 mM IdU,10 min
+/- UVC (0, 1J/m2)
Add 10 mls of reserved media containing 100mM
CldU, 20 min
+/- Knockdown of ATR or CHK1
Count new origins (CldU tracks)
0%
50%
100%
150%
Non
targ
etin
gR
NAi
, Sha
m
Non
targ
etin
gR
NAi
, UV
ATR
RN
Ai,
Sham
ATR
RN
Ai,
UV
NTC
RN
AiSh
am
NTC
RN
AiU
V
Chk
1 R
NAi
Sham
Chk
1 R
NAi
UV
Treatment
Rel
ativ
e %
of
New
Orig
inLog Population of HeLa Cells
ATR RNAi Chk1 RNAi
Timeless (Human) is required for activation of Chk1 and inhibition of DNA synthesis by low dose UVC (Űnsal-Kaçmaz et al., 2005, Mol Cell Biol).
Timeless-interacting protein (Tipin) discovered in yeast two-hybrid analysis of murine Timeless (Gotter, 2003, J Mol Biol)
Timeless and Tipin are homologs of S. pombe Swi/Swi3 which serves as a replication fork protection complex (e.g. Noguchi et al., 2004, Mol Cell Biol)
TimTip
Role of Timeless and Timeless-interacting protein (Tipin) in
Checkpoint Activation
Timeless and Tipin form a complex in human and insect cells
293T insect
Tipin has an RPA-binding domain similar to that seen in XPA, and binds to RPA34 in human cells
Tipin does not bind DNA directly but interacts with RPA/DNA complexes; RPA appears to load Tipin onto
DNA
Knockdown of Tipin reduces expression of Timeless and inhibits activation of Chk1 in response to replication
stress
Knockdown of Timeless and Tipin attenuates intra-S checkpoint response to UVC
1.Ongoing forks
2.Terminations
3.Newly Fired origins
4.Replicon Merging
IdU CldUA
B
0%
20%
40%
60%
80%
100%
120%
140%
Cnt siRNA,Sham
Cnt siRNA,UV
Tim siRNA,Sham
Tim siRNA,UV
Tip siRNA,Sham
Tip siRNA,UV
Rel
ativ
e %
of N
ew O
rigin
s
UVC inhibits DNA chain elongation; knockdown of Timeless inhibits DNA chain elongation; knockdown of Tipin reverses the
UVC-induced inhibition of chain elongation.
Red tracksa siRNAc Mean S.D. B/A p-value
μm (n)
A. NTC 5.3 2.9 (n=166) B. Tim 2.7 1.9 (n=225) 0.52 <0.0001
A. NTC 5.3 2.9 (n=166) B. Tipin 4.7 2.4 (n=201) 0.89 0.57
A. Tipin 4.7 2.4 (n=201) B. Tim 2.7 1.9 (n=225) 0.58 <0.0001
Green tracksb siRNAc Mean S.D. B/A p-value
μm (n)
A. NTC, Sham 5.8 3.0 (n=146) B. NTC, 2.5 J/m2 2.9 2.0 (n=139) 0.49 <0.0001
A. Tim, Sham 2.6 1.5 (n=158) B. Tim, 2.5 J/m2 2.2 1.1 (n=165) 0.82 0.153
A. Tipin, Sham 3.8 2.4 (n=162) B. Tipin, 2.5 J/m2 4.6 2.6 (n=161) 1.20 0.721
Analysis of DNA fibers reveals separation of function between Timeless and Tipin
Knockdown of either protein attenuates intra-S checkpoint response to UVC
Knockdown of Timeless inhibits DNA chain elongation in undamaged cells; knockdown of Tipin reverses this effect suggesting that Tipin may be responsible for the inhibition
Knockdown of Tipin reversed the UVC-induced inhibition of DNA chain elongation indicating that the inhibition of chain elongation includes active checkpoint signaling (see Wang et al.(2004) Nucl. Acids Res)
Tim/Tipin complex brings Chk1 to ATR/ATRIP/TopBP1 at stalled replication
forks
T<>T
T<>TRPA
ATR/ATRIP/TopBP1
Timeless
Chk1
Tipin
PO4
PO4 Phospho-Chk1
PO4 PO4
PO4
RPA loads Tipin onto DNA; this may inhibit DNA chain elongation
T<>T
T<>T
RPA
ATR/ATRIP/TopBP1
Timeless
Chk1
Tipin
Phospho-Chk1PO4
Intra-S Checkpoint Response to UVCRPAUV
photoproducts
TimTip
Claspin
Hus1 Rad1Rad9 Rad17
Hus1 Rad1Rad9 Rad17
Inhibit Replicon Initiation
Chk1
ATRATRIP
Inhibit Chain Elongation
ReplicationComplex
TopBP1
Dbf4Cdc7
?
?
Acknowledgements• Marila Cordeiro-Stone• Aziz Sancar• David Kaufman• Paul Chastain• Keziban Űnsal-Kaçmaz• Timothy Heffernan• Dennis Simpson• Yingchun Zhou• Ping-Ping Qu• Kathleen Nevis
• Cyrus Vaziri• Stuart Schreiber• Robert Weinberg• Parvis Minoo• Hisao Masai• Richard Paules• Alexandra Heinloth