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