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Transcript of The human intra-S checkpoint response to UV-induced DNA ... The human intra-S checkpoint response to

  • The human intra-S checkpoint response to UV-induced DNA

    damage

    William Kaufmann Department of Pathology and Laboratory

    Medicine University 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

    NER p53 XPC/XPE

    ?

    ?

  • Velocity Sedimentation Separates DNA replication intermediates based on size and is used to quantify the inhibition of DNA synthesis in classes of

    nascent DNA molecules.

    5%

    20%

    Linear Sucrose Gradient

    •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

    N or

    m al

    iz ed

    3 H

    C PM

    Sham Treated

    DNA Profile

    0.00%

    2.50%

    5.00%

    7.50%

    10.00%

    0 10 20 30

    Fraction Number

    Pe rc

    en t 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 effect N

    or m

    al iz

    ed 3 H

    C PM

    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

    rTTA Dox

    ATRWT ATRKD

    Dox- + - +

    rTTA Dox

    rTTA Dox

    rTTA Dox

    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

    0.10 0.20

    0.30

    0.40 0.50

    0.60 0.70

    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

    N or

    m al

    iz ed

    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 - - - + + + Dox

    U2OS 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 GFPD N

    A S

    yn th

    es is

    (P er

    ce nt

    o f C

    on tr

    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

    63 42

    5 7 63 42

    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

    N or

    m al

    iz ed

    3 H C

    PM

    UVC (1 J/m2) IR (5 Gy)

  • UVC IR Sham 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 10 Fluence (J/m2)

    0% 100% 200% 300% 400% 500%

    Sham 1 10 Fluence (J/m2)

    R el

    at iv

    e %

    R ed

    - O

    nl y

    tr ac

    ks R

    el at

    iv e

    % G

    re en

    - O

    nl y

    tr ac

    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 Caffeine Sham

    Caffeine, 1J/m2

    Treatment

    R el

    at iv

    e %

    o f

    N ew

    O rig

    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%

    N on

    ta rg

    et in

    g R

    N Ai

    , S ha

    m

    N on

    ta rg

    et in

    g R

    N Ai

    , U V

    AT R

    R N

    Ai ,

    Sh am

    AT R

    R N

    Ai ,

    U V

    N TC

    R N

    Ai Sh

    am

    N TC

    R N

    Ai U

    V

    C hk

    1 R

    N Ai

    Sh am

    C hk

    1 R

    N Ai

    U V

    Treatment

    R el

    at iv

    e %

    o f

    N ew

    O rig

    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