the DNA Damage Response

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the DNA Damage Response
an investigation of Pol? and ATM

• Johanna Steinbrecher
• Dr.John Hays
• Oregon State University

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The effect of UV light on DNA
Cyclobutane Pyrimidine Dimer
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-Activated by DNA damage

• DNA Damage Response

-Signals for transcription factors -Stalls the
cell during replication -stimulates repair
process or apoptosis -Cell avoids necrosis
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(No Transcript)
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Terminology

• Translesion polymerase helps to bypass damaged
  DNA so that replication can continue

Kinase phosphorylates proteins to alter their
structure- can activate or deactivate proteins
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How to study the DDR
-Create a model cell deficient in the proteins of
interest -Observe how the cell responds when DNA
damage is induced
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Bigger Picture
Four components of the DDR are being  studied

•  
• Translesion polymerases
• Pol?- bypasses CPD
• Pol?- can bypass CPDs
• -main role is to extend

Five double mutants of various combinations of
these four components have been constructed.

• Kinases
• ATR- activated by single stranded DNA
• ATM- activated by double strand breaks

6 polh atm
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How do ATM and Pol? function in DDR?
ATM (Ataxia telangiectasia mutated) -Double
role- signals for stalled fork recovery and also
for apoptosis (Similar to ATR) -Activated by
double strand breaks
Pol?
Pol? -Translesion Polymerase - Specifically
designed to bypass CPDs
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Methods
Arabidopsis thaliana -Plant model used to better
understand the process in both plant and animal
cells
T-DNA- Agrobacterium T-DNA insertion renders
genes inactive -single mutants deficient in Pol?
and ATM were obtained -F2 generation produced-
1/16 chance of a double mutant
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Identification
-PCR- polymerase chain reaction
ATM gene
Yes product mutant allele

T-DNA
ATM gene
Yes product wild-type allele
Polh gene
Yes product mutant allele
T-DNA
Polh gene
Yes product wild-type allele
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PCR
-DNA was amplified and run on gels -used to
genotype plants and identify and confirm
mutant -polh X atm identified by
genotyping the F2 generation
1 2 3 4 5 6 7 8
WT
1 2 3 4 5 6 7 8
T-DNA
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Quantifying Stem Cell Death
-Root tips irradiated with various gradients of
UV light - Dead cells stained with Propidium
Iodide and analyzed with microscopy -Stem cell
specific
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Activated by DNA damage
-avoid necrosis -signals for transcription
factors -stalls the cell during replication and
stimulates repair process or for apoptosis
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ATR and POLh
ATM and POLz
double mutant
0.3 kJ m-2 UV-B
0.03 kJ m-2 UV-B
No UV-B
No UV-B
single mutants
single mutants
double mutant
Mean dead stem cells per root
Mean dead stem cells per root
Wt atr- polh- atr-
polh-
Wt atm- polz- atm-
polz-
ATR and POLz
ATM and POLh
double mutant
0.3 kJ m-2 UV-B
?
No UV-B
single mutants
Mean dead stem cells per root
Wt atr- polz- atr-
polz-
Wt atm- polh atm- polh
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Hypothesis -Absence of pol Eta will create more
double strand breaks -The path to PCD will be
limited by the absence of ATM
Predictions 1) Increased stem cell death goes up
indicates ATM and Pol Eta work together on the
same pathway in the DDR- indicates a cooperation
between the two in the cell pathway 2) Stem cell
death in Pol?/ATM mutant does not exceed stem
cell death of ATM single mutant- indicates
importance of ATM in PCD signaling
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Present Findings Future work
Mutant identified! -Out of 29 plants screened,
one was found to be a double mutant -Plant
produced a total of seven seeds Next
steps -Continue screening -Create an F3
generation of double mutants -Collect seed -gt
root assay
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Acknowledgements

• Dr. John Hays
• Entirety of Hays' Lab
•         with special thanks to
•     -Dr. Marc Curtis
•     -Colin Tominey
•     -Dr. Peter Hoffman
•     -Buck Wilcox
• Chris Steinbrecher Nancy Hart
• Kevin Ahern
• Howard Hughes Medical Institute