the DNA Damage Response
description
Transcript of the DNA Damage Response
the DNA Damage Response
Johanna SteinbrecherDr.John Hays
Oregon State University
an investigation of Polη and ATM
The effect of UV light on DNA
Cyclobutane Pyrimidine Dimer
-Activated by DNA damage
DNA Damage Response
-Signals for transcription factors
-Stalls the cell during replication -stimulates repair process or apoptosis
-Cell avoids necrosis
Translesion polymerase: helps to bypass damaged DNA so that replication can continue
Kinase: phosphorylates proteins to alter their structure- can activate or deactivate proteins
Terminology
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
Bigger Picture
Translesion polymerases •Polη- bypasses CPD• Polζ- 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
Four components of the DDR are being studied:
#6: pol¯ atm¯
How do ATM and Polη function in DDR?
Polη
Polη-Translesion Polymerase- Specifically designed to bypass CPDs
ATM (Ataxia telangiectasia mutated) -Double role- signals for stalled fork recovery and also for apoptosis (Similar to ATR)-Activated by double strand breaks
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 Polη and ATM were obtained-F2 generation produced- 1/16 chance of a double mutant
Identification
T-DNA
Pol gene
Yes product = mutant allele
ATM gene
T-DNA
ATM gene Yes product = mutant allele
Pol gene
Yes product = wild-type allele
Yes product = wild-type allele
-PCR- polymerase chain reaction
PCR-DNA was amplified and run on gels -used to genotype plants and identify and confirm mutant
-polh¯ X atm¯ identified by genotyping theF2 generation
1 2 3 4 5 6 7 8WT
T-DNA
1 2 3 4 5 6 7 8
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
-avoid necrosis
-signals for transcription factors
-stalls the cell during replication and stimulates repair process or for apoptosis
Activated by DNA damage
Wt atr− pol− atr− pol−
doublemutant
single mutants
Mea
n de
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tem
cel
ls p
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No UV-B
0.3 kJ m-2 UV-B
ATR and POL
Mea
n de
ad s
tem
cel
ls p
er r
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singlemutants
No UV-B
0.3 kJ m-2 UV-B
ATM and POL
doublemutant
Wt atm− pol− atm− pol−
Mea
n de
ad s
tem
cel
ls p
er r
oot
singlemutants
No UV-B
0.03 kJ m-2 UV-B
ATR and POL doublemutant
Wt atr− pol− atr− pol−
ATM and POL
?Wt atm− pol atm− pol
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 Polη/ATM mutant does not exceed stem cell death of ATM single mutant- indicates importance of ATM in PCD signaling
Hypothesis
-Absence of pol Eta will create more double strand breaks-The path to PCD will be limited by the absence of ATM
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 -> root assay
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 HartKevin Ahern
Howard Hughes Medical Institute