Transcript DNA Damage Response - Oregon State University

the
DNA Damage Response
an investigation of Polη and
ATM
Johanna Steinbrecher
Dr.John Hays
Oregon State University
The effect of UV light on DNA
Cyclobutane Pyrimidine Dimer
DNA Damage Response
-Activated by DNA damage
-Signals for transcription factors
-Stalls the cell during replication
-stimulates repair process or apoptosis
-Cell avoids necrosis
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
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
Four components of the DDR are
being studied:
Translesion polymerases
•Polη- bypasses CPD
• Polζ- can bypass CPDs
-main role is to extend
Kinases
•ATR- activated by single
stranded DNA
•ATM- activated by double
strand breaks
Five double mutants of
various combinations of
these
four components have been
constructed.
#6: polh¯ atm¯
How do ATM and Polη 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
Polη
Polη
-Translesion Polymerase
- Specifically designed to
bypass CPDs
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
-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
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
WT
T-DNA
1 2 3 4
5 6 7 8
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
Activated by DNA damage
-avoid necrosis
-signals for transcription factors
-stalls the cell during replication
and stimulates repair process or for
apoptosis
ATM and POL
double
mutant
0.03 kJ m-2 UV-B
No UV-B
single
mutants
atr−
Wt
polh
atr− polh
0.3 kJ m-2 UV-B
Mean dead stem cells per root
Mean dead stem cells per root
ATR and POLh
No UV-B
single
mutants
Mean dead stem cells per root
double
mutant
0.3 kJ m-2 UV-B
Wt
single mutants
atr−
atm− pol
ATM and POLh
ATR and POL
No UV-B
pol
atm−
Wt
double
mutant
pol
?
atr− pol
Wt
atm−
polh
atm− polh
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 Polη/ATM mutant does not exceed stem
cell death of ATM single mutant- indicates importance of ATM
in PCD signaling
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 Hart
Kevin Ahern
Howard Hughes Medical Institute