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

ad s

tem

cel

ls p

er r

oot

No UV-B

0.3 kJ m-2 UV-B

ATR and POL

Mea

n de

ad s

tem

cel

ls p

er r

oot

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