the DNA Damage Response

1. theDNA Damage Response an investigation of Polη and ATM Johanna Steinbrecher Dr.John Hays Oregon State University

2. The effect of UV light on DNA Cyclobutane Pyrimidine Dimer

3. -Activated by DNA damage DNA Damage Response -Signals for transcription factors -Stalls the cell during replication -stimulates repair process or apoptosis -Cell avoids necrosis

5. 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

6. 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

7. Bigger Picture Four components of the DDR are being  studied: • 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 #6: polh¯ atm¯

8. 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

9. Arabidopsis thaliana -Plant model used to better understand the process in both plant and animal cells Methods 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

10. 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

11. 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

12. 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

13. Activated by DNA damage -avoid necrosis -signals for transcription factors -stalls the cell during replication and stimulates repair process or for apoptosis

14. ATR and POLh ATM and POLz double mutant 0.03 kJ m-2 UV-B 0.3 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− ATM and POLh ATR and POLz 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

15. 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 upindicates 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/ATMmutant does not exceed stem cell death of ATM single mutant- indicates importance of ATM in PCD signaling

16. 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

17. 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