Mutation Frequency Analysis in Arabidopsis thaliana: A Study of Mismatch Repair Inhibition

1. Mutation Frequency Analysis in Arabidopsis thaliana: A Study of Mismatch Repair Inhibition PI: Dr. John Hays Ana Brar

2. DNA Mismatch Repair • Evolution • Lynch Syndrome and human cancers • Plant Breeding

3. Small genome Short life cycle Thousands of progeny Genome sequenced Extensive collection of mutants available Plant mismatch repair pathway is similar to animal mismatch repair Arabidopsis thaliana: A Model System

4. Background • DNA integrity is challenged by endogenous and exogenous chemical mutagens, radiation, and replication errors • Avoidance and repair of DNA damage requires: • Accurate DNA replication • DNA repair pathways

5. Highly conserved Post-DNA replication Triggered by the mismatch of noncomplementary base pairs and short insertion/deletion loop-outs Mismatch repair proteins recognize DNA mismatches, remove the nascent DNA strand, and resynthesize through the resulting gap MutSα (MSH2-MSH6 heterodimer) MutSβ (MSH2-MSH3 heterodimer) MutSγ (MSH2-MSH7 heterodimer) MutLα (MLH1-PMS2 heterodimer) Mismatch Repair (MMR)

6. Mismatch Repair (MMR) Highly conserved Post-DNA replication Triggered by the mismatch of noncomplementary base pairs and short insertion/deletion loop-outs Mismatch repair proteins recognize DNA mismatches, remove the nascent DNA strand, and resynthesize through the resulting gap MutSα (MSH2-MSH6 heterodimer) MutSβ (MSH2-MSH3 heterodimer) MutSγ (MSH2-MSH7 heterodimer) MutLα (MLH1-PMS2 heterodimer)

8. Disruption of MMR genes • Disrupting MSH2 with T-DNA knocks out MMR • Dominant negative proteins • Plants deficient in MMR accumulate mutations more rapidly than do wild type (WT) • Insertion/deletion (indel) mutations in microsatellite repeats (SSRs) are a hallmark of MMR deficiency

9. 10 10 10 9 9 10 repeats 9 10 10 repeats 10 10 10 10 10 Microsatellite Mutation

10. Hypothesis Novel traits may be obtained in plants for breeding purposes by transiently debilitating MMR

11. Prediction • Arabidopsis plants expressing dominant negative proteins that interrupt MMR will display increased levels of microsatellite mutation levels relative to WT controls

12. Experimental Methods Plant seeds and collect seedlings DNA extraction DNA Quantification Analytical PCR at several microsatellite loci with fluorescently labeled primers

13. Experimental Methods Plant seeds and collect seedlings DNA extraction DNA Quantification Analytical PCR at several microsatellite loci with fluorescently labeled primers

14. Experimental Methods Plant seeds and collect seedlings DNA extraction DNA Quantification Analytical PCR at several microsatellite loci with fluorescently labeled primers

15. Experimental Methods Plant seeds and collect seedlings DNA extraction DNA Quantification Analytical PCR at several microsatellite loci with fluorescently labeled primers

16. Gel electrophoresis Capillary electrophoresis Calculation of mutation frequencies Experimental Methods: continued

17. Experimental Methods: continued Gel electrophoresis Capillary electrophoresis Calculation of mutation frequencies

18. Experimental Methods: continued Gel electrophoresis Capillary electrophoresis Calculation of mutation frequencies

19. Results – pending Capillary electrophoresis traces of PCR products

20. Results – pending

21. Acknowledgements • The Howard Hughes Medical Institute • URISC • Cripps Scholarship Fund • Dr. John Hays • Buck Wilcox • Colin Tominey • Peter Hoffman • Dr. Kevin Ahern