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

DNA Mismatch Repair Evolution Lynch Syndrome and human cancers Plant Breeding

Arabidopsis thaliana: A Model System 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

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

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)

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)

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

Microsatellite Mutation 10 repeats

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

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

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

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

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

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

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

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

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

Results – pending Capillary electrophoresis traces of PCR products

Results – pending

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