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