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 loopouts
• 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 loopouts
• 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
10
10
9
9
10 repeats
9
10
10 repeats
10
10
10
10
10
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 extraction
DNA Quantification
Analytical PCR at several microsatellite loci
with fluorescently labeled primers
Experimental Methods
Plant seeds and collect seedlings
DNA extraction
DNA Quantification
Analytical PCR at several microsatellite loci
with fluorescently labeled primers
Experimental Methods
Plant seeds and collect seedlings
DNA extraction
DNA Quantification
Analytical PCR at several microsatellite loci
with fluorescently labeled primers
Experimental Methods
Plant seeds and collect seedlings
DNA extraction
DNA Quantification
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
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Dr. John Hays
Buck Wilcox
Colin Tominey
Peter Hoffman
Dr. Kevin Ahern