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

PI: Dr. John HaysAna 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

10 repeats

10

10

10

10

10

1010

99

9

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 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