High Throughput Genome
Sequencing: A Test of
Functional Overlap in
Mismatch Repair Proteins
Ana Brar
PI: Dr. John Hays
Background
 Endogenous and exogenous sources of mutation
challenge fidelity of DNA
 DNA damage response (DDR) systems minimize
accumulation of mutations
 Mismatch repair
Mismatch Repair (MMR)
 Highly conserved
 Post-DNA replication
 Triggered by the mismatch of noncomplementary base
pairs and short insertion/deletion loop-outs
 Recognize pre-mutagenic DNA lesions, recruit necessary
proteins, remove the nascent DNA strand, and
subsequently resynthesize through the resulting gap
MMR Pathway
Mismatch
recognition
G
T
Strand choice
G
T
Excision
Resynthesis
• MutS family
*
• MutL family
T
• PCNA
• RPA
• Exonucleases
A
T
• Replicative DNA
polymerase
MMR Heterodimers
MSH MSH
2
6
MutS α
MSH MSH
2
3
MutSβ
MSH MSH
2
7
MutSγ
Arabidopsis thaliana: A Model
System
 Small genome (250 Mb)
 Genome sequenced
 Short life cycle (6 weeks)  Extensive collection of
mutants available
 Thousands of progeny
 Plant mismatch repair
pathway is similar to
animal mismatch repair
Hypothesis
 AtMSH6 and AtMSH7 are non-redundant and are
responsible for the correction of different mismatches
Prediction 1
 If MSH2/MSH6 and MSH2/MSH7 are responsible for
correction of different mismatches, then plants deficient
in these protein activities that are propagated
generation to generation will accumulate different
spectra of mutations as analyzed by genome
resequencing
Prediction 2
 If MSH6 and MSH7 activities are redundant then a
MSH6/MSH7 double mutant will display a more
pronounced phenotype than either single knock-out
when propagated generation to generation
Experimental Set-up
Experimental Set-up
Experimental Set-up
Experimental Set-up
Methods
 Mutation Accumulation (MA) line propagation for five
generations
 Illumina high throughput sequencing
 MSH6-/ MSH7-/ MSH6-/-/MSH7-/-
 Programs BWA and CASHX to parse, map, align, and
identify mutations
Methods
 To assess MSH6/MSH7 redundancy, existing MSH6-/- and
MSH7-/- lines have been crossed and 18 independent lines
propagated generation to generation while scoring
phenotypes:
 Germination frequencies
 Vegetative growth
 Seed set
 Mutation rate and spectrum
Results - pending
Significance
 At the cutting edge of bioinformatics technology
 An increasingly affordable and prompt technique
 In vivo information about what lesions various mismatch
repair proteins are responsible for correcting
 This data will complement and verify in vitro biochemical
DNA lesion binding studies
 Defective mismatch repair machinery has been
associated with an increased risk of certain cancers (i.e.
Human non-polyposis colorectal cancer)
 Strengthen the utility of Arabidopsis thaliana as a model
for the study of mismatch repair and DDR
Acknowledgements
 The Howard Hughes Medical Institute
 Cripps Research Scholarship
 Dr. John Hays
 Buck Wilcox
 Dr. Marc Curtis
 Peter Hoffman
 Dr. Kevin Ahern