high throughput genome sequencing: a test of functional overlap in mismatch repair proteins ana brar...
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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
Strand choice
Excision
Resynthesis
GT
T
TA
GT
• MutS family
• MutL family
• Exonucleases
• Replicative DNApolymerase
*• PCNA• RPA
MMR Heterodimers
MSH6
MSH3
MSH2
MSH7
MSH2
MSH2
MutSβ
MutS α
MutSγ
Arabidopsis thaliana: A Model System
Small genome (250 Mb)
Short life cycle (6 weeks)
Thousands of progeny
Genome sequenced
Extensive collection of mutants available
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
Genotype putative
MSH6-/- and MSH7-/- plants
Creategenomic library,Whole genome
sequencing
Analyze short sequence reads
of entire genome
Cross MSH6-/- andMSH7-/-,
Propagate MA lines
for 5 generations
Experimental Set-up
Genotype putative
MSH6-/- and MSH7-/- plants
Analyze short sequence reads
of entire genome
Cross MSH6-/- andMSH7-/-,
Propagate MA lines
for 5 generations
Creategenomic library,Whole genome
sequencing
Experimental Set-up
Genotype putative
MSH6-/- and MSH7-/- plants
Analyze short sequence reads
of entire genome
Cross MSH6-/- andMSH7-/-,
Propagate MA lines
for 5 generations
Creategenomic library,Whole genome
sequencing
Experimental Set-up
Genotype putative
MSH6-/- and MSH7-/- plants
Analyze short sequence reads
of entire genome
Cross MSH6-/- andMSH7-/-,
Propagate MA lines
for 5 generations
Creategenomic library,Whole genome
sequencing
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