High Throughput Genome Sequencing: A Test of Functional Overlap in Mismatch Repair Proteins Ana Brar PI: Dr. John Hays

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



of entire genome


Propagate MA lines

for 5 generations


sequencing

Experimental Set-up

Genotype putative



of entire genome


Propagate MA lines

for 5 generations


sequencing

Experimental Set-up

Genotype putative



of entire genome


Propagate MA lines

for 5 generations


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

Documents

High Throughput Genome Sequencing: A Test of Functional Overlap in Mismatch Repair Proteins Ana Brar PI: Dr. John Hays