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

2. Background  Endogenous and exogenous sources of mutation challenge fidelity of DNA  DNA damage response (DDR) systems minimize accumulation of mutations  Mismatch repair

3. 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

4. MMR Pathway Mismatch recognition Strand choice Excision Resynthesis G T T T A G T MutS family MutL family Exonucleases Replicative DNA polymerase * PCNA RPA

5. MMR Heterodimers MSH 6 MSH 3 MSH 2 MSH 7 MSH 2 MutSβ MutS α MutS γ

6. 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

7. Hypothesis  AtMSH6 and AtMSH7 are non-redundant and are responsible for the correction of different mismatches

8. 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

9. 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

10. Experimental Set-up Genotype putative MSH6 -/- and MSH7 -/- plants

11. Experimental Set-up Genotype putative MSH6 -/- and MSH7 -/- plants Cross MSH6 -/- and MSH7 -/-, Propagate MA lines for 5 generations

12. Experimental Set-up Genotype putative MSH6 -/- and MSH7 -/- plants Cross MSH6 -/- and MSH7 -/-, Propagate MA lines for 5 generations Create genomic library, Whole genome sequencing

13. Experimental Set-up Genotype putative MSH6 -/- and MSH7 -/- plants Analyze short sequence reads of entire genome Cross MSH6 -/- and MSH7 -/-, Propagate MA lines for 5 generations Create genomic library, Whole genome sequencing

14. 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

15. 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

16. Results - pending

17. 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

18. Acknowledgements  The Howard Hughes Medical Institute  Cripps Research Scholarship  Dr. John Hays  Buck Wilcox  Dr. Marc Curtis  Peter Hoffman  Dr. Kevin Ahern