1. Repression of Mismatch Repair (MMR) in Arabidopsis by Dominant-negative MMR Proteins Aly Mohamed Aly Mohamed Working Under Professor John B. Hays John B. Hays

2. DNA Mismatch Repair Consists of protein machines that are highly conserved in eukaryotes and prokaryotes Consists of protein machines that are highly conserved in eukaryotes and prokaryotes Corrects errors in the genome that result from DNA replication Corrects errors in the genome that result from DNA replication Elicit cell response to cytotoxic DNA lesions (e.g. O6 methyl guanine) Elicit cell response to cytotoxic DNA lesions (e.g. O6 methyl guanine) Reduces spontaneous mutation rates by 100 to 1000 times Reduces spontaneous mutation rates by 100 to 1000 times

3. MutS Protein Comparison

4. Mechanisms of DNA MMR The E. coli paradigm Recognition of mismatched base pairs Recognition of mismatched base pairs MutS  DNA base-mismatches MutS  DNA base-mismatches Determination of the incorrect base. Determination of the incorrect base. Resolving the unmethylated strand by detection of the GATC sequence Resolving the unmethylated strand by detection of the GATC sequence MutL + MutS  MutH protein MutL + MutS  MutH protein MutH specifically nicks the unmethylated strand MutH specifically nicks the unmethylated strand iii) Excision of the incorrect base and repair synthesis. iii) Excision of the incorrect base and repair synthesis. 3' to 5' or 5' to 3' exonucleases 3' to 5' or 5' to 3' exonucleases DNA Synthesis via Polymerase I DNA Synthesis via Polymerase I DNA Ligase DNA Ligase

6. Eukaryotic MMR System MutS genes in prokaryotes, synonymous MutS homolog (MSH) proteins in eukaryotes MSH1~Mitochondrial stability MSH1~Mitochondrial stability MSH2, MSH3, MSH6, MSH7~Mediate error correction MSH2, MSH3, MSH6, MSH7~Mediate error correction Form hetrodimers; MSH2●MSH3, MSH2●MSH6, and MSH2●MSH7(found only in plants) MSH4, MSH5~Play essential roles in meiosis MSH4, MSH5~Play essential roles in meiosis MutL similarly diverged in eukaryotic systems as MLH proteins. MLH1●PMS2 couples mismatch recognition to excision of DNA. MLH1●PMS2 couples mismatch recognition to excision of DNA. No MutH homolog No MutH homolog

7. Why don’t plants show mutational loading? Plants lack reserved germ lines; gametes arise from meristem cells Plants lack reserved germ lines; gametes arise from meristem cells Replication errors, environmental mutagens Replication errors, environmental mutagens Haplosufficiency quality checking  plants take advantage of haploidy in gametophytes. Haplosufficiency quality checking  plants take advantage of haploidy in gametophytes.

8. Hypothesis Genome maintenance is essential for plant genome integrity Genome maintenance is essential for plant genome integrity Primary defense in prevention of mutagenesis during diploid growth by rigorous DNA maintenance/repair Primary defense in prevention of mutagenesis during diploid growth by rigorous DNA maintenance/repair Haplosufficiency quality checking is an important backup Haplosufficiency quality checking is an important backup

9. Approach to Nonfunctional MMR Proteins The Dominant Negative Phenotype Create two mutated MSH2 gene constructs Create two mutated MSH2 gene constructs Construct one  mutation in ATPase domain Construct one  mutation in ATPase domain Construct two  Mutation in Helix turn Helix domain Attach CMYC tag at 3’ end of MSH2 constructs and transfer construct into super expression vector (p1803) Attach CMYC tag at 3’ end of MSH2 constructs and transfer construct into super expression vector (p1803) Transform constructs into Arabidopsis via Agrobacterium Transform constructs into Arabidopsis via Agrobacterium Overproduced negative MSH2 protein consumes most MSH6, MSH3, and MSH7  masks functional positive protein Overproduced negative MSH2 protein consumes most MSH6, MSH3, and MSH7  masks functional positive protein Gene silencing Gene silencing

10. Agrobacterium Tumefaciens

11. Screening for putative transformants Kanamycin resistance conferred in insertion construct Kanamycin resistance conferred in insertion construct Perform PCR specific to insertion construct Perform PCR specific to insertion construct Check for protein expression in plants by immunoblotting Check for protein expression in plants by immunoblotting

12. Microsatellite Sequences Repeat sequence loci in genome Repeat sequence loci in genome Susceptible to insertion/deletion mutations by replication machinery Susceptible to insertion/deletion mutations by replication machinery Repair of sequences facilitated by MMR Repair of sequences facilitated by MMR Allele shift “fingerprints” in microsatellite sequence loci indicative of defunct MMR system Allele shift “fingerprints” in microsatellite sequence loci indicative of defunct MMR system

13. AT NNNATATAT ATATAT NNNTATATA TATATATATATANNN NNNATATATATATAT NNNTATATATATATATATATANNN NNNATATAT ATATAT NNNTATATA TATATATATATANNN TA MMR: MSH2, MSH3, MSH6, MLH1, PMS2 MMR +2 insertion no insertion or deletion -2 deletion replication MMR Correction of Slip-Mispairing

14. WTMSH2::TDNA Electrophoretic analyses of individual progeny seeds TATATATATATATATATATATA ATATATATATATATATATATAT PCR fluorescent tag shifted allele ParentProgeny Microsatellite Instability Assay

15. Many Thanks to…. Dr. Kevin Ahern and the HHMI Program Dr. Kevin Ahern and the HHMI Program Dr. John B. Hays Dr. John B. Hays Dr. Walt Ream Dr. Walt Ream Wanda Crannell Wanda Crannell The Hays laboratory The Hays laboratory The EMT department The EMT department