1. Repression of Mismatch Repair (MMR) by Dominant-negative MMR Proteins Aly Mohamed Under Supervision of Dr. John Hays and Mrs. Stephanie Bollmann

2. DNA Mismatch Repair What is DNA Mismatch Repair? Consists of protein machines that are highly conserved in eukaryotes and prokaryotes Corrects errors in the genome, that result from DNA replication Reduces spontaneous mutation rates by 100 to 1000 times Promotes gene conversion during homologous recombination Prevents chromosomal "scrambling" between diverged members of gene families

3. Crucial Mechanisms Of DNA MMR The E. coli paradigm Recognition of mismatched base pairs MutS  DNA base-mismatches Determination of the incorrect base. Resolving the unmethylated strand by detection of the GATC sequence MutL + MutS  MutH protein MutH specifically nicks the unmethylated strand iii) Excision of the incorrect base and repair synthesis. 3' to 5' or 5' to 3' exonucleases DNA Synthesis via Polymerase 1 DNA Ligase

5. 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

6. Eukaryotic MMR System MutS genes in prokaryotes, synonymous MutS homolog (MSH) proteins in eukaryotes MSH1~Mitochondrial stability MSH2, MSH3, MSH6, MSH7~Mediate error correction MSH4, MSH5~Play essential roles in meiosis MutL similarly diverged in eukaryotic systems as MLH proteins

7. Experimental approach to Nonfunctional MMR Proteins The Dominate Negative Phenotype Deliberately mutated MSH2 gene, to create defects in ATPase domain or Helix turn Helix domain of protein Wild type and mutated MSH2 proteins form separate heterodimer complexes with MSH6 Overproduced negative MSH2 protein consumes most MSH6, and masks functional positive protein

8. Methodology Insert mutated MSH2 gene into intermediate vector for sequencing Transfer mutated MSH2 gene into super expression vector Include an epitope tag on MSH2 to verify production of the protein by antibody staining Employ a microsatellite instability assay to determine MMR deficiency Use GUS mutagenesis reporter to determine mutation rate in plant

9. WTMSH2::TDNA Electrophoretic analyses of individual progeny seeds TATATATATATATATATATATA ATATATATATATATATATATAT PCR fluorescent tag shifted allele ParentProgeny Microsatellite instability assay

10. Intermediate Vector Easy to work with because of small size High copy number vector Ease in ability to sequence gene prior to its insertion into the binary vector

11. +1 Out-of-Frame GUS atg ggg ggg gag t... … taa M G G E … … STOP CaMV 35S  -Glucuronidase In-Frame GUS atg ggg ggg agt... M G G S  Glucuronidase Single base deletion restores correct reading frame CaMV 35S ß-Glucuronidase (GUS) Mutagenesis Reporter GUS cleaves X-Gluc which turns blue after it is cut Mutations in catalitically necessary domains render GUS unable to cleave X-Gluc Blue spots represent a mutation likely due to a decrease in mismatch repair Histochemical staining shows spots of reverted wild type GUS activity arising from frame shift pathway, transition (A to G), or transversion (A to C, or T) mutations in catalytically necessary domains

12. Many thanks to…. Dr. Kevin Ahern and the HHMI Program The URISC program Dr. John B. Hays Mrs. Stephanie Bollmann Mr. Peter Hoffman The entire Hays laboratory