Repression of Mismatch Repair (MMR) by Dominant-negative MMR Proteins

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 RepairWhat 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 MMRThe 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. replication +2 insertion AT NNNATATAT ATATAT NNNTATATA TATATATATATANNN MMR: MSH2, MSH3, MSH6, MLH1, PMS2 no insertion or deletion NNNATATATATATAT NNNTATATATATATATATATANNN MMR NNNATATAT ATATAT NNNTATATA TATATATATATANNN TA -2 deletion 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 toNonfunctional MMR ProteinsThe 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. Electrophoretic analyses of individual progeny Parent Progeny WT MSH2::TDNA seeds shifted allele PCR fluorescent tag TATATATATATATATATATATA ATATATATATATATATATATAT 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 CaMV 35S -Glucuronidase M G G E … … STOP atg ggg ggg gag t ... … taa Single base deletion restores correct reading frame In-Frame GUS -Glucuronidase CaMV 35S M G G S atg ggg ggg agt ... 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