Repression of Mismatch Repair MMR by Dominantnegative MMR Proteins

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

• Aly Mohamed
• Under Supervision of
• Dr. John Hays and
• Mrs. Stephanie Bollmann

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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

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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

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(No Transcript)
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MMR Correction of Slip-Mispairing
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Eukaryotic MMR System

• MutS genes in prokaryotes, synonymous MutS
  homolog (MSH) proteins in eukaryotes
• MSH1Mitochondrial stability
• MSH2, MSH3, MSH6, MSH7Mediate error correction
• MSH4, MSH5Play essential roles in meiosis
• MutL similarly diverged in eukaryotic systems as
  MLH proteins

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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

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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

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Microsatellite instability assay
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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

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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

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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