repression of mismatch repair mmr by dominant negative 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. DNA Mismatch Repair What is DNA Mismatch Repair?. Consists of protein machines that are highly conserved in eukaryotes and prokaryotes

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repression of mismatch repair mmr by dominant negative mmr proteins

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

Aly Mohamed

Under Supervision of

Dr. John Hays and

Mrs. Stephanie Bollmann

dna mismatch repair what is dna mismatch repair
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
crucial mechanisms of dna mmr the e coli paradigm
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
slide5

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

eukaryotic mmr system
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

experimental approach to nonfunctional mmr proteins the dominate negative phenotype
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
methodology
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
slide9

Electrophoretic analyses

of individual progeny

Parent

Progeny

WT

MSH2::TDNA

seeds

shifted

allele

PCR

fluorescent

tag

TATATATATATATATATATATA

ATATATATATATATATATATAT

Microsatellite instability assay

intermediate vector
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
glucuronidase gus mutagenesis reporter

+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
many thanks to

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

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