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
 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
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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

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

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
 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


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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

Parent
Microsatellite instability assay
Progeny
Electrophoretic analyses of individual progeny
WT MSH2::TDNA seeds shifted allele
PCR fluorescent tag
TATATATATATATATATATATA
ATATATATATATATATATATAT

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

ß-Glucuronidase (GUS) Mutagenesis
Reporter
M G G E … …
STOP atg ggg ggg g ag t ... … taa
CaMV 35S

-Glucuronidase
+1 Out-of-Frame GUS
M G G S atg ggg ggg agt ...
Single base deletion restores correct reading frame



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CaMV 35S
-
Glucuronidase
In-Frame GUS
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

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