Transcript Aly Mohamed - Oregon State University

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?
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Consists of protein machines that are highly conserved in
eukaryotes and prokaryotes
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Corrects errors in the genome, that result from DNA replication
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Reduces spontaneous mutation rates by 100 to 1000 times
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Promotes gene conversion during homologous recombination
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Prevents chromosomal "scrambling" between diverged
members of gene families
Crucial Mechanisms Of DNA MMR
The E. coli paradigm
Recognition of mismatched base pairs
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MutS  DNA base-mismatches
Determination of the incorrect base.
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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.
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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
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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
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Deliberately mutated MSH2 gene, to create defects
in ATPase domain or Helix turn Helix domain of
protein
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Wild type and mutated MSH2 proteins form
separate heterodimer complexes with MSH6
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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
Microsatellite instability assay
Parent
Progeny
Electrophoretic analyses
of individual progeny
WT
MSH2::TDNA
seeds
shifted
allele
fluorescent
tag
PCR
TATATATATATATATATATATA
ATATATATATATATATATATAT
Intermediate Vector
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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 gag t ... … taa
CaMV 35S
-Glucuronidase
M G G S
atg ggg ggg agt ...
CaMV 35S
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+1 Out-of-Frame GUS
Single base deletion restores
correct reading frame
-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
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