Transcript Repression of Mismatch Repair Arabidopsis Dominant-negative MMR Proteins

Repression of Mismatch Repair
(MMR) in Arabidopsis by
Dominant-negative MMR
Proteins
Aly Mohamed
Working Under Professor
John B. Hays
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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Elicit cell response to cytotoxic DNA lesions (e.g. O6 methyl
guanine)
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Reduces spontaneous mutation rates by 100 to 1000 times
MutS Protein Comparison
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 I
DNA Ligase
Eukaryotic MMR System
MutS genes in prokaryotes, synonymous MutS homolog (MSH)
proteins in eukaryotes
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MSH1~Mitochondrial stability
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MSH2, MSH3, MSH6, MSH7~Mediate error correction
Form hetrodimers; MSH2●MSH3, MSH2●MSH6, and MSH2●MSH7(found
only in plants)
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MSH4, MSH5~Play essential roles in meiosis
MutL similarly diverged in eukaryotic systems as MLH proteins.
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MLH1●PMS2 couples mismatch recognition to excision of DNA.
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No MutH homolog
Why don’t plants show
mutational loading?
Plants lack reserved germ lines; gametes
arise from meristem cells
 Replication errors, environmental
mutagens
 Haplosufficiency quality checking  plants
take advantage of haploidy in
gametophytes.
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Hypothesis
Genome maintenance is essential for plant
genome integrity
 Primary defense in prevention of
mutagenesis during diploid growth by
rigorous DNA maintenance/repair
 Haplosufficiency quality checking is an
important backup
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Approach to
Nonfunctional MMR Proteins
The Dominant Negative Phenotype
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Create two mutated MSH2 gene constructs
Construct one  mutation in ATPase domain
Construct two Mutation in Helix turn Helix domain
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Attach CMYC tag at 3’ end of MSH2 constructs and transfer
construct into super expression vector (p1803)
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Transform constructs into Arabidopsis via Agrobacterium
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Overproduced negative MSH2 protein consumes most MSH6,
MSH3, and MSH7  masks functional positive protein
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Gene silencing
Agrobacterium
Tumefaciens
Screening for putative
transformants
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Kanamycin
resistance conferred
in insertion construct
Perform PCR specific
to insertion construct
Check for protein
expression in plants
by immunoblotting
Microsatellite Sequences
Repeat sequence loci in genome
 Susceptible to insertion/deletion mutations
by replication machinery
 Repair of sequences facilitated by MMR
 Allele shift “fingerprints” in microsatellite
sequence loci indicative of defunct MMR
system
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MMR Correction of Slip-Mispairing
replication
AT
NNNATATAT ATATAT
NNNTATATA TATATATATATANNN
MMR: MSH2, MSH3,
MSH6,
MLH1, PMS2
NNNATATATATATAT
NNNTATATATATATATATATANNN
+2 insertion
no insertion
or deletion
MMR
NNNATATAT ATATAT
NNNTATATA TATATATATATANNN
TA
-2 deletion
Microsatellite Instability Assay
Parent
Progeny
Electrophoretic analyses
of individual progeny
WT
MSH2::TDNA
seeds
shifted
allele
fluorescent
tag
PCR
TATATATATATATATATATATA
ATATATATATATATATATATAT
Many Thanks to….
Dr.
Kevin Ahern and the HHMI Program
Dr. John B. Hays
Dr. Walt Ream
Wanda Crannell
The Hays laboratory
The EMT department