Repression of Mismatch Repair (MMR) in Arabidopsis by Dominant-negative MMR Proteins Aly Mohamed Aly...
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Transcript of Repression of Mismatch Repair (MMR) in Arabidopsis by Dominant-negative MMR Proteins Aly Mohamed Aly...
Repression of Mismatch Repair Repression of Mismatch Repair (MMR) in (MMR) in ArabidopsisArabidopsis by by Dominant-negative MMR Dominant-negative MMR
ProteinsProteins
Aly MohamedAly Mohamed Working Under ProfessorWorking Under Professor
John B. HaysJohn B. Hays
DNA Mismatch RepairDNA Mismatch Repair
Consists of protein machines that are highly conserved in Consists of protein machines that are highly conserved in eukaryotes and prokaryoteseukaryotes and prokaryotes
Corrects errors in the genome that result from DNA replicationCorrects errors in the genome that result from DNA replication
Elicit cell response to cytotoxic DNA lesions (e.g. O6 methyl Elicit cell response to cytotoxic DNA lesions (e.g. O6 methyl guanine) guanine)
Reduces spontaneous mutation rates by 100 to 1000 timesReduces spontaneous mutation rates by 100 to 1000 times
MutS Protein Comparison MutS Protein Comparison
Mechanisms of DNA MMRMechanisms of DNA MMRTheThe E. coli E. coli paradigm paradigm
Recognition ofRecognition of mismatched base pairs mismatched base pairs
MutS MutS DNA base-mismatches DNA base-mismatches
Determination of the incorrect base.Determination of the incorrect base.
Resolving the unmethylated strand by detection of the GATC sequenceResolving the unmethylated strand by detection of the GATC sequence MutL + MutS MutL + MutS MutH protein MutH protein MutH specifically nicks the unmethylated strand MutH specifically nicks the unmethylated strand
iii) iii) Excision of Excision of the incorrect base and repair synthesis.the incorrect base and repair synthesis.
3' to 5' or 5' to 3' exonucleases 3' to 5' or 5' to 3' exonucleases DNA Synthesis via Polymerase IDNA Synthesis via Polymerase I DNA LigaseDNA Ligase
Eukaryotic MMR SystemEukaryotic MMR System
MutS genes in prokaryotes, synonymous MutS homolog (MSH) MutS genes in prokaryotes, synonymous MutS homolog (MSH) proteins in eukaryotesproteins in eukaryotes
MSH1~Mitochondrial stabilityMSH1~Mitochondrial stability
MSH2, MSH3, MSH6, MSH7~Mediate error correctionMSH2, MSH3, MSH6, MSH7~Mediate error correction
Form hetrodimers; MSH2Form hetrodimers; MSH2●MSH3, MSH2●MSH6, and MSH2●MSH7(found only ●MSH3, MSH2●MSH6, and MSH2●MSH7(found only in plants)in plants)
MSH4, MSH5~Play essential roles in meiosisMSH4, MSH5~Play essential roles in meiosis
MutL similarly diverged in eukaryotic systems as MLH proteins. MutL similarly diverged in eukaryotic systems as MLH proteins. MLH1MLH1●PMS2 couples mismatch recognition to excision of DNA.●PMS2 couples mismatch recognition to excision of DNA.
No MutH homologNo MutH homolog
Why don’t plants show Why don’t plants show mutational loading?mutational loading?
Plants lack reserved germ lines; gametes Plants lack reserved germ lines; gametes arise from meristem cellsarise from meristem cells
Replication errors, environmental Replication errors, environmental mutagensmutagens
Haplosufficiency quality checking Haplosufficiency quality checking plants plants take advantage of haploidy in take advantage of haploidy in gametophytes. gametophytes.
HypothesisHypothesis
Genome maintenance is essential for plant Genome maintenance is essential for plant genome integritygenome integrity
Primary defense in prevention of Primary defense in prevention of mutagenesis during diploid growth by mutagenesis during diploid growth by rigorous DNA maintenance/repairrigorous DNA maintenance/repair
Haplosufficiency quality checking is an Haplosufficiency quality checking is an important backupimportant backup
Approach toApproach toNonfunctional MMR ProteinsNonfunctional MMR Proteins
The Dominant Negative PhenotypeThe Dominant Negative Phenotype Create two mutated MSH2 gene constructsCreate two mutated MSH2 gene constructs Construct one Construct one mutation in ATPase domain mutation in ATPase domain
Construct two Construct two Mutation in Helix turn Helix domain Mutation in Helix turn Helix domain
Attach CMYC tag at 3’ end of MSH2 constructs and transfer Attach CMYC tag at 3’ end of MSH2 constructs and transfer construct into super expression vector (p1803)construct into super expression vector (p1803)
Transform constructs into Transform constructs into ArabidopsisArabidopsis via via AgrobacteriumAgrobacterium
Overproduced negative MSH2 protein consumes most MSH6, Overproduced negative MSH2 protein consumes most MSH6, MSH3, and MSH7 MSH3, and MSH7 masks functional positive protein masks functional positive protein
Gene silencingGene silencing
AgrobacteriumAgrobacterium Tumefaciens Tumefaciens
Screening for putative Screening for putative transformants transformants
Kanamycin Kanamycin resistance conferred resistance conferred in insertion constructin insertion construct
Perform PCR specific Perform PCR specific to insertion constructto insertion construct
Check for protein Check for protein expression in plants expression in plants by immunoblottingby immunoblotting
Microsatellite SequencesMicrosatellite Sequences
Repeat sequence loci in genomeRepeat sequence loci in genomeSusceptible to insertion/deletion mutations Susceptible to insertion/deletion mutations
by replication machinery by replication machinery Repair of sequences facilitated by MMRRepair of sequences facilitated by MMRAllele shift “fingerprints” in microsatellite Allele shift “fingerprints” in microsatellite
sequence loci indicative of defunct MMR sequence loci indicative of defunct MMR systemsystem
ATNNNATATAT ATATATNNNTATATA TATATATATATANNN
NNNATATATATATATNNNTATATATATATATATATANNN
NNNATATAT ATATATNNNTATATA TATATATATATANNN TA
MMR: MSH2, MSH3, MSH6, MLH1, PMS2
MMR
+2 insertion
no insertion or deletion
-2 deletion
replication
MMR Correction of Slip-Mispairing
WT MSH2::TDNA
Electrophoretic analyses of individual progeny
seeds
TATATATATATATATATATATAATATATATATATATATATATAT
PCRfluorescent tag
shiftedallele
Parent Progeny
Microsatellite Instability Assay
Many Thanks to….Many Thanks to….Dr. Kevin Ahern and the Dr. Kevin Ahern and the HHMIHHMI Program ProgramDr. John B. HaysDr. John B. HaysDr. Walt ReamDr. Walt ReamWanda CrannellWanda CrannellThe Hays laboratory The Hays laboratory The EMT departmentThe EMT department