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