Transcript Phenotypes of Pathogenic Mismatch Repair Mutants

Determining Phenotypes of
Pathogenic Mismatch Repair
Mutants
Brett Palama
Lab of Andrew Buermeyer, Ph.D.
Dept. of Environmental/Molecular Toxicology
Research Goals
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Determine functional phenotypes of
cells containing certain mutations in
a key DNA mismatch repair protein
which lead to human cancer
Aid the development of relevant
treatment and prevention methods
DNA Mismatch Repair (MMR)
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Highly conserved pathway which
helps ensure preservation of
genomic integrity
May also play role in recognition of
excessive DNA damage and
subsequent signaling of apoptosis
Well-defined in prokaryotes; still
lacking mechanistic information in
eukaryotes
Mechanism for Mismatch Repair
Recognition
G
T
Selection
G
T
Excision
Resynthesis
T
A
T
!
Eukaryotic MMR Proteins
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MMR carried out by two MutS homologs and three MutL
homologs, each with varying heteroduplex specificity
MLH1
MLH3
Substitutions
MSH2
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MLH1
PMS2
MLH1
Short Loops
MSH6
PMS1
Long Loops
MSH2
MSH3
Also dependent upon several replicative proteins (e.g.
PCNA); excision mechanism still unknown
Loss of MMR in Human Cancer
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Germline mutations in one copy of a MMR protein
are evident in a large majority of patients with
Lynch Syndrome (HNPCC)
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Accounts for 2-5% of annual colorectal cancer cases
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Characterized by microsatellite instability (MSI)
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70% of cases involve mutations of mlh1 or msh2 gene
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Greatly increases likelihood of developing other cancers
(endometrial, ovarian, etc.)
Pathogenic germline mutations in both copies of a
MMR protein result in severe early-onset leukemia or
lymphoma (lifespan < 5 yrs)
Inactivation of MMR (evident from MSI) also
apparent in 13% of sporadic colorectal cancers
Pathogenic Mutations in hMLH1
Specific interest of study is the hMLH1 protein (the nonredundant member in human MutL complexes)
ATP-binding/
hydrolysis
Dime r interface
ss DNA binding
3
241
492
621
711
756
Linker
PMS2, EX O1
inte raction
C-terminal
homology
Map of hMLH1 protein, tentative domains, and pathogenic mutations
Questions to Address
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Which cellular phenotypes (error correction
and DNA damage surveillance) are
present/deficient in each mutant?
Mutator phenotype = higher mutation frequency = increased
chance of mutation in tumor suppressor genes/oncogenes = cancer
DNA damage tolerance = higher DNA damage threshold =
increased likelihood of propagation of damaged cells = cancer
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Are there pathogenic mlh1 mutants in
which only one MMR function is inactivated
(“separation of function” mutant)?
What is the biochemical mechanism for the
deficiencies introduced by mutations in
mlh1?
L749ter and the CTH Domain
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Mutation which inserts a premature stop
codon in the hMLH1 sequence, deleting the
final eight amino acids of the polypeptide
Previous data shows that L749ter has a
mutator phenotype, high DNA damage
tolerance, and inability to stabilize PMS2
Two mutations identified in human cancer
are in the CTH domain (K751R and R755W)
Will these two mutations show phenotypes
similar to those of L749ter?
Project Outline
Site-directed mutagenesis of hMlh1 cDNA
Subcloning into mammalian expression vector
Transfection into mlh1- (and pms2- in transient
transfections) mouse embryo fibroblasts
Functional Assays
Mutagenesis/Subcloning
hMLH1
Site-directed
mutagenesis
hMLH1
Sequencing of cDNA
pBS
SK-
pBS
SK-
Removal of cDNA by
restriction digestion;
blunting of cDNA; gel
isolation of cDNA
hMLH1
pCMV
BamNeo
Sequencing
of cDNA
Ligation into linear, blunted
pCMVBamNeo vector;
hMLH1
Transformation into E.Coli
Large-scale plasmid
preparation
DNA for use in
functional assays!
Functional Assays
After subcloning into pCMVBamNeo and transfection into
MEF cells, assays are done to determine functional
characteristics of mutant MLH1 protein
Functions To Be Studied:
Error Correction
DNA Damage Recognition
PMS2 Stabilization
PMS2 Subcellular Localization
PMS2 Binding
PMS2 Stabilization
Evidence suggests that MLH1 has a stabilizing effect on PMS2.
Question: Which pathogenic mutants are
incapable of stabilizing PMS2?
Approach: Transiently transfect double-knockout
cells with mutant hMLH1 and PMS2; use SDS-PAGE
and Western Blot to determine protein levels.
PMS2 PMS2
PMS2
alone wt MLH1 L749t
PMS2 PMS2
K751R R755W
MSH6
PMS2
MLH1
Concluding Questions
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Why do the mutations K751R and R755W
show stabilization of hPMS2 while L749ter
does not?
Are the mutations at 751 and 755
pathogenic by some other means? Or are
they neutral polymorphisms?
Will other mutations in the CTH behave
similar to 751 and 755?
Acknowledgements
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Buermeyer Lab
Hays Lab
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URISC
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HHMI
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