Determining Phenotypes of Pathogenic Mismatch Repair Mutants Brett Palama
Determining Phenotypes of
Pathogenic Mismatch Repair
Mutants
Brett Palama
Lab of Andrew Buermeyer, Ph.D.
Dept. of Environmental/Molecular Toxicology
Research Goals
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)
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
MMR carried out by two MutS homologs and three MutL homologs, each with varying heteroduplex specificity
MLH1 MLH3 MLH1 PMS2 MLH1 PMS1
Substitutions Short Loops Long Loops
MSH2 MSH6 MSH2 MSH3
Also dependent upon several replicative proteins (e.g.
PCNA); excision mechanism still unknown
Loss of MMR in Human Cancer
Germline mutations in one copy of a MMR protein are evident in a large majority of patients with
Lynch Syndrome (HNPCC)
Accounts for 2-5% of annual colorectal cancer cases
Characterized by microsatellite instability (MSI)
70% of cases involve mutations of mlh1 or msh2 gene
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 inter face
3 ss DNA binding
241
Linker
492 621
PMS2, EX O1 inte raction
711 756
C-terminal homology
Map of hMLH1 protein, tentative domains, and pathogenic mutations
Questions to Address
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
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
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 pBS
SKhMLH1 pCMV
BamNeo
Site-directed mutagenesis
Sequencing of cDNA hMLH1 pBS
SK-
Ligation into linear, blunted pCMVBamNeo vector;
Transformation into E.Coli
hMLH1
Removal of cDNA by restriction digestion; blunting of cDNA; gel isolation of cDNA
Sequencing of cDNA
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 alone
PMS2 wt MLH1
PMS2
L749t
PMS2
K751R
PMS2
R755W
MSH6
PMS2
MLH1
Concluding Questions
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
Buermeyer Lab
Hays Lab
URISC
HHMI
