DNA Mismatch Repair - Oregon State University
DNA Mismatch Repair - Oregon State University
Phenotypic consequences of specific
mutations in human MLH1
Dr. Andrew Buermeyer
Department of Environmental and Molecular
Oregon State University
HHMI Program, Summer 2008
Image courtesy of: www.uniklinikum-saarland.de/.../forschung/hnpcc
DNA Mismatch Repair
Mismatch repair (MMR) is an excision repair pathway that
prevents mutations by correcting errors made in the DNA
strand during replication.
Errors result from:
Exogenous DNA damaging agents
MMR is also promotes apoptosis (cell death) in the presence
of excessive DNA damage
IMPORTANCE: COLON CANCER
- Linked to deficiencies in DNA mismatch repair
Colorectal cancers (CRC) affect 150,000 people in the United States
annually, and causes ~50,000 deaths per year.
Unfortunately, the mortality rate has shown modest reductions over the
last 50 years.
This highlights the
importance of preventative
lifestyle choices and
©2005 American Cancer Society, National Cancer
LYNCH SYNDROME AND MMR
Lynch Syndrome, also known as hereditary nonpolyposis colorectal cancer (HNPCC), is one of the
most common cancer predispositions, representing
2-5% of colorectal cancer cases in the US.
Lynch Syndrome is a disease where
inactivating mutations in a single gene, that is
essential for DNA mismatch repair, are
People who have Lynch Syndrome have a very
high lifetime risk of developing CRC, about
Since most CRC is late onset, the elevated risk
of sporadic CRC is hypothesized to be from
modest defects in MMR genes.
MutLα and MMR
MutLα is a heterodimer, made up of MHL1 and PMS2
MutLα facilitates and coordinates events in MMR.
In absence of MutLα, cells show:
-increased mutation rates
-decreased apoptotic response to genotoxins
Mutations in MLH1 account for 30-40% of Lynch
The Big Picture:
To determine the extent to which different phenotypes, caused by MMRdeficiency, contribute to the development of human cancer.
To analyze the pathogenicity of many single amino acid substitution mutations
identified in human patients with CRC.
Disease causing (or pathogenic) mutations in MLH1 cause measurable
decreases in cells’ ability to perform MMR.
Such changes may affect MLH1-dependant error correction and/or cytotoxic
responses to DNA damage
Determine if the L607H variant hMLH1
gene is capable of restoring a cytotoxic
response and decreasing spontaneous
mutation frequencies to a level
comparable to wild-type transfected
Analyze phenotypic consequences for
L607H is a mutation that was identified in two Lynch Syndrome family cases.
-Limited clinical genetic analysis, therefore co-segregation of this
mutation with HNCCP is unknown
-Shown to have no apparent microsatellite instability
-Found functionally to have no reduced MMR capacity.
It is a single amino acid substitution, located at 607, changing a leucine to a
This is a change from nonpolar, neutral amino acid to polar, weakly basic one.
Images courtesy of
Expression of hMLH1 in MLH1deficient mouse fibroblast cells
Select with G418
Positive control (WT
Pooled Assay General Outline
Frequency of ouabain R
Freeze cell lines
Cytotoxic response to 6-TG
Maintenance of cell lines
Grown in 15% complete media (CM)
Passed 1:10 when 80% confluent into 38⁰C incubator with 5% CO2
Cytotoxic response Assay:
300 cells plated onto four 57 cm2 plates
Following day 2 plates received 7 mL dose of 1μM 6-Thioguanine. After 24 hours, four plates refed with 10
mL 15% CM. Colonies fixed and stained with 1X methylene blue stain
Plates that didn’t receive 6-TG used as negative control for plating efficiency
300 cell plated onto twelve 57 cm2 plates
Next day exposed 1 hour to 7mL dose of varying doses of MNNG (between 100μM to 0.1μM, with 0 dose
control for plating efficiency)
Forward mutation assay:
Plated density of 1.0∙106 cells per 142 cm2 plate with 30mL 1mM Ouabain in 15% CM.
Fixed and stained with 1X methylene blue stain 12-14 days after plating
Colonies counted and mutation rates calculated based on survival of colonies exposed to Ouabain and the
estimated cell survival determined from the efficiency plates
In 12 well plate, 1.0∙105 cells plated per well in 1mL media. 2 days media aspirated and cells fixed with 10%
MLH1-dependent suppression of spontaneous mutations
* - new data
The MLH1 deficient vector +
hMLH1 and the MLH1 deficient
vector behaved within the range
of our historical values.
MLH1-dependent cytotoxic response to
* - New data
MNNG Induced Cytotoxicity
Similar to 6-TG, clearly a
Significance of the
difference in WT and
vector is not clear,
Wild-type hMLH1 and the empty vector controls are consistent with historical values.
The hMLH1 cultures with the L607H mutation have elevated mutation frequencies
compared to wild-type hMLH1, with some ability to reduce spontaneous mutation.
The L607H variant culture had percent survival within range of wild-type hMLH1.
The differences you see in the L607H mutations might be from how the DNA substrate was
prepared, and further testing should be done.
The assay has ability to determine capacity for DNA damage response.
More trials are needed to analyze differences in functionality
Did not obtain results. Cells peeled from the plates in multiple trials:
-Formalin that was used to set the cells for IHC went bad
-The cells were too confluent when setting for IHC
L607H has some capacity for MMR, most seen in cytotoxic response.
More experimentation is needed to determine pathogenicity of this
Future Work and Significance
To determine if L607H is likely to be pathogenic or not, more tests are needed to profile
The next step would be to run a western blot analysis to quantify active MLH1 proteins.
Transfecting MLH1 deficient mice with L607H variant hMLH1 would be another approach to
determining phenotypic consequences and their effect on pathogenicty.
Determining hMLH1 variant behavior and their pathogenicity is an important step in
understanding and evaluating individual risk.
Image courtesy of: studenthealth.sa.ucsb.edu/HealthEducation/
Howard Hughes Medical Institute
Dr. Andrew Buermeyer, mentor
Dr. Kevin Ahern, program coordinator