Transcript Functional consequences of NLS mutations in mlh1

Functional consequences of NLS
mutations in human MLH1
Alex Dukes
Dr. Andrew Buermeyer
Department of Environmental & Molecular
Toxicology
Oregon State University
HHMI Program, Summer 2007
The Importance of Mismatch Repair
(MMR)

Cellular mechanism for preventing DNA mutations, resulting from:




Replication errors
Recombination pathways
Exogenous DNA damaging agents
Necessary to trigger apoptosis in event of extensive DNA damage
MMR and Colon Cancer
Colorectal cancers account
for 10% of all new cancers
 Lynch Syndrome, a type of
hereditary colon cancer
(HNPCC)
 Caused by genetic defects in
MMR genes

©2005 American Cancer Society, National Cancer Institute
A Component of MMR: MutLα
Heterodimer composed of MLH1 and PMS2
proteins
 In the absence of MMR, cells display:

 Increased
mutation rate
 Decreased apoptotic response to genotoxins

Mutations in MLH1 account for 30-40% of
Lynch Syndrome cases
Two Mutations of Interest


Missense mutations both identified in cancer patients
Nuclear localization sequence (NLS):


Acts as a cellular “password” to allow protein into nucleus via the
nuclear pore
amino acids 470 – 474 in MLH1
Image courtesy of: Wu, X., et al, Dimerization of MLH1 and PMS2 Limits
Nuclear Localization of MutL. Molecular and Cellular Biology, 2003. 23(9):
p. 3320–3328
Image courtesy of : www.scripps.edu/.../proj/NPC/NucleusWhite.gif
Mutation #1
R474Q



Amino acid 474
Replaces arginine (R) with glutamine (Q)
Charged  Charged
Images courtesy of: www.contexo.info/.../images/aminoacidsweb.gif
Mutation #2
R472I
Amino
acid 472
Replaces arginine (R) with isoleucine (I)
Charged  Non-polar (more deleterious?)
Images courtesy of: www.contexo.info/.../images/aminoacidsweb.gif
Project Objectives

Examine two NLS mutations in MLH1 and identify the
phenotypic consequences of each
Experimental Methods


Transient transfection
Stable transfection
The Transient Transfection
Is the mutant MutLα heterodimer stable?
Does the mutation interfere with normal localization patterns?


Plasmids encoding mutated MLH1 and PMS2 are
introduced into MutLα- deficient cells
Cells assessed to determine protein expression and
localization pattern
Nuclear
MutLα present
MutLα absent
Cytoplasmic
The Transient Transfection
Lipid-based transfection
The Stable Transfection
Does the mutation result in reduced MMR function?
Plasmid encoding mutated MLH1 is introduced
into MLH1- deficient cells
 Cells exhibiting reduced MMR function
identified by:

 Increased
mutation levels
 Loss of apoptotic response
This assay also can be used to confirm the heterodimer stability
and localization pattern found in the transient transfection.
The Stable Transfection
Electroporation
Selection with cytotoxic G418
Positive Control
Negative Control
R472I
R474Q
Plating from the Stable Transfection
Ouabain Assay
6-TG Assay
Mutation frequency
Survival following DNA
damage response
Pass 1:10
Pass 1:10
Freeze cell lines
Interpretation of possible outcomes
Hypothesis: Both mutations interfere with localization and
cause MMR deficiency.
Localization
MMR
Phenotype
Interpretation of Results
Cytoplasmic
Deficient
Mutation interferes with localization causing
MMR deficiency
Cytoplasmic
Proficient
Low levels of MutLα enter nucleus and
are sufficient for MMR phenotype
Nuclear
Deficient
Mutation interferes with enzymatic function
or other protein activity, not localization
Nuclear
Proficient
Mutation does not interfere with normal
localization
Transient Transfection Results
1
2
3
4
MSH6
PMS2
MLH1
Both R474Q and
R472I mutants
stabilize PMS2
Stable Transfection Results:
Protein Expression Levels
MLH1 accumulates
similar to wild type
MLH1 stabilizes PMS2
similar to wild type
Stable Transfection Results:
Cellular Localization
Mutation #1: R474Q
Mutation #2: R472I
Both mutants exhibit nuclear localization
Stable Transfection Results: Mutation
Frequency
MMR Deficient
(HIGH mutant
frequency)
MMR Proficient
(LOW mutant
frequency)
Stable Transfection Results:
Cytotoxic Response
MMR Deficient
(HIGH survival)
MMR Proficient
(LOW survival)
Summary of Results
Localization?
NUCLEAR
 Mismatch repair phenotype?
PROFICIENT

Possible Explanations
1 The mutation in MLH1 did not critically interfere with the NLS
2
A redundant NLS sequence allowed the protein into the
nucleus (Another in MLH1? In PMS2?)
Colon Cancer Implications
Our functional assays show no reduced MMR
activity
 Other lab groups obtained similar results for
R474Q
 Clinical data is not conclusive in linking
mutations to Lynch Syndrome

CONCLUSIONS
1. The mutations are not pathogenic.
2. The mutations are moderately pathogenic and our
assays are not sensitive enough.
Acknowledgements



Howard Hughes Medical Institute
Dr. Andrew Buermeyer, mentor
Dr. Kevin Ahern, program coordinator
Generously funded by grants from the
HHMI Program