Pathogenic Mechanisms of Cancer Causing hMLH1 Mutations Functional Relationship between DNA Mismatch Repair and Tumor Formation - PowerPoint PPT Presentation

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Pathogenic Mechanisms of Cancer Causing hMLH1 Mutations Functional Relationship between DNA Mismatch Repair and Tumor Formation

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Eddie O'Donnell. Laboratory of Dr. Andrew B. Buermeyer ... Excludes basal and squamous cell skin cancers and in situ carcinomas except urinary bladder. ... – PowerPoint PPT presentation

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Title: Pathogenic Mechanisms of Cancer Causing hMLH1 Mutations Functional Relationship between DNA Mismatch Repair and Tumor Formation


1
Pathogenic Mechanisms of Cancer Causing hMLH1
Mutations Functional Relationship between DNA
Mismatch Repair and Tumor Formation
Eddie ODonnell Laboratory of Dr. Andrew B.
Buermeyer Department of Environmental and
Molecular Toxicology
2
2003 Estimated US Cancer Deaths
Men 285,900
Women 270,600
  • 25 Lung bronchus
  • 15 Breast
  • 11 Colon rectum
  • 6 Pancreas
  • 5 Ovary
  • 4 Non-Hodgkin lymphoma
  • 4 Leukemia
  • 3 Uterine corpus
  • 2 Brain/ONS
  • 2 Multiple myeloma
  • 23 All other sites

Lung bronchus 31 Prostate 10 Colon
rectum 10 Pancreas 5 Non-Hodgkin 4 lymphoma Le
ukemia 4 Esophagus 4 Liver/intrahepatic 3 bile
duct Urinary bladder 3 Kidney 3 All other
sites 22
ONSOther nervous system. Excludes basal and
squamous cell skin cancers and in situ carcinomas
except urinary bladder. Source American Cancer
Society, 2003.
3
Colon Cancer
  • Causes of Cancer
  • Mutations within cells cause uncontrolled cell
    growth
  • Cancer may be inherited or sporadic
  • Colorectal Cancer
  • 15 - Mismatch Repair (MMR) deficiency
    observed
  • 90 of Sporadic cases linked to MMR deficiency
    are mlh1
  • deficient (loss of expression)
  • 2-5 - Hereditary Non-Polyposis Colorectal
    Cancer (HNPCC)
  • Recent Discoveries involving HNPCC
  • 1993 MSH2 mutations linked to 35 of HNPCC
  • 1994 hMHL1 mutations linked to 35 of HNPCC

HNPCC, FAP
Non Hereditary
4
DNA Mismatch Repair
  • DNA Mismatches arise from errors during DNA
    Replication
  • MMR is an essential process for maintaining
    genomic integrity
  • Basic Mechanism


Mismatch

recognition


Strand choice

Excision

Resynthesis
5
Mutation Types
  • DNA Synthesis Error Mutation
  • Base Mismatches ? Base Substitution Mutations
  • Insertion / Deletion Loops ? Microsatellite
    Instability (MSI)
  • Microsatellite loci are common and unique to a
    persons genome
  • Looking specifically at dinucleotide repeated
    sequences
  • Deficient MMR can lead to increased probability
    of replication errors

No Repair, Continued Replication
Incorrect DNA sequence, mutation in genome copy
Incorrect Placement of Base
A T
A T
G T
Successful Repair
G C
Incorrect DNA sequence, mutation in genome copy
Dinucleotide Loop Insertion
No Repair, Continued Replication
AC TG
Successful Repair
6
Clinical relevance of the hMLH1 gene
  • hMLH1 significance
  • hMLH1 is an essential protein for the prevention
    of
  • mutations. Exact function is unknown.
  • Treatments
  • MMR deficient cancers may respond differently
  • to chemotherapeutic drugs.
  • Detection
  • MMR deficient cancers are commonly detected
  • through screening for MSI, however
  • Several hMLH1 Mutations have been implicated in
    HNPCC cases showing only high levels of base
    substitution mutations not initially identified
    as MMR deficient cancers.

E578G K618A D132H V716M
hMLH1 amino acid site 578 changed from E
(glutamic acid) to G (Glycine)
7
Preliminary Research with hMLH1 E578G
  • Data from analysis of cells expressing E578G
    demonstrate
  • No MSI, consistent with observed cancers
  • Increased levels of base substitution mutations
  • E578G mutation affects repair of base base
  • mismatches but not dinucleotide loops
  • Increased base substitutions may explain
    pathogenicity of mutations
  • suggests a possible novel role for MMR repair
    genes, containing
  • MLH1, in substrate recognition and / or
    commitment to initiate repair



8
Research Questions Goals
  • Are mutations of hMLH1 responsible for the
    observed molecular phenotypes?

The goal of the research is to determine the
mutation prevention capabilities of the MLH1
mutants E578G, K618A, V716M, and D132H using
biochemical assays
9
Project Overview
Generation of substrates to model DNA mismatches
Stable transfections create extracts containing
repair factors including mutant hMLH1 protein
Biochemical Assays will elucidate the repair
efficiency of the mutant hMLH1 protein
10
Biochemical Assay Procedure
Length of repair products will be measured with
Analytical gel electrophoresis.
11
Cytoplasmic Extracts
Stable Transfection
  • Mutant hMLH1 gene introduced via electroporation
  • A specific sequence , unrelated to the hMLH1
    gene, which enables resistance to the drug G418
    is also introduced to allow for selection of
    cells that were succesfuly transfected
  • Cells expressing the desired MLH1 mutation
    provide the source for MLH1 mutant protein and
    other repair proteins
  • Selection of cell lines expressing sufficient
    levels of MLH1 mutants relative to wildtype MHL1
    using western blotting

Extracts
E578G Cell Line Selection
250 kD
150 kD
MSH6 (140.1 kD)
100 kD
PMS2
75 kD
MLH1
ß-Tubulin (50.9 kD)
50 kD
12
Substrate Formation
  • Research will involve in-vitro MMR reactions and
    different plasmid
  • substrates, that model presumed replication
    errors, with either
  • -single base pair mismatches model base
    substitutions
  • -dinucleotide insertion loops model MSI
    mutations

Contaminating Plasmid
T G
-

Purified Substrate
13
Substrate Formation
A circular piece of DNA, known as a plasmid,
serves as the starting material for the mismatch
repair substrate.
A nicking enzyme, which cuts one strand of a
double stranded sequence, cuts at two sites on
the plasmid DNA. The Plasmid is heated to remove
the cut fragment, and the complementary strand is
added to create a pure gap molecule
A DNA oligo, similar to removed fragment, is
introduced to the gap molecule, and DNA Ligase is
used to anneal the mismatch oligo to form the
mismatch substrate.
14
Substrate Formation
Diagnostics used at each step to ensure quality
of substrate
  • Does the starting plasmid cut correctly?
  • How efficient was the nicking of the DNA
  • Purity of gap molecule ?
  • Is the gap molecule resistant to cutting ?
  • Is the mismatch substrate resistant to cutting ?

Gap Molecule
Mismatch Substrate
Starting Plasmid
Nicked Plasmid
Nicked DNA
Linearized DNA
Supercoiled DNA
Linear DNA from double digest
Uncut Plasmid
Restriction Endonuclease Cut
15
Preliminary Repair Assays
  • Cellular extracts without MLH1 protein and
    extract with functional (wildtype) MLH1
  • protein will serve as experimental controls

Test Repair Assays
Extracts A HeLa cells (positive control) B
WT22 cells (positive control) C E578G D
CMV2 (negative control) E No extract (negative
control)
16
Further Research
  • Questions
  • - Do any of the MLH1 mutants show ability to
    differentiate
  • Between different types of mismatches
  • At what efficiency relative to wildtype MLH1 do
    the
  • mutants repair mismatches

17
Thank You
  • HHMI
  • URISC
  • Dr. Andrew Buermeyer
  • The Buermeyer Lab Group. Good People
  • Dr. Kevin Ahern
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