August 25, 2005
Measuring the persistence
of MutS at a mismatch site
after binding
Nikki O’Donnell
Department of Environmental and Molecular Toxicology
Mentors: Dr. John Hays and Pete Hoffman
DNA Repair Systems
•
One major concern for an organism’s survival and fecundity
is genomic stability
•
Errors in DNA can occur during synthesis or post replication
from environmental factors (i.e. UV radiation)
•
This is a major problem because errors lead to mutations,
which can lead to a variety of problems including cancer
•
Organisms have multiple repair systems
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–
–
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Direct Reversal
Base Excision
Recombination
Nucleotide Excision
Mismatch Repair
Relevance of Mismatch Repair
• Evolutionary conserved process
• Essential for correcting errors during
DNA replication
• Increases genomic stability 100 to
1,000 fold
• Lack of MMR has been linked to
several forms of cancer
Overview

Relevance

Outline of Mismatch Repair

Question and Hypothesis

Project Design


Objectives
Project Methodology

Experimental Setup

Experimental protocol

Mutant Purification

Assay Validation

Key Findings / Results

Acknowledgements
DNA Mismatch Repair in Prokaryotes
Question and Hypothesis

Question


Does MutS stay fixed at a mismatch site after
binding or move away?
Hypothesis

MutS remains near the mismatch site and
does not migrate
Experiment Setup
Wild Type MutS
G G A T C C
G G A T C C
C C T A G G
C C T A G G
DNA is cleaved
MutS stays at the
mismatch site
G G A T C C
C C T A G G
Project Designs
1. Create substrate
with mismatch
2. Test wild type and
mutant in presence
and absence of ATP
3. Test wild type and mutant
in the presence of BAMHI
restriction enzyme
Experiment Protocol
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Start with an E.coli plasmid with two specific single stranded nicking sites
Heat to denature bonds
Make oligomer and run the reaction with a complementary strand
‘Gapped Plasmid’
Introduce new oligomer
Ligate
Final product is plasmid with a mismatched base pair
Mutant MutS Protein
2
1
3
• Mutation found in yeast made the
mutS protein non responsive to ATP
• Testing in E.coli MutS protein.
• Validate by sequencing, expression of
the protein and analysis of its
biochemical characteristics
•
4
5
Strand Synthesis
1)
Thermal cycle to denature DNA
template
2)
Anneal mutagenic primers containing
desired mutation
3)
Extend primers with PfuUltra DNA
polymerase
4)
Digest Parental DNA
5)
Transform mutated molecule into
competent cells for nick repair
* Specific site was found by another lab and was published in a paper.
Protein expression over time
1
2
3
4
5
 Induction in E.coli
 Samples taken at hour intervals to check for
expression
 Check for correct size
Assay Validation
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Two restriction sites- one near and one farther removed from the mismatch-- BAMHI site
Test binding and protection
Do this in quick time and add BAMH1
Determine if cleavage happens
Key findings and
Results
uncut
Cut
• Possible outcomes
• Separate wild type
and mutant on a
gel to separate
protected DNA and
possible cleaved
segments
• DNA shift indication
Time
Time
Acknowledgements
• The Howard Hughes Medical
Institute
• Dr. Kevin Ahern
• Dr. John Hays
• Pete Hoffman
• The Hays Lab Crew: Gerrick, Laurel, Aly,
Mark, Stephanie, Huixian