Kinetics of MutSα-Mediated DNA Mismatch Repair
William Gibson
Dr. John Hays
Oregon State University
Summer 2010
The Importance of DNA Mismatch
Repair (MMR)


A poorly working MMR system can
cause many problems such as accelerated
cell aging, cell death, and cancer.
Diseases caused by defects in MMR
systems are costly and damaging because
of their severity and the lack of treatment
options available.
MutSα-MMR System




MutSα-MMR system 3
main components
Mut-Sα
Mut-Lα
MutSα – Two subunits which
form a dimer to bind DNA
mismatches or adducts.
MutLα – Two subunits which
assist MutSα and exhibit a
nuclease function.
PCNA – A trimeric clamp
which helps guide MutSα and
MutLα along the DNA strand.
PCNA
DNA Damage from PAH Alkylation


Polycyclic aromatic
hydrocarbons (PAHs)
attach to DNA and
cause lesions.
PAH's are produced by
the combustion of
complex carbon
compounds (ex. coal).
What I Want to Do
• Examine MMR initiation at gaps vs. nicks.
• Examine how the MMR system responds to adducts
such as a PAH-guanine (PAH-G) adduct.
• Determine if PAH-G mismatches provoke MMR
excision similar to standard base mismatches.
Plasmid Model for MMR
•Initiation: MutSα complexes with MutLα and PCNA.
•Proximal Excision: MutSα-MMR complex excises to the
mismatch.
•Distal Excision: MutSα-MMR complex excises past
mismatch before dissociating.
Plasmid Construct


The plasmid construct
has multiple nicking and
gapping sites to initiate
repair.
PAH-Guanine is
opposite to any of the 4
bases (A, G, T, C)
Experimental Design
Growth of Plasmid
↓
Addition of Gap and Mismatch Sites
↓
MMR Assay (Using Radio-Labeled Reporter
Oligos)
↓
Results Analysis (Compare Gapped vs. Nicked and
3 Different Mismatches vs. Controls)
Growth of Plasmid
• The plasmid is modified from
the highly used pUC19
plasmid.
• The plasmid contains a drug
resistance gene.
Growth of
Plasmid
Addition of
Gap and
Mismatch Sites
MMR Assays
Results
Analysis
Gapping and Nicking
• Multiple unique restriction
sites allow different areas
to be targeted.
• Modified restriction
enzymes allow nicking of a
single strand at nick sites.
Growth of
Plasmid
Addition of
Gap and
Mismatch Sites
MMR Assays
Results
Analysis
MMR Assay 1
• Excision Assay: Measure Gapped vs. Nicked initiation.
Repair
Growth of
Plasmid
Addition of
Gap and
Mismatch Sites
No Repair
MMR Assays
Results
Analysis
No Repair
Growth of
Plasmid
Repair
MMR Assay 1
Addition of
Gap and
Mismatch Sites
Time 0
Time 2
MMR Assays
Time 4
Time 6
Results
Analysis
MMR Assay 2
• PAH Assay: Measure relative abundance of the radiolabeled probes.
Excision
past PAH
Excision
but not
past PAH
No excision triggered
Growth of
Plasmid
Addition of
Gap and
Mismatch Sites
MMR Assays
Results
Analysis
Results Still Pending
• The first assay will provide a definitive answer on gap vs.
nick initiation, which will allow future assays to use the
most efficient initiation method.
• By exploring the behavior of the MMR system in regards to
PAH-G adducts, specific kinetic models can be built to
further understand the MMR process at a molecular level.
Growth of
Plasmid
Addition of
Gap and
Mismatch Sites
MMR Assays
Results
Analysis
Acknowledgments
Dr. John Hays and Peter Hoffman
Colin Tominey
Dr. Mark Curtis, Johanna Steinbrecher, Robert Ursu, Ana Brar,
Taylor Hoffman, Buck Wilcox
Figures: ATSDR (slide 2), JBC (slide 4), myself (slide
3,6,7,11,12,13), NEBcutter (slide 10), David Brooks (slide 9)
Special thanks to Kevin Ahern and the HHMI program
for letting me take part!