Transcript Investigation of the Premutagenic Lesion 8-oxo dGTP and its Repair Mechanism Mut T Howard Hughes Medical Institute (HHMI) Daniel Bai Dr.

Investigation of the Premutagenic
Lesion 8-oxo dGTP and its Repair
Mechanism Mut T
Howard Hughes Medical Institute (HHMI)
Daniel Bai
Dr. Christopher K. Mathews
Biochemistry and Biophysics
Oregon State University
Objectives of the Mathews Lab
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Nucleic Acid Enzymology (DNA replication, repair and
mutation)
Understanding regulation of DNA synthesis in
mitochondria
Investigate the enzymes that regulate nucleotide
incorporation into DNA
Project Introduction
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Mitochondria are powerhouse of eukaryotic cells
Mitochondrial DNA mutations are linked to
neurodegenerative disorders, cardiomyopathies, and
cancer
Rate of mitochondria mutations are two orders
greater than in nuclear genomes
The suspect: oxidative damage from reactive species
(ROS)
Introduction cont.
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Deoxynucleotide-triphosphates (dNTP’s) are the
building blocks of DNA
Investigate both the damage to mitochondrial
dNTP’s and their built in repair mechanisms
Our Perpetrator
Oxidation
8-oxo-G
Early Trail Blazers
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Mut T mutator genes were discovered in 1954
Cells without Mut T enzyme have elevated mutation
rates of 1000 fold
Mut T minus strains of bacteria contained a mystery
metabolite
The mystery metabolite has never been described
before
HPLC Conclusions
Mut T- Strain
Mystery Metabolite
Low Amount of 8-oxo G
HPLC
Abs
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Time
Substrate for Something Else
Earlier Student
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Jordan Boutilier worked in the Mathews lab
demonstrated that there were minimal amounts of
8-oxo-dGTP
I am going to determine how damaging this small
amount is to mitochondria
Cells grown without damaging reagents did not
have reduced concentration of the Mut T enzyme
My Aims
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Compare cells absent of Mut T with wild
types in terms of oxidative damage
Qualitatively analyze 8-oxo-dGTP pools to
understand their role in damaged
nucleotides
Identify the Mystery Metabolite using Mass
Spectrometry
Research Design
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Compare cellular extract of Mut T wild type and
mutant strains that are absent of Mut T
Use HPLC and Electrochemical analysis to investigate
nucleotide pools of the wild and mutant strains
Use both TLC to separate nucleotides and radioactive
counter to quantitatively analyze oxidized dNTP
Time
Results
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Investigation of enzyme activity failed
Use of new buffers BICINE MOPS
TLC proved to be insensitive to enzyme
activity
Work involving other Labs
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Dr. Fred Steven’s mass spectrometer
Dr. Tory Hagen’s HPLC
Investigation of Nucleoside
Diphosphate Kinase
Abnormalities
Howard Hughes Medical Institute (HHMI)
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Daniel Bai
Dr. Christopher K. Mathews
Biochemistry and Biophysics
Oregon State University
What is Nucleoside Diphosphate
Kinase?
ATP + (d)NDP
ADP + (d)NTP
(d)NTP
ATP
NH
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ADP
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OH
(d)NDP
OH
Nucleoside diphosphate kinase (NDP kinase) is a multifunctional
enzyme that provides a pathway for both dNTP synthesis and
DNA replication.
Studies have shown that absence of NDP kinase does not
interfere with cell growth.
However, NDP kinase abnormalities have demonstrated highly
imbalanced dNTP pool levels and ultimately mutagenesis.
Medical Importance
Breast cancer patients who have abnormal NDP kinase activity
also have a greater probability of cancer metathesis.
In a mismatch repair-defective background, an excess of dCTP
or dCTP, increases the chance of insertion errors. (AT -> GC)
Past Work
Previous researchers in the Mathews Lab have discovered enlargement of
dCTP by twentyfold as well as increases in CTP, and dGTP pools in the NDP
kinase absence E-coli cells.
How are we sure that pool imbalances were caused by NDP kinase
abnormalities and not by loss of protein-protein interaction resulting from
absence of NDP kinase?
A mutant strain with structurally intact, but catalytically inactive form of NDP
kinase was tested for pool levels. The results were identical to untransformed
mutants. Which suggests that loss of NDP kinase activity is responsible for
pool imbalances.
My Role
Spent part of past summer successfully isolating a mutant
NDP kinase gene from plasmids of Dr. Edith Postel.
This summer’s work involves inserting each mutant gene into
a suitable plasmid by gene cloning techniques and then
creating transformed E. coli cells.
Future Experiment
The ultimate aim is to determine whether the mutator phenotype of
an ndk mutant results more directly from altered dNTP pools or
from loss of a DNA repair activity associated with the enzyme.
After successful transformation, the mutation rates will be
measured.
An assay for dNTP pool levels will be performed using a
scintillation counter.
NDP kinase enzyme activity will be analyzed using Western
Blotting for protein expression.
Acknowledgements
Howard Hughes Medical Institute
Dr. Christopher K. Mathews
Linda Benson
Dr. Rongkun Shen
Dr. Edith Postel