Expanded Precursor Pools and
DNA Methylation as
Determinants of Mutagenesis
Howard Hughes Medical Institute
Summer 2004
Nancy Jade Lee
Dr. Christopher K. Mathews’ Laboratory
Department of Biochemistry & Biophysics
Oregon State University
Imbalances in Nucleotide Levels:
Mutagenesis by Expanded DNA
Precursor Pools of Mammalian
Cells
 To analyze DNA building block concentrations
(Deoxyribonucleoside triphosphates or dNTPs)
 To understand the relationship between intracellular dNTP
concentration and mutagenesis
 To study the effects of hydroxyurea on ribonucleotide reductase
(rNDP reductase) in mammalian cells
Background: dNTPs
 dNTPs are necessary
for the biosynthesis of
DNA
dATP
dTTP
dCTP
dGTP
*The amount of each dNTP contained in a cell is referred to as a “pool”
dNTP Pools
 Normal cells have balanced pool sizes
 Unbalanced pool sizes can stimulate mutagenesis
 Example
dATP pool
dTTP pool
dGTP pool
dCTP pool
= more mutations
However…
In E. coli cells balanced increases in dNTP pools also
stimulates mutagenesis
dATP pool
dTTP pool
dGTP pool
dCTP pool
= more mutations
dNTP Biosynthesis
 To make dNTPs
ribonucleoside
diphosphate (NDP) must
convert to
deoxyribonucleoside
diphosphate (dNDP)
Ribonucleotide Reductase (rNDP reductase)
Hydroxyurea-resistant Cell Lines
 Hydroxyurea-resistant cell lines carry elevated levels of
ribonucleotide reductase
Wild Type Cell
Hydroxyurearesistant Cell 
Question
 Do hydroxyurea-resistant mammalian cells
exhibit enlarged dNTP pools?
 If so, do these cells also have elevated
spontaneous mutation rates?
Measuring dNTP Pools
• Radiolabeled dNTP (3H
3H
dTTP and
dATP) are
counted in a scintillation
counter
• This tells us how much
regular dNTP a sample
contains
Example
 To measure dATP (analyzed with 3H dTTP)
 Template  A A A T A A A T…
 Base pair 
T* T* T* A T* T* T* A…
dNTP Pool Assay Results
Comparison of wild type and hydroxyurea treated cell
dNTP pools
Percent difference
(pmol/106 cells)
250%
200%
V79 wild type
150%
.35 mM HU-Res clone 1
100%
.35 mM HU-Res clone 2
1.3 mM HU-Res clone A
50%
0%
dATP
dGTP
dTTP
dCTP
dNTPs
 Hydroxyurea-resistant cells exhibit balanced increases in dATP,
dGTP, and dTTP pools
 decrease in dCTP pool size
 Balanced increase in all four dNTP pools not observed
Measuring Mutagenesis
 Known quantity of cells plated in the presence of 6thioguanine
 Targets hypoxanthine-guanine phosphoribosyltransferase
(HGPRT)
Wild type cells
Mutagenic cells
6-Thioguanine Assay Results
Surviving Colonies (per plate)
(plated 1 X 106 cells per plate)
Trial #1
Trial #2
Trial #3
V79 Wild Type
0,3
2,0
1,1
.35 mM HU-Res clone 1
0,3
3,2
0,0
.35 mM HU-Res clone 2
2,1
0,0
0,2
1.3 mM HU-Res clone A
0,0
0,0
0,1
 No significant difference in the number surviving
colonies (wild type vs. hydroxyurea-resistant)
 Higher rate of mutagenesis not detected in hydroxyurearesistant clones
Summary
 Balanced increase in all four dNTP pools of hydroxyurearesistant cells not observed
 Higher rate of mutagenesis in hydroxyurea-resistant cells not
observed
Further Research
 Investigate and analyze other hydroxyurea-resistance clones
available in the lab
 Develop and test a model to explain the dNTP pool changes and
rate of mutagenesis seen in the hydroxyurea-resistance cells
dATP pool
dTTP pool
dGTP pool
dCTP
pool
Chemical Reactions of DNA Bases:
Mutagenesis as a Result of
Deamination at
5-Methylcytosine
 To understand the complex relationship among methylation of a
base, deamination and the rate of mutagenesis
 To examine the rate of deamination at methylcytosine-cytosineguanine (mCCG) regions versus cytosine-methylcytosine-guanine
(CmCG) regions
Importance
• Link to cancer
• Point mutations
• “hotspot”
Background: DNA Bases
Methylation
AATCCGGTAT
Deamination of Cytosine and 5methylcytosine
-------------------------------------------------------------------------------
Base Excision Repair
 Cytosine (C)
CG

UG

CG
= repaired
 5-methylcytosine (mC)
mCG

TG
= not repaired
mC – G  T – A
= point
mutation
Question
 Why does deamination occur?
 Which factor has the most effect on the high rate of
deamination seen at the 5-methylcytosine base?
 Methylation
 Adjacent to Guanine
 Structural change
mCG
Methods
Control
CCGG
GGCC
Outer C
methylated
CCGG
GGCC
= high rate of mutagenesis due to
structural changes
Inner C
methylated
CCGG
GGCC
= high rate of mutagenesis due to
methylation
Analyzing deamination at this site: C  T or U
Acknowledgements
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Howard Hughes Medical Institute (HHMI)
Christopher Mathews
Linda Wheeler
Indira Rajagopal
Kevin Ahern
Stephanie Junell
Carolyn Wiesner
Department of Biochemistry & Biophysics