Mutagenesis by Expanded DNA
Precursor Pools of Mammalian
Cells
Howard Hughes Medical Institute (HHMI)
Summer 2003
Nancy Jade Lee
Dr. Christopher K. Mathews’ Laboratory
Department of Biochemistry & Biophysics
Oregon State University
Lab Objectives

To understand mechanisms of mutagenesis caused by
perturbations of nucleotide metabolism

To understand the source and regulation of DNA
precursor pools in mitochondria and eukaryotic cells

To understand how enzymes of DNA precursor
synthesis interact within cells to facilitate the flow of
nucleotides into DNA
My Objectives

To examine DNA precursors (Deoxyribonucleotides or
dNTPs) and their ability to stimulate mutagenesis

To understand the relationship between intracellular
DNA precursor concentration and mutagenesis

To study the effects of hydroxyurea on ribonucleotide
reductase (rNDP reductase) in mammalian cells in
order to understand the role of rNDP reductase
Importance

Cancer results from mutations that accumulate
in pre-cancerous cells (Loeb, 1998)
• Tumor cells in culture tend to have higher
levels of dNTPs than non-tumor cells
(Martomo & Mathews, 2002)
Background

Deoxyribonucleotides
(dNTPs) are necessary
for biosynthesis of
DNA
dATP
dTTP
dCTP
dGTP
*The amount of each dNTP contained in a cell is referred to as a “pool”
(Images courtesy of www.fermentas.com)
dNTP Pools



Regular cells have balanced pool sizes
Unbalanced dNTP pools can stimulate mutagenesis (Kunz et
al, 1994)
Example
dATP pool
dTTP pool
dGTP pool
dCTP pool
= more mutations
Meanwhile…
In E. coli cells balanced increases in dNTP pools also
stimulates mutagenesis
(Wheeler & Rajagopal, 2002)
dATP pool
dTTP pool
dGTP pool
dCTP pool
= more mutations
dNTP Biosynthesis

To make dNTPs, the
conversion from
ribonucleoside
diphosphate (NDP) to
deoxyribonucleoside
diphosphate (dNDP) must
occur
Ribonucleotide Reductase (rNDP reductase)
(Images courtesy of Biochemistry, 3rd ed.)
Ribonucleotide Reductase



R1
R2

Discovered by Peter
Reichard
The single enzyme that
reduces NDP to dNDP
(Jordan & Reichard, 1998)
Regulates the amount of
dNTP produced in a cell
Hetero-tetramer shape
• R1 & R2 subunits
dNTP Pools (cont.)


Increasing rNDP
reductase activity in a
cell can lead to
increased dNTP pool
sizes
Adding hydroxyurea is
a convenient way to
enlarge pool sizes
Ball-Stick Model of Hydroxyurea
Hydroxyurea



Also known as hydroxy
carbamide
Commonly used to treat
Hydroxyurea
certain types of cancer
(leukemia), Sickle Cell
--------------------------------------------Anemia and HIV & AIDS
When added to rNDP
reductase
• Destroys the free
radical portion of the
enzyme, inhibiting its
function
rNDP reductase
R2 subunit
(Images courtesy of Biochemistry, 3rd ed. & www.cancerquest.org)
Hydroxyurea-resistant Cells

Hydroxyurea-resistant cell lines carry elevated levels of
ribonucleotide reductase
Regular Cell
Hydroxyurearesistant Cell 

Has not been established in mammalian cells whether or
not over-expression of the enzyme leads to increased
dNTP pools
Question

Do hydroxyurea-resistant mammalian
cells exhibit enlarged dNTP pools?
• If so, do these cells also have elevated
spontaneous mutation rates?
Methods



Culture V79 hamster lung cells so that they
become resistant to hydroxyurea
Extract dNTPs
Analyze dNTP pool sizes through assays
Cell Cultures


Culture hydroxyurea-resistant V79 cells
Two methods
• Hydroxyurea-resistant cells from liquid nitrogen stock in
lab
• Treat normal V79 cells with hydroxyurea and isolate
resistant cells
Liquid nitrogen freezer
dNTP Extraction

dNTPs are separated from the cell
• Cells washed with 1X PBS and followed by treatment
by methanol
• Boiled, centrifuged, and speed-vacuumed
Speed vaccuum
dNTP Pool Assays


A method for measuring dNTP pool sizes
Uses synthetic DNA polymers, DNA
polymerase, and an excess of radiolabeled dNTPs
dNTP Pool Assays (cont.)

Watson-Crick base pair:
dATP = dTTP
dGTP = dCTP
A=T

G=C
Example
• To measure dATP (analyzed with 3H dTTP)

Template 
A A

Base pair 
T* T* T* A T* T* T* A…
• Radio-labeled dNTP (3H
3H
dTTP and
dATP) are
counted in a scintillation
counter
A
T A A
A
T…
• This tells us how much
regular dNTP a sample
contains
dNTP Pool Assays (cont.)
Cell dNTP compared to a standard
curve
Example dATP Standard Curve
y = 5800.9x + 390
7000
6000
5000
cpm

4000
3000
2000
1000
0
0
0.5
1
pmol
1.5
Research Timeline

Cell culture complications
• Hydroxyurea-resistant V79 cells from liquid
nitrogen stock failed to grow on culture plates

Used smaller 6-well plates
Instead of

Added extra fetal bovine serum
• Regular V79 cells treated with hydroxyurea

Concentration of added hydroxyurea steadily increased
Research Timeline (cont.)
Meanwhile…

Other projects
• dNTP pool assay protocol
• Mammalian cells & bacteria cells

Treated V79 cells with thymidine
• Effects on dCTP and dTTP pool
Research Timeline (cont.)
Thymidine results
• Tested varying concentrations of thymidine
• Results:
Thymidine: dCTP
uM Thy vs. pmol/10^6 cells
Thymidine: dTTP
uM Thy vs. pmol/10^6 cells
140
Average pmol/ 10^6 cells
Average pmol/10^6 cells

120
100
80
60
40
20
0
0
10
20
30
40
60
uM Thymidine
80
100 500
450
400
350
300
250
200
150
100
50
0
0
10
20
30
40
60
uM Thymidine
80
100
500
Research Timeline (cont.)


Hydroxyurea-resistant V79 project
From liquid nitrogen storage: extracted
4 sets of V79 cells
• 1 set of regular V79 cells
• 1 set of .35 mM HU-res V79 cells
• 2 sets of 1.3 mM HU-res V79 cells
Data

dTTP

dCTP
dCTP
70
300
60
250
pmol/ 10^6 cells
pmol/ 10^6 cells
dTTP
50
40
30
20
10
150
100
50
0
0
V79
0.35 HU-res
1.3 HU-res Set
#1
V79
1.3 HU-res Set
#2
dATP

pmol/10^6 cells
0.35 HU-res
1.3 HU-res Set
#1
1.3 HU-res Set
#2
dGTP
80
70
60
50
40
30
20
10
0
V79
0.35 HU-res
dGTP
dATP
pmol/ 10^6 cells

200
1.3 HU-res Set
#1
1.3 HU-res Set
#2
18
16
14
12
10
8
6
4
2
0
V79
0.35 HU-res
1.3 HU-res Set
#1
1.3 HU-res Set
#2
Data (cont.)
Hydroxyurea-resistant cell dNTP: Comparison Graph
Percent Difference
(pmol/10^6 cells)
500%
400%
V79
0.35 HU-res
300%
1.3 HU-res Set #1
200%
1.3 HU-res Set #2
100%
0%
dTTP
dATP
dCTP
dNTPs
dGTP
Research Timeline (cont.)

Regular V79 cells
• One cell line treated with increasing levels of
hydroxyurea
• Still in culture
• No data
Summary



Three hydroxyurea-resistant cell lines were
grown and analyzed
One new hydroxyurea-resistant cell lines was
developed but has not yet been analyzed
Results of dNTP pool analyses do not support
the expectation of dNTP accumulation in the
mutant cells
Further Research


Develop and test a model to explain the dNTP
pool changes seen in the hydroxyurearesistant mutants
Determine whether any of the hydroxyurearesistant mutants shows increased
spontaneous mutagenesis
Acknowledgements







Howard Hughes Medical Institute (HHMI)
Undergraduate Research Innovation Scholarship
Creativity (URISC)
Christopher Mathews
Linda Wheeler
Kevin Ahern
Indira Rajagopal
Department of Biochemistry & Biophysics