Transcript Mutagenesis by Expanded DNA Precursor Pools of Mammalian Cells Howard Hughes Medical Institute (HHMI) Summer 2003 Nancy Jade Lee Dr.

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
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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
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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

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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

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R1
R2
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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.)
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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
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
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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
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Culture V79 hamster lung cells so that they
become resistant to hydroxyurea
Extract dNTPs
Analyze dNTP pool sizes through assays
Cell Cultures

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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
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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

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A method for measuring dNTP pool sizes
Uses synthetic DNA polymers, DNA
polymerase, and an excess of radiolabeled dNTPs
dNTP Pool Assays (cont.)
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Watson-Crick base pair:
dATP = dTTP
dGTP = dCTP
A=T
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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
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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
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Concentration of added hydroxyurea steadily increased
Research Timeline (cont.)
Meanwhile…

Other projects
• dNTP pool assay protocol
• Mammalian cells & bacteria cells
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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.)
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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
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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
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Howard Hughes Medical Institute (HHMI)
Undergraduate Research Innovation Scholarship
Creativity (URISC)
Christopher Mathews
Linda Wheeler
Kevin Ahern
Indira Rajagopal
Department of Biochemistry & Biophysics