1. 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

2. 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
Dr. Mathews’ laboratory deals with abnormalities in the metabolism of DNA precursors and how they instigate errors during DNA replication, eventually leading to mutations. Therefore, instead of studying a certain mutation, like many other research labs, Dr. Mathews’ lab focuses on conditions that lead to mutations and their metabolic consequences.

3. 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
Main Goal: To determine if increased dNTP pool sizes produced by hydroxyurea-resistant mammalian cells lead to more mutations

4. 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)
The study of dNTPs and ribonucleotide reductase is important because it deals with cancer research
Loeb  spontaneous mutation rate has to increase in cells that are destined to be cancer cells
It has been found that tumor cells contain higher levels of dNTPs in comparison to non-tumor cells
The purpose of this project is to test whether or not increased levels of dNTPs is a direct cause of cancerous mutations

5. Background
Deoxyribonucleotides (dNTPs) are necessary for biosynthesis of DNA
dATP
dTTP
dCTP
dGTP
For future reference, *…..
*The amount of each dNTP contained in a cell is referred to as a “pool”
(Images courtesy of

6. Does this also occur in mammalian cells?
dNTP Pools
Regular cells have balanced pool sizes
Unbalanced dNTP pools can stimulate mutagenesis (Kunz et al, 1994)
Example
dCTP pool
dATP pool
dTTP pool
dGTP pool
= more mutations
Meanwhile…
In E. coli cells balanced increases in dNTP pools also stimulates mutagenesis
(Wheeler & Rajagopal, 2002)
Regular cells have balanced pool sizes according to cell needs
Unbalanced dNTP pools can stimulate mutatagenesis: unbalanced relative to its normal size, the needs of the cell
One mechanism is: a nucleotide in excess can compete to form a non-Watson-Crick base pair
Meanwhile, An unexpected discovery….
Does this also occur in mammalian cells?
dATP pool
dTTP pool
dGTP pool
dCTP pool
= more mutations

7. dNTP Biosynthesis
To make dNTPs, the conversion from ribonucleoside diphosphate (NDP) to deoxyribonucleoside diphosphate (dNDP) must occur
Ribonucleotide Reductase (rNDP reductase)
Ribonucleotide reductase is responsible for this conversion
(Images courtesy of Biochemistry, 3rd ed.)

8. Ribonucleotide Reductase
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
Although R1 contains the activity site, R2 is important in maintaining the activity of R1 R1 R2

9. 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
Since ribonucleotide reductase is responsible for maintaining the amount of dNTP produced by a cell, increasing its activity can lead to increased dNTP pool sizes

10. Hydroxyurea When added to rNDP reductase
Also known as hydroxy carbamide
Commonly used to treat 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
Hydroxyurea
rNDP reductase
R2 subunit
(Images courtesy of Biochemistry, 3rd ed. &

11. Hydroxyurea-resistant Cells
Hydroxyurea-resistant cell lines carry elevated levels of ribonucleotide reductase
Has not been established in mammalian cells whether or not over-expression of the enzyme leads to increased dNTP pools
Regular Cell
Hydroxyurea-
resistant Cell 
HOWEVER,
Elevated synthesis of R2 subunit  in mammalian cells this is normally limiting

12. Question
Do hydroxyurea-resistant mammalian cells exhibit enlarged dNTP pools?
If so, do these cells also have elevated spontaneous mutation rates?
Do spontaneous increases occur?

13. Methods
Culture V79 hamster lung cells so that they become resistant to hydroxyurea
Extract dNTPs
Analyze dNTP pool sizes through assays

14. 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
Dulbecco’s minimal essential median
Cell lines generated in lab by a graduate student vs. generating own
Mutant cells isolated and grown on new plates
Liquid nitrogen freezer

15. 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
1X PBS- phosphate buffer saline  washes off all media and serum
Methanol  makes the cell wall more permeable so the dNTPs can escape
Boiled  to denature any protein that may have been soluble in MeOH

16. dNTP Pool Assays A method for measuring dNTP pool sizes
Uses synthetic DNA polymers, DNA polymerase, and an excess of radio-labeled dNTPs

17. 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 A T A A A T…
dATP from cell extraction sample and dTTP radio-labeled
Synthetic template
Base pair from solution
Radio-labeled dNTP denoted with an asterick
Polymerized product that 3H is incorporated- spotted on DEA paper
Ion exhcnage medium bind polymerized nucleotides
Base pair  T* T* T* A T* T* T* A…
Radio-labeled dNTP (3H dTTP and 3H dATP) are counted in a scintillation counter
This tells us how much regular dNTP a sample contains

18. dNTP Pool Assays (cont.)
Cell dNTP compared to a standard curve
Example -
Data from dNTP extracted from V79 cells compared to a standard curve  for every pmol of dNTP in a sample you should get a corresponding cpm.

19. Research Timeline Cell culture complications
Hydroxyurea-resistant V79 cells from liquid nitrogen stock failed to grow on culture plates
Used smaller 6-well plates
Added extra fetal bovine serum
Regular V79 cells treated with hydroxyurea
Concentration of added hydroxyurea steadily increased
Instead of
Fetal bovine serum  contains nutrients that the cells need to multiply, a boost
Beginning of summer
At the end of this slide mention how it took a while to grow the cells

20. 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
Therefore, meanwhile, while waiting for cells to grow
Worked on other projects which followed the exact same dNTP pool assay protocol as used in hydroxyurea project
Middle of summer

21. Research Timeline (cont.)
Thymidine results
Tested varying concentrations of thymidine
Results:
The greater the concentration of thymidine the smaller dCTP pool and the larger the dTTP pool
Middle of summer

22. 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
End of Summer
Determine the pool sizes of these cells
Explain difference between .35 and 1.3 mM

23. Data
dTTP
dATP
dCTP
dGTP

24. Data (cont.)

25. Research Timeline (cont.)
Regular V79 cells
One cell line treated with increasing levels of hydroxyurea
Still in culture
No data

26. 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

27. Further Research
Develop and test a model to explain the dNTP pool changes seen in the hydroxyurea-resistant mutants
Determine whether any of the hydroxyurea-resistant mutants shows increased spontaneous mutagenesis

28. Acknowledgements Howard Hughes Medical Institute (HHMI)
Undergraduate Research Innovation Scholarship Creativity (URISC)
Christopher Mathews
Linda Wheeler
Kevin Ahern
Indira Rajagopal
Department of Biochemistry & Biophysics