Title: Mutagenesis by Expanded DNA Precursor Pools of Mammalian Cells
1Mutagenesis 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
2Lab 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
3My 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
4Importance
- 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)
5Background
- Deoxyribonucleotides (dNTPs) are necessary for
biosynthesis of DNA
The amount of each dNTP contained in a cell is
referred to as a pool
(Images courtesy of www.fermentas.com)
6dNTP 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)
7dNTP Biosynthesis
- To make dNTPs, the conversion from ribonucleoside
diphosphate (NDP) to deoxyribonucleoside
diphosphate (dNDP) must occur
(Images courtesy of Biochemistry, 3rd ed.)
8Ribonucleotide 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
9dNTP 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
10Hydroxyurea
- 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
---------------------------------------------
(Images courtesy of Biochemistry, 3rd ed.
www.cancerquest.org)
11Hydroxyurea-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
12Question
- Do hydroxyurea-resistant mammalian cells exhibit
enlarged dNTP pools? - If so, do these cells also have elevated
spontaneous mutation rates?
13Methods
- Culture V79 hamster lung cells so that they
become resistant to hydroxyurea - Extract dNTPs
- Analyze dNTP pool sizes through assays
14Cell 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
15dNTP Extraction
- dNTPs are separated from the cell
- Cells washed with 1X PBS and followed by
treatment by methanol - Boiled, centrifuged, and speed-vacuumed
16dNTP Pool Assays
- A method for measuring dNTP pool sizes
- Uses synthetic DNA polymers, DNA polymerase, and
an excess of radio-labeled dNTPs
17dNTP 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
- Base pair ? T T T A T T T A
18dNTP Pool Assays (cont.)
- Cell dNTP compared to a standard curve
19Research 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
20Research Timeline (cont.)
Meanwhile
- Other projects
- dNTP pool assay protocol
- Mammalian cells bacteria cells
- Treated V79 cells with thymidine
- Effects on dCTP and dTTP pool
21Research Timeline (cont.)
- Thymidine results
- Tested varying concentrations of thymidine
- Results
22Research 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
23Data
24Data (cont.)
25Research Timeline (cont.)
- Regular V79 cells
- One cell line treated with increasing levels of
hydroxyurea - Still in culture
- No data
26Summary
- 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
27Further 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
28Acknowledgements
- Howard Hughes Medical Institute (HHMI)
- Undergraduate Research Innovation Scholarship
Creativity (URISC) - Christopher Mathews
- Linda Wheeler
- Kevin Ahern
- Indira Rajagopal
- Department of Biochemistry Biophysics