Mutagenesis by Expanded DNA Precursor Pools of Mammalian Cells

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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

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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

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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

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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)

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Background

• 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)
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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)
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dNTP Biosynthesis

• To make dNTPs, the conversion from ribonucleoside
  diphosphate (NDP) to deoxyribonucleoside
  diphosphate (dNDP) must occur

(Images courtesy of Biochemistry, 3rd ed.)
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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

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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

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Hydroxyurea

• 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)
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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

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Question

• Do hydroxyurea-resistant mammalian cells exhibit
  enlarged dNTP pools?
• If so, do these cells also have elevated
  spontaneous mutation rates?

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Methods

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

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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
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dNTP Extraction

• dNTPs are separated from the cell
• Cells washed with 1X PBS and followed by
  treatment by methanol
• Boiled, centrifuged, and speed-vacuumed

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dNTP Pool Assays

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

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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

• Base pair ? T T T A T T T A

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dNTP Pool Assays (cont.)

• Cell dNTP compared to a standard curve

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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

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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

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Research Timeline (cont.)

• Thymidine results
• Tested varying concentrations of thymidine
• Results

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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

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Data

• dTTP
• dATP

• dCTP
• dGTP

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Data (cont.)
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Research Timeline (cont.)

• Regular V79 cells
• One cell line treated with increasing levels of
  hydroxyurea
• Still in culture
• No data

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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

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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

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Acknowledgements

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