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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’

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Presentation on theme: "Mutagenesis by Expanded DNA Precursor Pools of Mammalian Cells Howard Hughes Medical Institute (HHMI) Summer 2003 Nancy Jade Lee Dr. Christopher K. Mathews’"— Presentation transcript:

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 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 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 To understand how enzymes of DNA precursor synthesis interact within cells to facilitate the flow of nucleotides into DNA

3 My Objectives To examine DNA precursors (Deoxyribonucleotides or dNTPs) and their ability to stimulate mutagenesis 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 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 To study the effects of hydroxyurea on ribonucleotide reductase (rNDP reductase) in mammalian cells in order to understand the role of rNDP reductase

4 Importance Cancer results from mutations that accumulate in pre-cancerous cells (Loeb, 1998) 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)Tumor cells in culture tend to have higher levels of dNTPs than non-tumor cells (Martomo & Mathews, 2002)

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

6 dNTP Pools Regular cells have balanced pool sizes Regular cells have balanced pool sizes Unbalanced dNTP pools can stimulate mutagenesis (Kunz et al, 1994) Unbalanced dNTP pools can stimulate mutagenesis (Kunz et al, 1994) Example Example In E. coli cells balanced increases in dNTP pools also stimulates mutagenesis (Wheeler & Rajagopal, 2002) Meanwhile… dATP pooldTTP pooldGTP pool dCTP pool = more mutations dATP pooldTTP pooldGTP pooldCTP pool = more mutations

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

8 Ribonucleotide Reductase Discovered by Peter Reichard Discovered by Peter Reichard The single enzyme that reduces NDP to dNDP (Jordan & Reichard, 1998) The single enzyme that reduces NDP to dNDP (Jordan & Reichard, 1998) Regulates the amount of dNTP produced in a cell Regulates the amount of dNTP produced in a cell Hetero-tetramer shape Hetero-tetramer shape R1 & R2 subunits R1 R2

9 dNTP Pools (cont.) Increasing rNDP reductase activity in a cell can lead to increased dNTP pool sizes Increasing rNDP reductase activity in a cell can lead to increased dNTP pool sizes Adding hydroxyurea is a convenient way to enlarge pool sizes Adding hydroxyurea is a convenient way to enlarge pool sizes Ball-Stick Model of Hydroxyurea

10 rNDP reductase Hydroxyurea Also known as hydroxy carbamide Also known as hydroxy carbamide Commonly used to treat certain types of cancer (leukemia), Sickle Cell Anemia and HIV & AIDS Commonly used to treat certain types of cancer (leukemia), Sickle Cell Anemia and HIV & AIDS When added to rNDP reductase When added to rNDP reductase Destroys the free radical portion of the enzyme, inhibiting its functionDestroys the free radical portion of the enzyme, inhibiting its function --------------------------------------------- Hydroxyurea R2 subunit (Images courtesy of Biochemistry, 3 rd ed. & www.cancerquest.org)

11 Hydroxyurea-resistant Cells Hydroxyurea-resistant cell lines carry elevated levels of ribonucleotide reductase 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 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 

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

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

14 Cell Cultures Culture hydroxyurea-resistant V79 cells Culture hydroxyurea-resistant V79 cells Two methods Two methods Hydroxyurea-resistant cells from liquid nitrogen stock in labHydroxyurea-resistant cells from liquid nitrogen stock in lab Treat normal V79 cells with hydroxyurea and isolate resistant cellsTreat normal V79 cells with hydroxyurea and isolate resistant cells Liquid nitrogen freezer

15 dNTP Extraction dNTPs are separated from the cell dNTPs are separated from the cell Cells washed with 1X PBS and followed by treatment by methanolCells washed with 1X PBS and followed by treatment by methanol Boiled, centrifuged, and speed-vacuumedBoiled, centrifuged, and speed-vacuumed Speed vaccuum

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

17 dNTP Pool Assays (cont.) Watson-Crick base pair: Watson-Crick base pair: dATP = dTTP dGTP = dCTP A = T G = C A = T G = C Example Example To measure dATP (analyzed with 3 H dTTP)To measure dATP (analyzed with 3 H dTTP) Template  A A A T A A A T… Template  A A A T A A A T… Radio-labeled dNTP ( 3 H dTTP and 3 H dATP) are counted in a scintillation counter Radio-labeled dNTP ( 3 H dTTP and 3 H dATP) are counted in a scintillation counter This tells us how much regular dNTP a sample contains This tells us how much regular dNTP a sample contains Base pair  T* T* T* A T* T* T* A… Base pair  T* T* T* A T* T* T* A…

18 dNTP Pool Assays (cont.) Cell dNTP compared to a standard curve Cell dNTP compared to a standard curve Example -

19 Research Timeline Cell culture complications Cell culture complications Hydroxyurea-resistant V79 cells from liquid nitrogen stock failed to grow on culture platesHydroxyurea-resistant V79 cells from liquid nitrogen stock failed to grow on culture plates Used smaller 6-well plates Used smaller 6-well plates Added extra fetal bovine serum Added extra fetal bovine serum Regular V79 cells treated with hydroxyureaRegular V79 cells treated with hydroxyurea Concentration of added hydroxyurea steadily increased Concentration of added hydroxyurea steadily increased Instead of

20 Research Timeline (cont.) Other projects Other projects dNTP pool assay protocoldNTP pool assay protocol Mammalian cells & bacteria cellsMammalian cells & bacteria cells Treated V79 cells with thymidine Treated V79 cells with thymidine Effects on dCTP and dTTP poolEffects on dCTP and dTTP pool Meanwhile…

21 Research Timeline (cont.) Thymidine results Thymidine results Tested varying concentrations of thymidineTested varying concentrations of thymidine Results:Results:

22 Research Timeline (cont.) Hydroxyurea-resistant V79 project Hydroxyurea-resistant V79 project From liquid nitrogen storage: extracted 4 sets of V79 cells From liquid nitrogen storage: extracted 4 sets of V79 cells 1 set of regular V79 cells1 set of regular V79 cells 1 set of.35 mM HU-res V79 cells1 set of.35 mM HU-res V79 cells 2 sets of 1.3 mM HU-res V79 cells2 sets of 1.3 mM HU-res V79 cells

23 Data dTTP dTTP dATP dATP dCTP dCTP dGTP dGTP

24 Data (cont.)

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

26 Summary Three hydroxyurea-resistant cell lines were grown and analyzed Three hydroxyurea-resistant cell lines were grown and analyzed One new hydroxyurea-resistant cell lines was developed but has not yet been 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 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 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 Determine whether any of the hydroxyurea- resistant mutants shows increased spontaneous mutagenesis

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


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