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Mutagenesis by Expanded DNA Precursor Pools of Mammalian Cells

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.

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Mutagenesis by Expanded DNA Precursor Pools of Mammalian Cells

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  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

  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

  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)

  5. dATP dTTP dCTP dGTP 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)

  6. dATP pool dTTP pool dGTP pool dCTP pool = more mutations 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)

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

  8. R1 R2 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

  9. Ball-Stick Model of Hydroxyurea 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

  10. rNDP reductase R2 subunit 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)

  11. Regular Cell Hydroxyurea- resistant Cell  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

  12. Question • Do hydroxyurea-resistant mammalian cells exhibit enlarged dNTP pools? • 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 • 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 Liquid nitrogen freezer

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

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

  17. Radio-labeled dNTP (3H dTTP and 3H dATP) are counted in a scintillation counter • This tells us how much regular dNTP a sample contains 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…

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

  19. Instead of 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

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

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

  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

  23. dTTP dATP dCTP dGTP Data

  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

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