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Mutagenesis by Expanded DNA Precursor Pools of Mammalian Cells

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Presentation on theme: "Mutagenesis by Expanded DNA Precursor Pools of Mammalian Cells"— 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 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

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