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Mutagenesis by Expanded DNA Mutagenesis by Expanded DNA Precursor Pools of Mammalian Precursor Pools of Mammalian
CellsCells
Howard Hughes Medical Institute (HHMI)Howard Hughes Medical Institute (HHMI)Summer 2003Summer 2003
Nancy Jade LeeNancy Jade LeeDr. Christopher K. Mathews’ LaboratoryDr. Christopher K. Mathews’ Laboratory
Department of Biochemistry & BiophysicsDepartment of Biochemistry & BiophysicsOregon State UniversityOregon State University
Lab ObjectivesLab Objectives
To understand mechanisms of mutagenesis caused by To understand mechanisms of mutagenesis caused by
perturbations of nucleotide metabolismperturbations of nucleotide metabolism
To understand the source and regulation of DNA To understand the source and regulation of DNA
precursor pools in mitochondria and eukaryotic cellsprecursor pools in mitochondria and eukaryotic cells
To understand how enzymes of DNA precursor To understand how enzymes of DNA precursor
synthesis interact within cells to facilitate the flow of synthesis interact within cells to facilitate the flow of
nucleotides into DNAnucleotides into DNA
My ObjectivesMy Objectives
To examine DNA precursors (Deoxyribonucleotides or To examine DNA precursors (Deoxyribonucleotides or
dNTPs) and their ability to stimulate mutagenesis dNTPs) and their ability to stimulate mutagenesis
To understand the relationship between intracellular To understand the relationship between intracellular
DNA precursor concentration and mutagenesisDNA precursor concentration and mutagenesis
To study the effects of hydroxyurea on ribonucleotide To study the effects of hydroxyurea on ribonucleotide
reductase (rNDP reductase) in mammalian cells in reductase (rNDP reductase) in mammalian cells in
order to understand the role of rNDP reductaseorder to understand the role of rNDP reductase
ImportanceImportance
Cancer results from mutations that accumulate Cancer results from mutations that accumulate in pre-cancerous cells (Loeb, 1998)in pre-cancerous cells (Loeb, 1998)• Tumor cells in culture tend to have higher Tumor cells in culture tend to have higher
levels of dNTPs than non-tumor cells levels of dNTPs than non-tumor cells (Martomo & Mathews, 2002)(Martomo & Mathews, 2002)
BackgroundBackground
Deoxyribonucleotides Deoxyribonucleotides (dNTPs) are necessary (dNTPs) are necessary for biosynthesis of for biosynthesis of DNADNA
dCTPdTTPdATP dGTP
*The amount of each dNTP contained in a cell is referred to as a “pool”
(Images courtesy of www.fermentas.com)
dNTP PoolsdNTP Pools Regular cells have balanced pool sizesRegular cells have balanced pool sizes Unbalanced dNTP pools can stimulate mutagenesis (Kunz et Unbalanced dNTP pools can stimulate mutagenesis (Kunz et
al, 1994)al, 1994) ExampleExample
In In E. coliE. coli cells cells balanced increasesbalanced increases in dNTP pools also in dNTP pools also stimulates mutagenesis stimulates mutagenesis
(Wheeler & Rajagopal, 2002)(Wheeler & Rajagopal, 2002)
Meanwhile…
dATP pool dTTP pool dGTP pool dCTP pool = more mutations
dATP pool dTTP pool dGTP pool dCTP pool = more mutations
dNTP BiosynthesisdNTP Biosynthesis To make dNTPs, the To make dNTPs, the
conversion from conversion from ribonucleoside ribonucleoside diphosphate (NDP) to diphosphate (NDP) to deoxyribonucleoside deoxyribonucleoside diphosphate (dNDP) must diphosphate (dNDP) must occuroccur
Ribonucleotide Reductase (rNDP reductase)(Images courtesy of Biochemistry, 3rd ed.)
Ribonucleotide ReductaseRibonucleotide Reductase Discovered by Peter Discovered by Peter
ReichardReichard The single enzyme that The single enzyme that
reduces NDP to dNDP reduces NDP to dNDP (Jordan & Reichard, 1998)(Jordan & Reichard, 1998)
Regulates the amount of Regulates the amount of dNTP produced in a celldNTP produced in a cell
Hetero-tetramer shapeHetero-tetramer shape• R1 & R2 subunitsR1 & R2 subunits
R1
R2
dNTP Pools (cont.)dNTP Pools (cont.)
Increasing rNDP Increasing rNDP reductase activity in a reductase activity in a cell can lead to cell can lead to increased dNTP pool increased dNTP pool sizessizes
Adding hydroxyurea is Adding hydroxyurea is a convenient way to a convenient way to enlarge pool sizesenlarge pool sizes
Ball-Stick Model of Hydroxyurea
rNDP reductase
HydroxyureaHydroxyurea Also known as hydroxy Also known as hydroxy
carbamidecarbamide Commonly used to treat Commonly used to treat
certain types of cancer certain types of cancer (leukemia), Sickle Cell (leukemia), Sickle Cell Anemia and HIV & AIDSAnemia and HIV & AIDS
When added to rNDP When added to rNDP reductasereductase• Destroys the free Destroys the free
radical portion of the radical portion of the enzyme, inhibiting its enzyme, inhibiting its functionfunction
---------------------------------------------
Hydroxyurea
R2 subunit(Images courtesy of Biochemistry, 3rd ed. & www.cancerquest.org)
Hydroxyurea-resistant CellsHydroxyurea-resistant Cells Hydroxyurea-resistant cell lines carry elevated levels of Hydroxyurea-resistant cell lines carry elevated levels of
ribonucleotide reductaseribonucleotide reductase
Has not been established in mammalian cells whether or Has not been established in mammalian cells whether or not over-expression of the enzyme leads to increased not over-expression of the enzyme leads to increased dNTP poolsdNTP pools
Regular Cell
Hydroxyurea-resistant Cell
QuestionQuestion
Do hydroxyurea-resistant mammalian Do hydroxyurea-resistant mammalian cells exhibit enlarged dNTP pools?cells exhibit enlarged dNTP pools?• If so, do these cells also have elevated If so, do these cells also have elevated
spontaneous mutation rates?spontaneous mutation rates?
MethodsMethods
Culture V79 hamster lung cells so that they Culture V79 hamster lung cells so that they become resistant to hydroxyureabecome resistant to hydroxyurea
Extract dNTPsExtract dNTPs Analyze dNTP pool sizes through assaysAnalyze dNTP pool sizes through assays
Cell CulturesCell Cultures Culture hydroxyurea-resistant V79 cellsCulture hydroxyurea-resistant V79 cells Two methodsTwo methods
• Hydroxyurea-resistant cells from liquid nitrogen stock in Hydroxyurea-resistant cells from liquid nitrogen stock in lablab
• Treat normal V79 cells with hydroxyurea and isolate Treat normal V79 cells with hydroxyurea and isolate resistant cellsresistant cells
Liquid nitrogen freezer
dNTP ExtractiondNTP Extraction
dNTPs are separated from the celldNTPs are separated from the cell• Cells washed with 1X PBS and followed by treatment Cells washed with 1X PBS and followed by treatment
by methanolby methanol• Boiled, centrifuged, and speed-vacuumedBoiled, centrifuged, and speed-vacuumed
Speed vaccuum
dNTP Pool AssaysdNTP Pool Assays
A method for measuring dNTP pool sizesA method for measuring dNTP pool sizes Uses synthetic DNA polymers, DNA Uses synthetic DNA polymers, DNA
polymerase, and an excess of radio-polymerase, and an excess of radio-labeled dNTPslabeled dNTPs
dNTP Pool Assays (cont.)dNTP Pool Assays (cont.) Watson-Crick base pair:Watson-Crick base pair:
dATP = dTTP dGTP = dCTPdATP = dTTP dGTP = dCTP A = TA = T G = C G = C
ExampleExample
• To measure dATP To measure dATP (analyzed with (analyzed with 33H dTTP)H dTTP) Template Template A A A T A A A T… A A A T A A A T…
• Radio-labeled dNTP (Radio-labeled dNTP (33H H dTTP and dTTP and 33H dATP) are H dATP) are counted in a scintillation counted in a scintillation countercounter
• This tells us how much This tells us how much regular dNTP a sample regular dNTP a sample containscontains
Base pair Base pair T* T* T*T* T* T* A A T* T* T*T* T* T* A… A…
dNTP Pool Assays (cont.)dNTP Pool Assays (cont.)
Cell dNTP compared to a standard Cell dNTP compared to a standard curvecurve
dATP Standard Curve
y = 5800.9x + 390
0
1000
2000
3000
4000
5000
6000
7000
0 0.5 1 1.5
pmol
cpm
Example -
Research TimelineResearch Timeline
Cell culture complicationsCell culture complications• Hydroxyurea-resistant V79 cells from liquid Hydroxyurea-resistant V79 cells from liquid
nitrogen stock failed to grow on culture platesnitrogen stock failed to grow on culture plates Used smaller 6-well plates Used smaller 6-well plates
Added extra fetal bovine serumAdded extra fetal bovine serum
• Regular V79 cells treated with hydroxyureaRegular V79 cells treated with hydroxyurea Concentration of added hydroxyurea steadily increasedConcentration of added hydroxyurea steadily increased
Instead of
Research Timeline (cont.)Research Timeline (cont.)
Other projectsOther projects• dNTP pool assay protocoldNTP pool assay protocol• Mammalian cells & bacteria cellsMammalian cells & bacteria cells
Treated V79 cells with thymidineTreated V79 cells with thymidine• Effects on dCTP and dTTP poolEffects on dCTP and dTTP pool
Meanwhile…
Research Timeline (cont.)Research Timeline (cont.)
Thymidine resultsThymidine results• Tested varying concentrations of thymidine Tested varying concentrations of thymidine • Results:Results:
Thymidine: dTTPuM Thy vs. pmol/10^6 cells
0
50
100
150
200
250
300
350
400
450
0 10 20 30 40 60 80 100 500
uM Thymidine
Average pmol/ 10^6 cells
Thymidine: dCTPuM Thy vs. pmol/10^6 cells
0
20
40
60
80
100
120
140
0 10 20 30 40 60 80 100 500
uM Thymidine
Average pmol/10^6 cells
Research Timeline (cont.)Research Timeline (cont.)
Hydroxyurea-resistant V79 projectHydroxyurea-resistant V79 project From liquid nitrogen storage: extracted 4 From liquid nitrogen storage: extracted 4
sets of V79 cellssets 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
DataData dTTPdTTP
dATPdATP
dCTP
0
50
100
150
200
250
300
V79 0.35 HU-res 1.3 HU-res Set#1
1.3 HU-res Set#2
pmol/ 10^6 cells
dTTP
0
10
20
30
40
50
60
70
V79 0.35 HU-res 1.3 HU-res Set#1
1.3 HU-res Set#2
pmol/ 10^6 cells
dATP
01020
30405060
7080
V79 0.35 HU-res 1.3 HU-res Set#1
1.3 HU-res Set#2
pmol/ 10^6 cells
dGTP
02468
1012141618
V79 0.35 HU-res 1.3 HU-res Set#1
1.3 HU-res Set#2
pmol/10^6 cells
dCTPdCTP
dGTPdGTP
Data (cont.)Data (cont.)
Hydroxyurea-resistant cell dNTP: Comparison Graph
0%
100%
200%
300%
400%
500%
dTTP dATP dCTP dGTP
dNTPs
Percent Difference(pmol/10^6 cells)
V79
0.35 HU-res
1.3 HU-res Set #1
1.3 HU-res Set #2
Research Timeline (cont.)Research Timeline (cont.)
Regular V79 cellsRegular V79 cells• One cell line treated with increasing levels of One cell line treated with increasing levels of
hydroxyureahydroxyurea• Still in cultureStill in culture• No dataNo data
SummarySummary
Three hydroxyurea-resistant cell lines were Three hydroxyurea-resistant cell lines were grown and analyzedgrown and analyzed
One new hydroxyurea-resistant cell lines was One new hydroxyurea-resistant cell lines was developed but has not yet been analyzeddeveloped but has not yet been analyzed
Results of dNTP pool analyses do not support Results of dNTP pool analyses do not support the expectation of dNTP accumulation in the the expectation of dNTP accumulation in the mutant cellsmutant cells
Further ResearchFurther Research
Develop and test a model to explain the dNTP Develop and test a model to explain the dNTP pool changes seen in the hydroxyurea-pool changes seen in the hydroxyurea-resistant mutantsresistant mutants
Determine whether any of the hydroxyurea-Determine whether any of the hydroxyurea-resistant mutants shows increased resistant mutants shows increased spontaneous mutagenesisspontaneous mutagenesis
AcknowledgementsAcknowledgements
Howard Hughes Medical Institute (HHMI)Howard Hughes Medical Institute (HHMI) Undergraduate Research Innovation Scholarship Undergraduate Research Innovation Scholarship
Creativity (URISC)Creativity (URISC) Christopher MathewsChristopher Mathews Linda WheelerLinda Wheeler Kevin AhernKevin Ahern Indira RajagopalIndira Rajagopal Department of Biochemistry & BiophysicsDepartment of Biochemistry & Biophysics