Mutagenesis by Expanded DNA Precursor Pools of Mammalian Cells Howard Hughes Medical Institute...

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…


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


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


1.3 HU-res Set#2

pmol/ 10^6 cells

dATP

01020

30405060

7080


1.3 HU-res Set#2

pmol/ 10^6 cells

dGTP

02468

1012141618


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


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

Mutagenesis by Expanded DNA Precursor Pools of Mammalian Cells Howard Hughes Medical Institute...

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