Investigation of the Investigation of the Premutagenic Lesion 8-oxo Premutagenic Lesion 8-oxo

dGTP and its Repair dGTP and its Repair Mechanism Mut TMechanism Mut T

Howard Hughes Medical Institute (HHMI)Howard Hughes Medical Institute (HHMI)

Daniel BaiDaniel BaiDr. Christopher K. MathewsDr. Christopher K. Mathews

Biochemistry and BiophysicsBiochemistry and BiophysicsOregon State UniversityOregon State University

Objectives of the Mathews LabObjectives of the Mathews Lab

Nucleic Acid Enzymology (DNA replication, Nucleic Acid Enzymology (DNA replication, repair and mutation)repair and mutation)

Understanding regulation of DNA synthesis in Understanding regulation of DNA synthesis in mitochondriamitochondria

Investigate the enzymes that regulate Investigate the enzymes that regulate nucleotide incorporation into DNAnucleotide incorporation into DNA

Project IntroductionProject Introduction

Mitochondria are powerhouse of eukaryotic cellsMitochondria are powerhouse of eukaryotic cells Mitochondrial DNA mutations are linked to Mitochondrial DNA mutations are linked to

neurodegenerative disorders, cardiomyopathies, neurodegenerative disorders, cardiomyopathies, and cancerand cancer

Rate of mitochondria mutations are two orders Rate of mitochondria mutations are two orders greater than in nuclear genomesgreater than in nuclear genomes

The suspect: oxidative damage from reactive The suspect: oxidative damage from reactive species (ROS)species (ROS)

Introduction cont.Introduction cont.

Deoxynucleotide-triphosphates (dNTP’s) are Deoxynucleotide-triphosphates (dNTP’s) are the building blocks of DNA the building blocks of DNA

Investigate both the damage to Investigate both the damage to mitochondrial dNTP’s and their built in mitochondrial dNTP’s and their built in repair mechanismsrepair mechanisms

Our PerpetratorOur Perpetrator

8-oxo-GOxidation

Early Trail BlazersEarly Trail Blazers

Mut T mutator genes were discovered in 1954Mut T mutator genes were discovered in 1954 Cells without Mut T enzyme have elevated Cells without Mut T enzyme have elevated

mutation rates of 1000 foldmutation rates of 1000 fold Mut T minus strains of bacteria contained a Mut T minus strains of bacteria contained a

mystery metabolite mystery metabolite The mystery metabolite has never been The mystery metabolite has never been

described beforedescribed before

Time

Ab

s

HPLC

Mut T- Strain

Mystery Metabolite Low Amount of 8-oxo G Substrate for Something Else

HPLC Conclusions

Earlier StudentEarlier Student

Jordan Boutilier worked in the Mathews lab Jordan Boutilier worked in the Mathews lab demonstrated that there were minimal demonstrated that there were minimal amounts of 8-oxo-dGTPamounts of 8-oxo-dGTP

I am going to determine how damaging this I am going to determine how damaging this small amount is to mitochondriasmall amount is to mitochondria

Cells grown without damaging reagents did not Cells grown without damaging reagents did not have reduced concentration of the Mut T have reduced concentration of the Mut T enzymeenzyme

My AimsMy Aims

Compare cellsCompare cells absent of Mut T with absent of Mut T with wild types in terms of oxidative wild types in terms of oxidative damagedamage

Qualitatively analyze 8-oxo-dGTP pools Qualitatively analyze 8-oxo-dGTP pools to understand their role in damaged to understand their role in damaged nucleotidesnucleotides

Identify the Mystery Metabolite using Identify the Mystery Metabolite using Mass SpectrometryMass Spectrometry

Research DesignResearch Design

Compare cellular extract of Mut T wild type and Compare cellular extract of Mut T wild type and mutant strains that are absent of Mut Tmutant strains that are absent of Mut T

Use HPLC and Electrochemical analysis to Use HPLC and Electrochemical analysis to investigate nucleotide pools of the wild and investigate nucleotide pools of the wild and mutant strainsmutant strains

Use both TLC to separate nucleotides and Use both TLC to separate nucleotides and radioactive counter to quantitatively analyze radioactive counter to quantitatively analyze oxidized dNTPoxidized dNTP

Time

ResultsResults

Investigation of enzyme activity Investigation of enzyme activity failed failed

Use of new buffers BICINE MOPSUse of new buffers BICINE MOPS TLC proved to be insensitive to TLC proved to be insensitive to

enzyme activityenzyme activity

Work involving other LabsWork involving other Labs

Dr. Fred Steven’s mass spectrometerDr. Fred Steven’s mass spectrometer Dr. Tory Hagen’s HPLCDr. Tory Hagen’s HPLC

Investigation of Nucleoside Investigation of Nucleoside Diphosphate Kinase Diphosphate Kinase

AbnormalitiesAbnormalitiesHoward Hughes Medical Institute (HHMI)Howard Hughes Medical Institute (HHMI)

Daniel BaiDaniel BaiDr. Christopher K. MathewsDr. Christopher K. MathewsBiochemistry and BiophysicsBiochemistry and Biophysics

Oregon State UniversityOregon State University

OH

OHOP

NO

O

OH

O

OH

O

P

N

NH

N

NH2

O

What is Nucleoside Diphosphate What is Nucleoside Diphosphate Kinase?Kinase?

Nucleoside diphosphate kinase (NDP kinase) is a multifunctional Nucleoside diphosphate kinase (NDP kinase) is a multifunctional enzyme that provides a pathway for both dNTP synthesis and enzyme that provides a pathway for both dNTP synthesis and DNA replication.DNA replication.Studies have shown that absence of NDP kinase does not Studies have shown that absence of NDP kinase does not interfere with cell growth.interfere with cell growth.However, NDP kinase abnormalities have demonstrated highly However, NDP kinase abnormalities have demonstrated highly imbalanced dNTP pool levels and ultimately mutagenesis. imbalanced dNTP pool levels and ultimately mutagenesis.

N

NH

N

N

N

N

P

O

OH

OH

ATP

ADP

(d)NTP

(d)NDP

ATP + (d)NDP ADP + (d)NTP

Medical ImportanceMedical Importance

Breast cancer patients who have abnormal NDP kinase activity Breast cancer patients who have abnormal NDP kinase activity also have a greater probability of cancer metathesis. also have a greater probability of cancer metathesis. In a mismatch repair-defective background, an excess of dCTP In a mismatch repair-defective background, an excess of dCTP or dCTP, increases the chance of insertion errors. (AT -> GC)or dCTP, increases the chance of insertion errors. (AT -> GC)

Past WorkPast Work

Previous researchers in the Mathews Lab have discovered enlargement of Previous researchers in the Mathews Lab have discovered enlargement of dCTP by twentyfold as well as increases in CTP, and dGTP pools in the NDP dCTP by twentyfold as well as increases in CTP, and dGTP pools in the NDP kinase absence kinase absence E-coliE-coli cells. cells.How are we sure that pool imbalances were caused by NDP kinase How are we sure that pool imbalances were caused by NDP kinase abnormalities and not by loss of protein-protein interaction resulting from abnormalities and not by loss of protein-protein interaction resulting from absence of NDP kinase?absence of NDP kinase?A mutant strain with structurally intact, but catalytically inactive form of NDP A mutant strain with structurally intact, but catalytically inactive form of NDP kinase was tested for pool levels. The results were identical to untransformed kinase was tested for pool levels. The results were identical to untransformed mutants. Which suggests that loss of NDP kinase activity is responsible for mutants. Which suggests that loss of NDP kinase activity is responsible for pool imbalances.pool imbalances.

My RoleMy Role

Spent part of past summer successfully isolating a mutant Spent part of past summer successfully isolating a mutant NDP kinase gene from plasmids of Dr. Edith Postel.NDP kinase gene from plasmids of Dr. Edith Postel.This summer’s work involves inserting each mutant gene into This summer’s work involves inserting each mutant gene into a suitable plasmid by gene cloning techniques and then a suitable plasmid by gene cloning techniques and then creating transformed creating transformed E. coliE. coli cells. cells.

Future ExperimentFuture Experiment

The ultimate aim is to determine whether the mutator phenotype of The ultimate aim is to determine whether the mutator phenotype of an an ndkndk mutant results more directly from altered dNTP pools or from mutant results more directly from altered dNTP pools or from loss of a DNA repair activity associated with the enzyme.loss of a DNA repair activity associated with the enzyme.After successful transformation, the mutation rates will be measured.After successful transformation, the mutation rates will be measured.An assay for dNTP pool levels will be performed using a scintillation An assay for dNTP pool levels will be performed using a scintillation counter.counter.NDP kinase enzyme activity will be analyzed using Western Blotting NDP kinase enzyme activity will be analyzed using Western Blotting for protein expression.for protein expression.

AcknowledgementsAcknowledgements

Howard Hughes Medical InstituteHoward Hughes Medical Institute

Dr. Christopher K. MathewsDr. Christopher K. Mathews

Linda BensonLinda Benson

Dr. Rongkun ShenDr. Rongkun Shen

Dr. Edith PostelDr. Edith Postel