Investigation of the Premutagenic Lesion 8-oxo dGTP and its Repair Mechanism Mut T Howard Hughes Medical Institute (HHMI) Daniel Bai Dr. Christopher K. Mathews Biochemistry and Biophysics Oregon State University

Objectives of the Mathews Lab Nucleic Acid Enzymology (DNA replication, repair and mutation) Nucleic Acid Enzymology (DNA replication, repair and mutation) Understanding regulation of DNA synthesis in mitochondria Understanding regulation of DNA synthesis in mitochondria Investigate the enzymes that regulate nucleotide incorporation into DNA Investigate the enzymes that regulate nucleotide incorporation into DNA

Project Introduction Mitochondria are powerhouse of eukaryotic cells Mitochondria are powerhouse of eukaryotic cells Mitochondrial DNA mutations are linked to neurodegenerative disorders, cardiomyopathies, and cancer Mitochondrial DNA mutations are linked to neurodegenerative disorders, cardiomyopathies, and cancer Rate of mitochondria mutations are two orders greater than in nuclear genomes Rate of mitochondria mutations are two orders greater than in nuclear genomes The suspect: oxidative damage from reactive species (ROS) The suspect: oxidative damage from reactive species (ROS)

Introduction cont. Deoxynucleotide-triphosphates (dNTP’s) are the building blocks of DNA Deoxynucleotide-triphosphates (dNTP’s) are the building blocks of DNA Investigate both the damage to mitochondrial dNTP’s and their built in repair mechanisms Investigate both the damage to mitochondrial dNTP’s and their built in repair mechanisms

Our Perpetrator 8-oxo-G Oxidation

Early Trail Blazers Mut T mutator genes were discovered in 1954 Mut T mutator genes were discovered in 1954 Cells without Mut T enzyme have elevated mutation rates of 1000 fold Cells without Mut T enzyme have elevated mutation rates of 1000 fold Mut T minus strains of bacteria contained a mystery metabolite Mut T minus strains of bacteria contained a mystery metabolite The mystery metabolite has never been described before The mystery metabolite has never been described before Time Abs HPLC Mut T- Strain Mystery Metabolite Low Amount of 8-oxo G Substrate for Something Else HPLC Conclusions

Earlier Student Jordan Boutilier worked in the Mathews lab demonstrated that there were minimal amounts of 8-oxo-dGTP Jordan Boutilier worked in the Mathews lab demonstrated that there were minimal amounts of 8-oxo-dGTP I am going to determine how damaging this small amount is to mitochondria I am going to determine how damaging this small amount is to mitochondria Cells grown without damaging reagents did not have reduced concentration of the Mut T enzyme Cells grown without damaging reagents did not have reduced concentration of the Mut T enzyme

My Aims Compare cells absent of Mut T with wild types in terms of oxidative damage Compare cells absent of Mut T with wild types in terms of oxidative damage Qualitatively analyze 8-oxo-dGTP pools to understand their role in damaged nucleotides Qualitatively analyze 8-oxo-dGTP pools to understand their role in damaged nucleotides Identify the Mystery Metabolite using Mass Spectrometry Identify the Mystery Metabolite using Mass Spectrometry

Research Design Compare cellular extract of Mut T wild type and mutant strains that are absent of Mut T Compare cellular extract of Mut T wild type and mutant strains that are absent of Mut T Use HPLC and Electrochemical analysis to investigate nucleotide pools of the wild and mutant strains Use HPLC and Electrochemical analysis to investigate nucleotide pools of the wild and mutant strains Use both TLC to separate nucleotides and radioactive counter to quantitatively analyze oxidized dNTP Use both TLC to separate nucleotides and radioactive counter to quantitatively analyze oxidized dNTP Time

Results Investigation of enzyme activity failed Investigation of enzyme activity failed Use of new buffers BICINE MOPS Use of new buffers BICINE MOPS TLC proved to be insensitive to enzyme activity TLC proved to be insensitive to enzyme activity

Work involving other Labs Dr. Fred Steven’s mass spectrometer Dr. Fred Steven’s mass spectrometer Dr. Tory Hagen’s HPLC Dr. Tory Hagen’s HPLC

Investigation of Nucleoside Diphosphate Kinase Abnormalities Howard Hughes Medical Institute (HHMI) Daniel Bai Dr. Christopher K. Mathews Biochemistry and Biophysics Oregon State University

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

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

Past Work 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 kinase absence E-coli cells. How are we sure that pool imbalances were caused by NDP kinase abnormalities and not by loss of protein-protein interaction resulting from absence of NDP kinase? A mutant strain with structurally intact, but catalytically inactive form of NDP kinase was tested for pool levels. The results were identical to untransformed mutants. Which suggests that loss of NDP kinase activity is responsible for pool imbalances.

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

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

Acknowledgements Howard Hughes Medical Institute Dr. Christopher K. Mathews Linda Benson Dr. Rongkun Shen Dr. Edith Postel