Investigation of the Premutagenic Lesion 8-oxo dGTP and its Repair Mechanism Mut T

1. 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

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

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

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

5. Our Perpetrator 8-oxo-G Oxidation Mut-T 8-oxo-G Adenine Transersion Mutation

6. Early Trail Blazers • Mut T mutator genes were discovered in 1954 • Cells without Mut T enzyme have elevated mutation rates of 1000 fold • Mut T minus strains of bacteria contained a mystery metabolite • The mystery metabolite has never been described before HPLC Abs HPLC Conclusions Time

7. Earlier Student • 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 • Cells grown without damaging reagents did not have reduced concentration of the Mut T enzyme

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

9. Research Design • 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 both TLC to separate nucleotides and radioactive counter to quantitatively analyze oxidized dNTP Time

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

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

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

13. 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.

14. 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)

15. 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.

16. 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.

17. 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.

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