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Investigation of Mutagenesis from Deoxyribonucleotide Pool Abnormalities

Investigation of Mutagenesis from Deoxyribonucleotide Pool Abnormalities. Howard Hughes Medical Institute (HHMI) Daniel Bai Dr. Christopher K. Mathews Biochemistry and Biophysics Oregon State University. Background. What is a dNTP? What is a dNTP pool?

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Investigation of Mutagenesis from Deoxyribonucleotide Pool Abnormalities

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  1. Investigation of Mutagenesis from Deoxyribonucleotide Pool Abnormalities Howard Hughes Medical Institute (HHMI) Daniel Bai Dr. Christopher K. Mathews Biochemistry and Biophysics Oregon State University

  2. Background • What is a dNTP? • What is a dNTP pool? • How are dNTP pool abnormalities related to mutagenesis? Normal dNTP Pool Abnormal dNTP pool dCTP pool dATP pool dTTP pool dGTP pool = more mutations Increase in dCTP pool dATP pool dTTP pool dGTP pool dCTP pool = more mutations Symmetric increase in all 4 pools

  3. Background Continued umuC, D dinF • What is the SOS response? • How does it work? • What is RecA? LexA RecA LexA LexA Upon DNA damage RecA uvrB rNDP reductase LexA suppressing repair systems SOS Activated

  4. Introduction • What does ribonucleotide (rNDP) reductase do? • How does rNDP reductase related to oxidative stress? rNDP reductase rNDP dNDP rNDP reductase

  5. Research Aims • To analyze the effects of oxidative stress on dNTP concentrations. • To understand the correlation between pool imbalances and the mechanisms of mutagenesis. • To study the activity of the enzyme ribonucleotide reductase under oxidative stress. • To measure rates of mutation after oxidative stress.

  6. Research Design • How do we analyze changes in dNTP pools from oxidative stress? • What is the treatment? P2 Phage RecA- mutant E-coli B E-coli B

  7. Questions to be answered • Questions to be answered: • Will RecA mutants contain different dNTP pools compared to the wild type? • What effects does this particular dNTP change have on mutagenesis? • How does the activity of ribonucleotide reductase change in response to oxidative stress? • In cells with abnormal dNTP pools, are the mutations resulting from DNA replication or DNA repair?

  8. Past Experiments John Stenberg • Worked with the enzyme Lex A • Little ribonucleotide reductase activity • Little to no difference in pools

  9. Methods • How are dNTP pools measured? • How are oligonucleotides used? • What is Klenow? • How does a scintillation counter work? Example • To measure dATP (analyzed with 3H dTTP) • Oligonucleotide A A A T A A A T… • Complementary Base pair  T* T* T* A T* T* T* A…

  10. Methods Continued • How is ribonucleotide reductase activity measured? • How are mutation rates measured? Scintillationcounter

  11. Current Results dNTP Pools Changes Under Oxidative Stress What are the treatments? Why is there a decrease in dTTP pools? Chart. 1 dNTP pool changes under 3 treatments, each with 2 runs

  12. Results Decreasing UV • Successfully transduced E-coli cells • Observable UV sensitive RecA- cells Test for RecA- UV sensitivity

  13. Future Work • Measure dNTP RecA mutant dNTP pools • Measure mutation rates • Perform ribonucleotide reductase assay

  14. Acknowledgements • Howard Hughes Medical Institute • Dr. Christopher K. Mathews • Linda Benson • Indira Rajagopal • Dr. Kevin Ahern

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