Expanded Precursor Pools and DNA Methylation as Determinants of Mutagenesis Howard Hughes Medical Institute Summer 2004 Nancy Jade Lee Dr. Christopher K. Mathews’ Laboratory Department of Biochemistry & Biophysics Oregon State University

Imbalances in Nucleotide Levels: Mutagenesis by Expanded DNA Precursor Pools of Mammalian Cells  To analyze DNA building block concentrations (Deoxyribonucleoside triphosphates or dNTPs)  To understand the relationship between intracellular dNTP concentration and mutagenesis  To study the effects of hydroxyurea on ribonucleotide reductase (rNDP reductase) in mammalian cells

Background: dNTPs  dNTPs are necessary for the biosynthesis of DNA dATP dTTP dCTPdGTP *The amount of each dNTP contained in a cell is referred to as a “pool”

dNTP Pools  Normal cells have balanced pool sizes  Unbalanced pool sizes can stimulate mutagenesis  Example dATP pooldTTP pooldGTP pool dCTP pool = more mutations However… In E. coli cells balanced increases in dNTP pools also stimulates mutagenesis dATP pooldTTP pooldGTP pooldCTP pool = more mutations

dNTP Biosynthesis  To make dNTPs ribonucleoside diphosphate (NDP) must convert to deoxyribonucleoside diphosphate (dNDP) Ribonucleotide Reductase (rNDP reductase)

Hydroxyurea-resistant Cell Lines  Hydroxyurea-resistant cell lines carry elevated levels of ribonucleotide reductase Hydroxyurea- resistant Cell   Wild Type Cell

Question  Do hydroxyurea-resistant mammalian cells exhibit enlarged dNTP pools?  If so, do these cells also have elevated spontaneous mutation rates?

Measuring dNTP Pools Example  To measure dATP (analyzed with 3 H dTTP)  Template  A A A T A A A T… Radiolabeled dNTP ( 3 H dTTP and 3 H dATP) are counted in a scintillation counter Radiolabeled dNTP ( 3 H dTTP and 3 H dATP) are counted in a scintillation counter This tells us how much regular dNTP a sample contains This tells us how much regular dNTP a sample contains Base pair  T* T* T* A T* T* T* A… Base pair  T* T* T* A T* T* T* A…

dNTP Pool Assay Results  Hydroxyurea-resistant cells exhibit balanced increases in dATP, dGTP, and dTTP pools  decrease in dCTP pool size  Balanced increase in all four dNTP pools not observed

Measuring Mutagenesis  Known quantity of cells plated in the presence of 6- thioguanine  Targets hypoxanthine-guanine phosphoribosyltransferase (HGPRT) Wild type cells Mutagenic cells

6-Thioguanine Assay Results  No significant difference in the number surviving colonies (wild type vs. hydroxyurea-resistant)  Higher rate of mutagenesis not detected in hydroxyurea- resistant clones (plated 1 X 10 6 cells per plate) Trial #1Trial #2Trial #3 V79 Wild Type 0,32,01,1.35 mM HU-Res clone 1 0,33,20,0.35 mM HU-Res clone 2 2,10,00,2 1.3 mM HU-Res clone A 0,0 0,1 Surviving Colonies (per plate)

Summary  Balanced increase in all four dNTP pools of hydroxyurea- resistant cells not observed  Higher rate of mutagenesis in hydroxyurea-resistant cells not observed Further Research  Investigate and analyze other hydroxyurea-resistance clones available in the lab  Develop and test a model to explain the dNTP pool changes and rate of mutagenesis seen in the hydroxyurea-resistance cells dATP pooldTTP pooldGTP pool dCTP pool

Chemical Reactions of DNA Bases: Mutagenesis as a Result of Deamination at 5-Methylcytosine  To understand the complex relationship among methylation of a base, deamination and the rate of mutagenesis  To examine the rate of deamination at methylcytosine-cytosine- guanine (mCCG) regions versus cytosine-methylcytosine-guanine (C m CG) regions Importance Link to cancer Point mutations “hotspot” “hotspot”

Background: DNA Bases AATCCGGTAT Methylation

Deamination of Cytosine and 5- methylcytosine

Base Excision Repair  Cytosine (C) CG  UG  CG  5-methylcytosine ( m C) m CG  TG = point mutation = repaired = not repaired m C – G T – A 

Question  Why does deamination occur?  Which factor has the most effect on the high rate of deamination seen at the 5-methylcytosine base?  Methylation  Adjacent to Guanine  Structural change mCG

Methods CCGG GGCC CCGG GGCC CCGG GGCC Control Outer C methylated Inner C methylated = high rate of mutagenesis due to methylation = high rate of mutagenesis due to structural changes Analyzing deamination at this site: C  T or U

Acknowledgements  Howard Hughes Medical Institute (HHMI)  Christopher Mathews  Linda Wheeler  Indira Rajagopal  Kevin Ahern  Stephanie Junell  Carolyn Wiesner  Department of Biochemistry & Biophysics