Analysis of 8-oxo-dGTP, a mutagenic nucleotide, at

physiological levels in E.coli

Jordan Kane Boutilier

Mentor: Dr. Christopher Mathews

Department of Biochemistry and Biophysics

Oregon State University

Reactive oxygen species (ROS)

• Generated via cellular respiration

• Most ROS are free radicals that contain unpaired electrons

• Oxidative Stress

• Oxidative damage

• Causes [nucleo]base modification

ROS induced mutagenesis: 7,8-dihydro-8-oxoguanine (8-oxodG)

87

9

5

43

2

16

•OH

dRDeoxyguanosine

Alternative base pairings observed for (8-oxo-G).

Normal G:C Watson Crick base pair

7,8-dihydro-8-oxoguanine forms stable base pair with adenine

MutT removes 8-oxo-dGTP from Nucleotide pools

Is 8-oxo-dGTP a critical substrate for MutT?

• mutT mutants can display a mutator phenotype during anaerobic growth

• 8-oxo-dGTP is poor substrate for DNA polymerase

• Physiological levels: dNTP precursor pools

Biosynthesis of precursor poolsIMP

AMP GMP

GDPADP

dADP dGDP

dATP 60μM dGTP 10μM

CTP UTP UMP

UDPCDP

dUDPdCDP

dCTP 30μM dTTP*60μM

rNDP reductase

NDP kinase

Hypothesis: 8-oxo-dGTP is not mutagenic at intracellular levels.

method

HPLC

UV detection EC detection

mutTmutT-

Extract nucleotides

mpA

Linear gradient

mpB

Identify/quantify nucleotides

52-278M E.coliB

Calibration technique

HPLC Elution Profile of Standards

Approach

• Comparison of crude extracts of E. coli mutT wild type and mutant strains

• preliminary quantification of 8-oxo-dGTP at physiological levels

Detection of 8-oxo-dGTP

52-278M

52-278M + 60fmol spike 8-oxo-dGTP

E.coliB +420mV

Significance

• 8-oxo-dGTP at physiological levels is extremely low

• Comparison to other dNTPs

• Most likely not mutagenic at this level

• Not the critical substrate for MutT

Acknowledgments

• Dr. Mathews and Lab

• Dr. Tory Hagen and Lab

• Dr. Kevin Ahern

• Mary Lynn Tassotto

• HHMI

• National Science Foundation (Undergraduate research supplement)