Investigation of Mutagenesis from Deoxyribonucleotide Pool

Abnormalities

Howard Hughes Medical Institute (HHMI)

Daniel BaiDr. Christopher K. Mathews

Biochemistry and BiophysicsOregon State University

Background

• What is a dNTP?

• What is a dNTP pool?

• How are dNTP pool abnormalities related to mutagenesis?

dATP pooldATP pool dTTP pooldTTP pool dCTP pooldCTP pooldGTP pooldGTP pool = more mutations

= more mutationsdATP pooldATP pool dTTP pooldTTP pool dCTP pooldCTP pooldGTP pooldGTP pool

Increase in dCTP pool

Symmetric increase in all 4 pools

Normal dNTP Pool

Abnormal dNTP pool

Background Continued

• What is the SOS response?

• How does it work?

• What is RecA?

rNDP reductaseuvrB

LexA

LexA

umuC, D

RecA

dinF

LexA suppressing repair systems

Upon DNA damage

LexARecA

SOS Activated

Introduction

• What does ribonucleotide (rNDP) reductase do?

• How does rNDP reductase related to oxidative stress?

rNDP dNDP

rNDP reductase

rNDP reductaserNDP reductase

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.

Research Design

• How do we analyze changes in dNTP pools from oxidative stress?

• What is the treatment?

P2 PhageP2 PhageE-coli BE-coli B RecARecA-- mutant E-coli B mutant E-coli B

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?

Past Experiments

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

Methods

• How are dNTP pools measured?

• How are oligonucleotides used?

• What is Klenow?

• How does a scintillation counter work?

Complementary Complementary Base pair Base pair T* T* T*T* T* T* A A T* T* T*T* T* T* A… A…

Example

– To measure dATP (analyzed with 3H dTTP)• Oligonucleotide A A A T A A A T…

Methods Continued

• How is ribonucleotide reductase activity measured?

• How are mutation rates measured?

ScintillationScintillation countercounter

Current Results

dNTP Pools Changes Under Oxidative Stress

What are the treatments?

Why is there a decrease in dTTP pools?

Chart. 1dNTP pool changes under 3 treatments, each with 2 runs

E-coli dNTP Pools After Oxidative Stress

0

0.2

0.4

0.6

0.8

1

1.2

1.4

no trt. growth w/ trt trt after 25 mingrowth

dN

TP

(p

mo

l)/p

rote

in (

ug

)

dATP

dTTP

dGTP

dCTP

Results

• Successfully transduced E-coli cells

• Observable UV sensitive RecA- cells

Test for RecA- UV sensitivity

Decreasing UV

Future Work

• Measure dNTP RecA mutant dNTP pools• Measure mutation rates• Perform ribonucleotide reductase assay

Acknowledgements

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