The Effect of DNA Methylation on
Deamination Rate and Mutagenesis
Analysis of Ribonucleotide Reductase
Activity in Yeast Mitochondria
Tuyen T. Dang
Mentor: Dr. Christopher K. Mathews
Biochemistry/Biophysics
Project I: The Effect of DNA Methylation
on Deamination Rate and Mutagenesis
Relevance
► One
cause of mutation is deamination.
► By knowing how methylation of DNA cytosine
affects deamination, we can better understand
what controls the rate of mutagenesis.
Deamination
Deamination is the DNA
removal of an amino
group in a DNA
nucleotide. The most
common deamination is
the change of cytosine to
uracil. Deamination
occurs when there is a
chemical instability in the
DNA.
NH2
O
N
HN
Deamination
N
H
O
N
H
O
Cytosine
Uracil
NH2
O
CH3
CH3
N
HN
Deamination
O
N
H
5-methylcytosine
O
N
H
Thymine
The lacZ gene
The lacZ gene was chosen because it is easy to monitor the
mutation of the gene by observing the change of white versus blue
plaques in an M13 phage/E.coli system.
The difference between white and blue plaques can be determined
by just one base difference.
White plaques
Control
CCGG
Experimental
CmCGG
Experimental
mCCGG
Deamination
Deamination
Deamination
Blue plaques
CUGG
CTGG
mCUGG
Research goals
Goal One: To determine the correlation between methylation of the second
cytosine in CCGG and the deamination rate as was reported by Xiaolin Zhang
and Christopher K. Mathews in 1994.
Basically comparing the deamination rate of CmCGG vs. CCGG.
Goal Two: Methylate the first cytosine in the sequence to test the structure
theory formulated by Vargason and et al. in 2000.
Essentially comparing the deamination rate of mCCGG vs. CCGG.
Experimental procedure
Methylate the sequence CCGG to either
mCCGG or CmCGG.
The M13 phage containing the modified sequence
is then incubated for a period of time.
At intervals the modified sequence would then be
Inserted into the E.coli via electroporation and then plated.
Results
►
The ratio of white versus dark blue
plaques over time would determine
the rate of mutagenesis.
My progress
►
What I have accomplished:
 Perfected my electroporation to get
even plaque distribution.
 Isolated, purified, and methylated
M13 phage.
►
Where I am today:
 Mass production of M13 phages.
 Practicing electroporation.
From: Xiaolin Zhang’s Effect of DNA Cytosine
Methylation upon Deamination-induced Mutagenesis in
a Natural Target Sequence in Duplex DNA
Summary
► Project
I: Determine if there is a correlation
between methylation and deamination rate
using the lacZ gene. Also, determine if the
correlation is related to the modified DNA
helix or is an intrinsic effect of methylating a
particular DNA base.
Project II: Analysis of Ribonucleotide
Reductase Activity in Yeast Mitochondria
Cellular replication
O
H
O
►
►
Deoxyribonucleotides or dNTPs
are formed via metabolic
methods. One of the steps in
the process uses ribonucleotide
reductase.
In this project I want to
understand how the dNTPs are
formed for mitochondrial DNA
replication.
O
O
H
Base
P
P
O
H
O
O
H
ribonucleotide
H O
H
H
H
H
OH
HO
Ribonucleotide
reductase
O
H
O
O
O
H
Base
P
P
O
H
O
O
H
H
deoxyribonucleotide
H O
H
H
H
OH
H
Relevance
►
►
There are some human diseases/conditions that are
affected by mitochondrial DNA replication and gene
expression. Ex. Aging.
By understanding how mitochondria get their dNTPs, we
may understand the relationship of some mitochondrial
diseases and mitochondrial replication.
Possible methods for dNTP transport
There are two major theories as to how dNTPs enter the
mitochondrion so that the mitochondrion can replicate
itself.
dNTP
Intake of dNTP
Synthesis of dNTP
within the
mitochondrion
Research goal
Goal: Determine whether or not dNTPs are synthesized within the
mitochondria for mitochondrial DNA replication.
Prediction: If dNTPs are synthesized within the mitochondria then
there should be detectable ribonucleotide reductase activity
inside the mitochondria.
Experimental procedure
►
►
►
►
►
►
Before experimenting on yeast mitochondria, I will be
experimenting on E.coli.
Break open the cells
Take enzyme extracts
Allow the enzyme to react with [3H]CDP
Isolate [3H]dCDP via thin layer chromatography
Determine the amount of enzymatic activity
E.coli
Yeast
Results
► Calculate
the
amount of
enzymatic activity
per hour.
► My standard
deviation is 0.06.
0.6
0.5
0.4
0.3
0.2
0.1
0
Enzymatic
Activity
(pmol/ug of
protein/hr)
Trial
Trial
Trial
Trial
1
3
4
5
Summary
► Project
II: Perfect the assay for
ribonucleotide reductase so that I can
perform the assay for yeast mitochondria.
By studying ribonucleotide reductase, I can
determine where the mitochondria get their
dNTPs.
Thank You
► Howard
Hughes Medical Institute
► National Science Foundation: Protein-protein
interaction and DNA precursor biosynthesis,
Research Experience for Undergraduates
► Dr.Christopher K. Mathews
► Dr. Kevin Ahern
► Dr. Indira Rajagopal
► Linda Benson