The Effect of DNA Methylation on The Effect of DNA Methylation on Deamination Rate and MutagenesisDeamination Rate and Mutagenesis

Tuyen T. DangTuyen T. Dang

Mentor: Dr. Christopher K. MathewsMentor: Dr. Christopher K. Mathews

Biochemistry/BiophysicsBiochemistry/Biophysics

Analysis of Ribonucleotide Reductase Activity in Yeast Mitochondria

RelevanceRelevance► One cause of mutation is deamination.One cause of mutation is deamination.► By knowing how methylation of DNA cytosine By knowing how methylation of DNA cytosine

affects deamination, we can better understand affects deamination, we can better understand what controls the rate of mutagenesis.what controls the rate of mutagenesis.

Project I: The Effect of DNA Project I: The Effect of DNA Methylation on Deamination Rate and Methylation on Deamination Rate and

MutagenesisMutagenesis

DeaminationDeamination

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.

O

NH2

N

NH O

O

NH

NH

CH3

O

NH2

N

NH

CH3

O

O

NH

NH

CytosineUracil

5-methylcytosine Thymine

D eam in a tio n

D eam in a tio n

The The lacZlacZ gene 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.

CCGG CUGGDeamination

CmCGG CTGGDeamination

mCCGG mCUGGDeamination

Control

Experimental

Experimental

White plaques Blue plaques

Research goalsResearch 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 procedureExperimental procedure

Methylate the sequence CCGG to eithermCCGG or CmCGG.

The M13 phage containing the modified sequenceis then incubated for a period of time.

At intervals the modified sequence would then beInserted into the E.coli via electroporation and then plated.

ResultsResults► The ratio of white versus dark The ratio of white versus dark

blue plaques over time would blue plaques over time would determine the rate of determine the rate of mutagenesis.mutagenesis.

My progressMy progress► What I have accomplished:What I have accomplished:

Perfected my electroporation to Perfected my electroporation to get even plaque distribution.get even plaque distribution.

Isolated, purified, and Isolated, purified, and methylated M13 phage.methylated M13 phage.

► Where I am today:Where I am today: Mass production of M13 Mass production of M13

phages.phages. Practicing electroporation.Practicing electroporation.

From: Xiaolin Zhang’s Effect of DNA Cytosine Methylation upon Deamination-induced Mutagenesis in a Natural Target Sequence in Duplex DNA

SummarySummary

►Project I:Project I: Determine if there is a Determine if there is a correlation between methylation and correlation between methylation and deamination rate using the deamination rate using the lacZlacZ gene. gene. Also, determine if the correlation is Also, determine if the correlation is related to the modified DNA helix or is related to the modified DNA helix or is an intrinsic effect of methylating a an intrinsic effect of methylating a particular DNA base.particular DNA base.

Cellular replicationCellular replication► Deoxyribonucleotides or Deoxyribonucleotides or

dNTPs are formed via dNTPs are formed via metabolic methods. One of metabolic methods. One of the steps in the process the steps in the process uses ribonucleotide uses ribonucleotide reductase.reductase.

► In this project I want to In this project I want to understand how the dNTPs understand how the dNTPs are formed for are formed for mitochondrial DNA mitochondrial DNA replication.replication.

Project II: Project II: Analysis of Ribonucleotide Analysis of Ribonucleotide Reductase Activity in Yeast MitochondriaReductase Activity in Yeast Mitochondria

OO

P

O

OO

P

O

O

O

HH

H

H

HBase

H

OH

HH

OH

H

OO

P

O

OO

P

O

O

O

HH

H

H

HBase

H

OH

HH

H

H

R ib o n u c le o tid e re d u c ta s e

ribonucleotide

deoxyribonucleotide

RelevanceRelevance

► There are some human diseases/conditions that are There are some human diseases/conditions that are affected by mitochondrial DNA replication and gene affected by mitochondrial DNA replication and gene expression. Ex. Aging.expression. Ex. Aging.

► By understanding how mitochondria get their By understanding how mitochondria get their dNTPs, we may understand the relationship of some dNTPs, we may understand the relationship of some mitochondrial diseases and mitochondrial mitochondrial diseases and mitochondrial replication.replication.

Possible methods for dNTP Possible methods for dNTP transporttransport

There are two major theories as to how dNTPs enter the mitochondrion so that the mitochondrion can replicate itself.

Intake of dNTP

Synthesis of dNTP within the

mitochondrion

dNTP

Research goalResearch goal

Goal:Goal: Determine whether or not dNTPs are synthesized Determine whether or not dNTPs are synthesized within the mitochondria for mitochondrial DNA within the mitochondria for mitochondrial DNA replication.replication.

Prediction:Prediction: If dNTPs are synthesized within the If dNTPs are synthesized within the mitochondria then there should be detectable mitochondria then there should be detectable ribonucleotide reductase activity inside the ribonucleotide reductase activity inside the mitochondria.mitochondria.

Experimental procedureExperimental procedure

► Before experimenting on yeast mitochondria, I will Before experimenting on yeast mitochondria, I will be experimenting on be experimenting on E.coliE.coli. .

► Break open the cellsBreak open the cells► Take enzyme extractsTake enzyme extracts► Allow the enzyme to react with [Allow the enzyme to react with [33H]CDPH]CDP► Isolate [Isolate [33H]dCDP via thin layer chromatographyH]dCDP via thin layer chromatography► Determine the amount of enzymatic activityDetermine the amount of enzymatic activity

YeastE.coli

ResultsResults

►Calculate the Calculate the amount of amount of enzymatic enzymatic activity per hour.activity per hour.

►My standard My standard deviation is 0.06.deviation is 0.06.

00.10.20.30.40.50.6

EnzymaticActivity

(pmol/ ug ofprotein/ hr)

Trial 1Trial 3Trial 4Trial 5

SummarySummary

►Project II:Project II: Perfect the assay for Perfect the assay for ribonucleotide reductase so that I can ribonucleotide reductase so that I can perform the assay for yeast perform the assay for yeast mitochondria. By studying mitochondria. By studying ribonucleotide reductase, I can ribonucleotide reductase, I can determine where the mitochondria get determine where the mitochondria get their dNTPs.their dNTPs.

Thank YouThank You

► Howard Hughes Medical InstituteHoward Hughes Medical Institute► National Science FoundationNational Science Foundation: Protein-protein : Protein-protein

interaction and DNA precursor biosynthesis, interaction and DNA precursor biosynthesis, Research Experience for UndergraduatesResearch Experience for Undergraduates

► Dr.Christopher K. MathewsDr.Christopher K. Mathews► Dr. Kevin AhernDr. Kevin Ahern► Dr. Indira RajagopalDr. Indira Rajagopal► Linda BensonLinda Benson