The Effect of DNA Methylation on Deamination Rate and Mutagenesis Tuyen T. Dang Mentor: Dr. Christopher K. Mathews Biochemistry/Biophysics Analysis of.

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Presentation transcript:

The Effect of DNA Methylation on Deamination Rate and Mutagenesis Tuyen T. Dang Mentor: Dr. Christopher K. Mathews Biochemistry/Biophysics Analysis of Ribonucleotide Reductase Activity in Yeast Mitochondria

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

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. CCGGCUGG Deamination CmCGGCTGG Deamination mCCGGmCUGG Deamination Control Experimental White plaques Blue plaques

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 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 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.

Cellular replication ► 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. Project II: Analysis of Ribonucleotide Reductase Activity in Yeast Mitochondria

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. Intake of dNTP Synthesis of dNTP within the mitochondrion dNTP

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 [ 3 H]CDP ► Isolate [ 3 H]dCDP via thin layer chromatography ► Determine the amount of enzymatic activity Yeast E.coli

Results ► Calculate the amount of enzymatic activity per hour. ► My standard deviation is 0.06.

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