Date of Award
Master of Science (MS)
John A. Duerre
The properties of DNA methyltransferase in various age rats were investigated to better characterize the enzyme. When purified chromatin from 5-day-old rat brain or liver nuclei was incubated with S-adenosyl-L-[methyl-3H]-methionine, 5-[methyl-3H]methyl-cytosine was the only product of methylation. The optimum pH of the DNA methyltransferase was approximately 8.0 with little or no change in activity over a pH range of 6.9 to 8.3. The Km value of the chromosomal bound DNA methyltransferase for S-adenosyl-L-methionine (AdoMet) was 3.5 ± 0.15 μm and 4.2 ± 0.13 μm, in the liver and brain, respectively. The Vmax values for the enzyme from liver and brain of 5-day-old rats were 0.17 ± 0.04 and 0.23 ± 0.09 pmol of 3H-methyl groups incorporated/mg DNA/min, respectively. S-adenosyl-L-homocysteine was found to inhibit competitively the DNA methyltransferase in the brain with a Ki value of 3.5 ± 1.6 μm. The enzyme was firmly bound to chromatin. After ten extractions with water or 20 mM potassium phosphate buffer, pH 7.0, only trace amounts of enzyme could be eluted from the chromatin. Measurable amounts of the enzyme could be extracted from chromatin with 0.4 M NaCl. However, measurable amounts of the DNA methyltransferase were still associated with the NaCl extracted chromatin.
The rate of the reaction catalyzed by the chromosomal-bound DNA methyltransferase was dependent on the age of the rat and the type of cell from which the chromatin was isolated. In the liver, the rate declined from 0.28 pmol of methyl-3H incorporated/mg DNA/min at birth to 0.13 pmol of methyl-3H incorporated/mg DNA/min at 30 days. Enzyme activity remained at this level throughout life. In the brain, the rate decreased rapidly from 0.48 pmol of methyl-3H incorporated/mg DNA/min at birth to 0.05 pmol of methyl-3H incorporated/mg DNA/min at 30 days. Little or no activity could be detected after 30 days.
Chromosomal DNA methyltransferase activity was found to be dependent on the availability of methyl acceptors and enzyme concentration. The number of methylation sites available for in vitro methylation in liver decreased from 3.5 pmol of 5-[methyl-3H]methylcytosine/mg DNA at birth to 1.5 pmol of 5-[methyl-3H]methylcytosine/mg DNA at 14 days, remaining constant thereafter. The number of methyl acceptors in the brain decreased from 8.5 pmol of 5-[methyl-3H]methylcytosine/mg DNA at birth to 2.5 pmol of 5-[methyl-3H]methylcytosine/mg DNA at 40 days, remaining constant thereafter.
The level of extractable enzyme from the cerebellum increased from 0.13 pmol of methyl-3H incorporated/mg protein/min at birth to 0.29 pmol of methyl-3H incorporated/mg protein/min at 10 days. The level of the enzyme decreased thereafter to 0.07 pmol of methyl-3H incorporated/mg protein/min in 30-day-old rats, remaining at this level throughout life. The amount of extractable enzyme from the cerebrum decreased from 0.13 pmol of methyl-3H incorporated/mg protein/min at birth to 0.07 methyl-3H incorporated/mg DNA/min in 30-day-old rats, remaining at this level of activity thereafter. The extractable enzyme from the liver increased from 0.45 pmol of methyl-3H incorporated/mg protein/min at birth to 0.60 pmol of methyl-3H incorporated/mg protein/min at 16 days. In 30-day-old rat liver the enzyme decreased to 0.10 pmol of methyl-3H incorporated/mg protein/min, remaining at this level until 100 days. At this time the extractable enzyme increased to 0.25 pmol of methyl-3H incorporated/mg protein/min at 300 days, remaining at approximately this level, thereafter.
The level of DNA methyltransferase and the number of in vitro methylation sites available on DNA correlated well with the rate of DNA synthesis in these organs. The synthesis of DNA methyltransferase appears to be coupled to DNA synthesis.
Quick, Douglas P., "In Vitro Studies on the Methylation of DNA in Rat Brain and Liver Chromatin" (1978). Theses and Dissertations. 620.