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DNA methylase from Pisum sativum
H M Yesufu1, A Hanley, A Rinaldi
1Department of Biochemistry, University of Glasgow, U.K.
Abstract:
DNA methylase activity was detected in nuclei from pea shoots. The enzyme can only be extracted by low-salt treatment if the nuclei are pretreated with micrococcal nuclease. Only a single enzyme was detected, and it was purified to a specific activity of 1620 units/mg of protein. It has an Mr of 160,000 on gel filtration and SDS/PAGE. Pea DNA methylase methylates cytosine in all four dinucleotides, and this is interpreted to show that it acts on CNG trinucleotides. Although it shows a strong preference for hemi-methylated double-stranded DNA, it is also capable of methylation de novo. Homologous DNA is the best natural substrate. In vitro the enzyme interacts with DNA to form a salt-resistant complex with DNA that is stable for at least 4 h.
Insights
Pea shoot nuclei contain a DNA methylase enzyme that can be extracted after micrococcal nuclease pretreatment. This purified enzyme methylates DNA, preferring hemi-methylated substrates and homologous DNA, with potential for de novo methylation.
Area of Science:
- Plant molecular biology
- Enzymology
- Epigenetics
Background:
- DNA methylation is a crucial epigenetic mechanism regulating gene expression in eukaryotes.
- Understanding DNA methyltransferases (DNMTs) is key to deciphering epigenetic regulation in plants.
Purpose of the Study:
- To detect, extract, and purify DNA methylase activity from pea (Pisum sativum) shoot nuclei.
- To characterize the biochemical properties and substrate specificity of the purified pea DNA methylase.
Main Methods:
- Nuclei isolation from pea shoots.
- Enzymatic extraction using micrococcal nuclease pretreatment and low-salt treatment.
- Protein purification using chromatography.
- Enzyme characterization via gel filtration and SDS-PAGE.
- DNA methylation assays using various DNA substrates.
Main Results:
- A single DNA methylase was purified from pea shoot nuclei with a specific activity of 1620 units/mg.
- The enzyme has an estimated molecular weight of 160,000 Da.
- Pea DNA methylase methylates cytosine in CNG trinucleotides, shows a preference for hemi-methylated DNA, and can perform de novo methylation.
- Homologous DNA serves as the optimal natural substrate in vitro.
- The enzyme forms a stable, salt-resistant complex with DNA.
Conclusions:
- A novel DNA methylase from pea shoots has been purified and characterized.
- The enzyme's properties suggest a role in plant epigenetic regulation, potentially targeting specific DNA sequences.
- Further studies can elucidate its in vivo function and interaction dynamics with DNA.